Thermal rectification is a fundamental phenomenon for active heat flow control. Significant thermal rectification is expected to exist in the asymmetric nanostructures, such as nanowires and thin films. As a one-atom-thick membrane, graphene has attracted much attention for realizing thermal rectification as shown by many molecular dynamics simulations. Here, we experimentally demonstrate thermal rectification in various asymmetric monolayer graphene nanostructures. A large thermal rectification factor of 26% is achieved in a defect-engineered monolayer graphene with nanopores on one side. A thermal rectification factor of 10% is achieved in a pristine monolayer graphene with nanoparticles deposited on one side or with a tapered width. The results indicate that the monolayer graphene has great potential to be used for designing high-performance thermal rectifiers for heat flow control and energy harvesting. Thermal rectification is instrumental to achieving active heat flow control and energy harvesting in nanoscale devices. Here, the authors demonstrate thermal rectification in asymmetric graphene nanostructures, achieving a large rectification factor up to 26%. One of the most attractive issues associated with thermal management in solid-state devices is thermal rectification124678911121314η is ∼26%, while in the other two structures, η is ∼10%. Theoretical and MD analysis show two different mechanisms for the varied thermal rectification behaviour in the different asymmetric graphene nanostructures.However, as far as we know, thermal rectification in suspended monolayer graphene has not been realized experimentally. This article reports the monolayer graphene thermal rectifiers with different asymmetric designs. The experiments were conducted with five suspended monolayer graphene devices, including three asymmetric nanostructures: graphene with nanopores on one side, graphene with nanoparticles deposited on one side and graphene with a tapered width. In the first structure, the measured thermal rectification factor The monolayer graphene thermal rectifiers were prepared based on our previously reported method for suspending large-area graphene20The H-type sensor method was used to measure the thermal rectification effect in the experiments. This method meets the two basic requirements for examining thermal rectification. First, the heat flow direction through the graphene sample is reversible. Second, the thermal conductivity of graphene can be measured with high accuracy. As shown in TM dual-beam system, where the ion beam was used to create defects and the electron beam was used to observe the sample. The defect-engineered graphene samples are shown in The nanomanufacturing of the suspended monolayer graphene used a FEI Versa 3D−4 Pa. In the experiments, one sensor was heated by a large direct current (DC) current, while the other sensor was used as a precise resistance thermometer to monitor the temperature rise caused by the heat conduction through the monolayer graphene. The thermal conductivity of graphene was then calculated based on a two-dimensional heat conduction model for the H-type sensor. The thermal analysis was performed using a commercial finite-element software COMSOL Multiphysics. Nine models were built in COMSOL (one model for sample #5 and two models for the other four samples) for thermal analysis of each graphene sample before and after nanomanufacturing. The contact thermal resistance, Rc, between the monolayer graphene and the sensor was analysed using a fin thermal resistance modelRc was ∼12% of the total thermal resistance of the pristine graphene and ∼3% of that of the modified graphene =0.148 for the sample #1. The estimated λ1 of the region with nanopores is independent of temperature, which is 230 Wm−1K−1, ∼10% of λP, the thermal conductivity of pristine grapheneλP. λ2 is temperature dependent, which is 380 Wm−1K−1 in case I and 530 Wm−1K−1 in case II of λcase I as 345 Wm−1K−1 and λcase II as 442 Wm−1K−1. The calculated thermal rectification factor, η, is then 28%, which agrees well with the experimental result. It should be mentioned that the thermal rectification factor is dependent on the temperature difference between both sides of the graphene sample. If the temperature difference is small, the thermal rectification caused by the asymmetric distribution of thermal conductivity in graphene will be weakened.where In addition to the defect-engineered samples, the asymmetric pristine graphene samples, #4 and #5, were also fabricated. The scanning electron microscopy images are shown in Graphene sample #4 had ten 200 nm diameter amorphous carbon nanoparticles deposited on one side. The nanoparticles can be clearly distinguished in the scanning electron microscopy image. For the sample #5, the graphene ribbon was designed with a tapered width. The asymmetric two-dimensional geometry can cause thermal rectification behaviour in the suspended graphene.To understand the physical mechanisms of thermal rectification in sample #5, we have performed MD simulation and computed the spatial energy distribution for the propagating phonon modes in the non-equilibrium steady-state for the trapezoid graphene sheet (Methods section). The calculation result is shown in L=17 nm, W1=22 nm, W2=2 nm and the total number of C atoms is 7,420. We fix the angle θ in the trapezoid graphene and increase both L and W proportionally, extending the width W1 to 200 nm, and 440 nm, respectively. There are 2,985,734 C atoms in the MD model for the largest width we considered. It is a formidable task to further extend the MD simulation size to the micrometre scale, due to the tremendously increased computational load at large scale  is relatively small. In other words, the narrow end of graphene at high temperature appears to be the bottleneck for the propagating phonon modes, where the edge scattering affects a larger number of phonons than that at the wide end. In this way, the thermal conductivity in the heat flux direction from the narrow end to the wide end is smaller than that in the opposite direction. see the . The MD  see the , followisp2 bonding, graphene has unusually long phonon mean free path, which ranges from sub-micrometre to hundreds of micrometres at room temperature30Originated from the ultra-strong Another approach to induce additional phonon scattering is via the mass loading to the graphene sheet, where the thermal conductivity can be suppressed. This is the case of graphene sample #4. To simulate the asymmetric structure of sample #4, we asymmetrically deposited heavy atoms on one side of the graphene in the MD model, as shown in The simulation result indicates that the heat flux in graphene is larger from the clean region to the other region with deposited heavy atoms, which is consistent with the experimental result of sample #4. We find that the deposited heavy atoms can cause significant phonon scattering in the graphene sheet. Similar to the narrow end in the trapezoid graphene sheet, the deposited region at high temperature appears to be the bottleneck for the propagating phonon modes in graphene, so that fewer propagating modes can reach the cold side. Hence, the local energy of propagating modes is smaller in the heat flux direction from the deposited region to the clean region, and the thermal conductivity is smaller as well. We have repeated the MD simulation by using different Lennard-Jones potential parameters and obtained similar results. More details about the MD simulation can be found in the This work reports on two different thermal rectification phenomena in suspended monolayer graphene. The asymmetric defect-engineered samples had measured thermal conductivities ∼26% higher for heat flow from the region with nanopores to the region without pores. Asymmetric pristine sample with tapered width/nanoparticles had measured thermal conductivity ∼10% higher for the heat flow from the wide/clean region to the narrow/deposited region. The inseparable dependence of the thermal conductivity on the temperature and space is found to be the mechanism for the first thermal rectifier type. The asymmetric phonon scattering varying with the heat flux direction is found to be responsible for the second thermal rectifier type. The suspended monolayer graphene provides an excellent platform for designing two-dimensional thermal rectifiers which have potential applications in autonomous heat flow control and efficient energy harvesting.2/Si substrate following a traditional polymethyl methacrylate (PMMA) method. Then the graphene was cut into micro ribbons by electron beam lithography and O2 plasma etching. Different shapes and sizes of graphene ribbon can be designed in this step. After that, the metallic sensor and electrode pads were created on graphene by using an electron beam physical vapour deposition method. A graphene ribbon bridged between two metallic thin film sensors, forming an H shape. Then, another protection resist layer was spin-coated on the sample, leaving several uncovered windows for etching the substrate underneath. The SiO2 layer and Si substrate were separately etched by buffered hydrofluoric acid and XeF2 gas. Due to the isotropic nature of XeF2 gas etching, the metallic sensors and graphene were completely suspended from the substrate. The gas etching time was controlled by observing the suspended area under an optical microscope. Finally, the protection resist layer and SiO2 layer at both sides of graphene were removed by wet etching. The whole device was dried following a standard supercritical point drying procedure to avoid rupture of graphene caused by liquid surface tension.We have developed a new method for preparing large-area suspended monolayer graphene. The graphene grown by chemical vapour deposition method was transferred onto SiO−4 Pa. In the steady-state, the thermal conductivity of graphene is directly related to the temperature difference between two sensors  was computed as:Non-equilibrium MD simulations were performed with the LAMMPSJ+ and J− are the steady-state heat flux in two directions. More details about the simulation protocol can be found in our previous publicationwhere i was defined as36The spatial energy distribution on atom n is the phonon occupation number given by the Bose-Einstein distribution and εiα,λ is the αth eigenvector component of eigen-mode λ for the ith atom. To measure the energy distribution, the participation ratio (Pλ) for each phonon mode λ was computed as3637where N is the total number of atoms. To select the propagating phonon modes, only phonons with Pλ>0.4 were included in the summation in Eq. 3. More details can be found in the where The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Wang, H. et al. Experimental study of thermal rectification in suspended monolayer graphene. Nat. Commun.8, 15843 doi: 10.1038/ncomms15843 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Tables.


Our measurement results have shown that bilayer graphene exhibits an unexpected sharp transition of the resistance value in the temperature region 200~250 K. We argue that this behavior originates from the interlayer ripple scattering effect between the top and bottom ripple graphene layer. The inter-scattering can mimic the Coulomb scattering but is strongly dependent on temperature. The observed behavior is consistent with the theoretical prediction that charged impurities are the dominant scatters in bilayer graphene. The resistance increase with increasing perpendicular magnetic field strongly supports the postulate that magnetic field induces an excitonic gap in bilayer graphene. Our results reveal that the relative change of resistance induced by magnetic field in the bilayer graphene shows an anomalous thermally activated property. There iπ –4. Recenπ [π –8. It haπ [π . In bilang model –15. Whatng model –18. Varing model . There aR-T property and an unexpected sharp transition of the resistance value in the temperature region 200~250 K. The longitudinal resistance decreases with increasing temperature and electric field, a behavior that is markedly different from the experimental reports of monolayer graphene. Our results reveal that the energy gap in the bilayer graphene shows an anomalous thermally activated property and increases with In this paper, we have systematically investigated the charge transport properties in bilayer graphene as a function of temperature, magnetic field, and electric field. Our measurement results have shown that bilayer graphene exhibits a semi-metallic SiO2. The doped silicon substrate and SiO2 were used as back-gate and gate dielectric, respectively. Graphene electrical electrodes were patterned using photolithography techniques. A pair of ohmic Cr/Au (5 nm/100 nm) contacts were deposited via thermal evaporation at a background pressure of 10−7 mbar and subsequently lifted off in warm acetone. Electronic transport measurements have been carried out on multiple samples, using PPMS (Quantum Design) with a fixed excitation current of 10 μA. Electrical measurements were performed in the temperature range 2~340 K, and a magnetic field up to 12 T was applied. In order to enhance electrical transport, the sample was cleaned in situ by the magnetic and electric field. Four-terminal electrical measurements were used for transport characterization.The bilayer graphene flakes in our study were prepared via mechanical exfoliation techniques from the bulk highly oriented pyrolytic graphite  and transferred onto the surface of a lightly doped silicon substrate covered with a 285-nm-thick layer of thermally grown G peak and 2D band. The two most intense features are the G peak and the 2D band which is sensitive to the number of layers of graphene. The position of the G peak and the shape of the 2D band confirm the number of layers of graphene. Additionally, the number of layers of graphene can be easily distinguished from the full-width half maximum of the 2D band, as its mode changes from a narrow and symmetric feature for monolayer graphene to an asymmetric distribution on the high-energy side for bilayer graphene [Rxx as a function of carrier density n, and the sample shows a pronounced peak at density 5.48 × 10−10 cm−2. Note that the sharp peak in resistance at low n is enhanced by the opening of the small energy gap owing to the disorder-induced differences in carrier density between the top and the bottom layers of the flake.It has been shown that Raman spectroscopy is a reliable, nondestructive tool for identifying the number of graphene layers and it can be done through the 2D band deconvolution procedure –22. The graphene . The 2D I)-voltage (V) characteristics of the bilayer graphene via four-terminal measurement, at different temperatures and magnetic fields. Shown in Fig. I-V curves for bilayer graphene under the application of various magnetic fields at three different temperatures: 2, 200, and 340 K. The magnetic field is applied in the perpendicular direction to the plane of the graphene. For all the temperatures and magnetic field strengths, the bilayer graphene exhibits a linear I-V curve. This implies that the graphene layer is ohmic in nature. We observed that for a fixed magnetic field, the I-V curve displays a larger gradient at a higher temperature than at lower temperature. Interestingly, the gradient of the I-V curve decreases with increasing magnetic field. In our structure, the gradient of the curve corresponds to the conductivity of the graphene layer. Such temperature- and magnetic field-dependent behavior of conductivity are characteristic of an intrinsic semiconductor. The decrease in the conductivity of the bilayer graphene with increasing magnetic field is attributed to the excitonic energy gap induced by the magnetic field. This conductivity dependence on the magnetic field suggests that the resistance We have characterized the current . The conductivity of the surface roughness model at the limit n → 0 at low temperature is ΔR ≈ (ℏ/e2)(Td/2πka)2, which yields ∆R~100 to 1000 Ω at T = 300 K. The model of quenched-ripple disorder [R-T curve rather than the sudden decrease in R-T as observed for our bilayer graphene. In the absence of a theory to explain the stronger temperature-dependent behavior, we propose that the behavior is consistent with the ripple effect interlayer scattering instead of interlayer scattering. Figure R-T above 200 K can be attributed to the transition between the low- and high-T limits in the interlayer ripple effect scattering. One should note that the effect of the ripple will be screened as the number layer increasing in our previous reports [Ripples are a common feature of cleaved graphene because it is never atomically flat, as it is placed on a substrate such as SiO ripples , 41. Des ripples –43. Alte ripples , 45. The ripples . Theoret ripples  show thabon atom . For lowRn→0~1n2 . As the disorder  suggeste reports , 48.T dependence could be explained by thermally excited surface polar phonons of the SiO2 substrate [2 optical phonons at the substrate-graphene interface induce an electric field which couples to the carriers in graphene due to it modulating the polarizability [Conversely, it was suggested that the observed strong ubstrate –37. The zability . Howeverzability . RecentlQ is the charge of impurities. Based on the Boltzmann transport theory, we can obtain the bilayer graphene resistivity with massless Dirac fermions (MDF) at low energies as E → kBT, we can obtain the bilayer graphene resistivity as Nss is the density of impurities per unit volume, εs is the permittivity of the semiconductor, and Zi is the charge state of the impurity. This shows that the resistance of bilayer graphene limited by Coulomb scattering increases as Nss increases and decreases with increasing temperature. Considering the above analysis, we deduce that the temperature dependence of resistance in bilayer graphene is mainly determined by Coulomb scattering. The short-range scattering is independent of temperature for bilayer graphene, as the density-of-states, the matrix element, and the screening function are all energy independent. As a result of the parabolic band structure of bilayer graphene, the energy averaging of the Coulomb scattering time can give rise to the resistivity decreasing proportionally to temperature: ρbilayer ∝ (kBT)− 1.We introduce a relaxation-time approximation and treat the unscreened Coulomb potential as mentclass2pt{minimeQ4πε0εr  where Q nℏεs2kBT , where NRbilayer = RC − bilayer + Rsr ‐ bilayer, where RC − bilayer and Rsr ‐ bilayer are the resistance due to the Coulomb and short-range scatterings, respectively. Figure Rxx ∝ exp(ΔE/kBT), where ∆E is the energy gap. The opening of the energy gap due to a potential difference between the two layers and Coulomb interactions could be a cause for this. These considerations explain qualitatively why the resistance of bilayer graphene decreases with increasing temperature. Note that the relative resistance change is a strong function of temperature. At temperatures of 2~180 and 220~250 K, the relative resistance ∆R strongly increases as temperature increases, indicating that an energy gap forms due to many-body correction in Landau Level. When the temperature increases to T > 250 K, the relative resistance ∆R is roughly independent of the increasing temperature; this indicates that the energy gap is mostly stable at high temperatures. On the other hand, with the temperature increase from 180 to 220 K, the relative resistance ∆R dependence of temperature shows a sharp decrease, which indicates that the energy gap shows an anomalous thermally activated behavior as a function of temperature.Based on the above discussion, we fit the measured resistance in Fig. Rxx as a function of applied perpendicular field B. Figure Rxx of bilayer graphene as a function of magnetic field at T = 2 K at the charge-neutrality point. We have plotted the resistance per square Rxx because it is independent of a size effect of the sample. As seen from Fig. Rxx increases nonlinearly with the magnetic field strength followed by a plateau-like phase. One should note that the plateau-like phase in Fig. kB is the Boltzmann constant. We found that our results are in good agreement with this equation. These considerations explain qualitatively why the nonlinear resistance Rxx increases with the magnetic field.For zero gate voltage , we measured changes in longitudinal resistance E) under different magnetic fields. The dependent Rxx-E characteristics are symmetric due to the chirality of graphene electrons when an applied electric field changes from E to −E. The normalized resistance Rxx-E curve describes the response under the applied magnetic field in the range of B = 0 T to B = 12 T and the temperatures of 2~340 K. The results demonstrate that when the magnetic field increases from 0 to 12 T at low temperatures (2~200 K) and low electric field (E < 0.001 V/μm), the resistance of bilayer graphene drops significantly. The larger slump in the resistance at lower temperature T = 2 K and low electric field as the increasing of the electric field is due to Coulomb scattering by impurities, which is a strong function of temperature. On the other hand, at high temperatures (T > 200 K) and electric fields (E > 0.01 V/μm), the resistance of bilayer graphene show a linear decrease. This can be explained by the scattering from thermally excited surface polar phonons of the SiO2 substrate being screened by the additional top graphene layers [Figure e layers . This fuIn conclusion, temperature and magnetic field dependence of resistance of bilayer graphene was investigated. Intrinsic semiconductor behavior at the range of temperature 2~340 K was observed. The strange sharp threshold-like decrease in resistance around 200 K is unexpected, and we attribute it to the presence of mesoscopic ripples between the top and bottom layer. Our results reveal that the energy gap in the bilayer graphene is thermally dependent. This potentially indicates the sublattice symmetry breaking and an energy gap formation due to Landau Level splits. The obtained results are important for the better understanding of magnetic field-induced high resistance and provide indications of a theoretically predicted magnetic field-induced excitonic gap.


PD-L1 expression in glioblastoma multiforme and to find associated pathway activation and genetic alterations. We show that PD-L1 is up-regulated in IDH1/2 wildtype glioblastoma multiforme compared to lower-grade gliomas. In addition, a strong association of PD-L1 with the mesenchymal expression subgroup was observed. Consistent with that, NF1 mutation and corresponding activation of the MAPK pathway was strongly connected to PD-L1 expression. Our findings may explain different response to PD-L1 inhibition of patients in ongoing trials and may help to select patients that may profit of immunotherapy in the future.Glioblastoma multiforme are highly malignant brain tumours with frequent genetic and epigenetic alterations. The poor clinical outcome of these tumours necessitates the development of new treatment options. Immunotherapies for glioblastoma multiforme including PD1/PD-L1 inhibition are currently tested in ongoing clinical trials. The purpose of this study was to investigate the molecular background of  Glioblastoma multiforme (WHO grade IV) is the most common type of brain tumours, which is characterised by poor clinical outcome and short survival time, rarely longer than 14 months , 3. UntiPD-L1) or the programmed death ligand protein 1 (PD-1) receptor and attempt to re-activate the immune system. Currently, PD-1/PD-L1 therapeutic antibodies are investigated for a large variety of different cancer types. For instance, in small-cell lung carcinoma and melanoma, PD-1 inhibitors were successfully tested in clinical trials and could significantly improve the overall survival [Glioblastoma multiforme contains a high frequency of genetic and epigenetic alterations with numerous potentially produced neoantigens , which asurvival –8.PD-L1 or CD274) plays a major role in preventing the immune response in many cancer types [PD-L1 in glioblastoma, which is targetable by prospective immunotherapies [PTEN and increased expression of PD-L1 in glioblastoma multiforme [The programmed death ligand 1 -kinase and phospho-S6-kinase in breast cancer [PD-L1 regulation as PI(3)Kinase and MAPK pathway [STAT3 transcriptional regulation was found [In other cancer types, melanoma . In addimelanoma . Anothert cancer . Chen et pathway . Also, aas found , 20.TCGA Data Platform and local tumour-bank in order to perform an integrative analysis of PD-L1 expression in glioblastoma multiforme. These results may improve further diagnostics and identify glioblastoma multiforme subgroups that could profit from PD-L1 targeted therapy and may explain different clinical courses of patients in ongoing clinical trials.The study reported here, combines data from both the PD-L1 was differently expressed according to the tumour grade   Figure , whereasPD-L1 expression  or low PD-L1 expression  with a Hazard ratio (HR) of 0.98 (p>0.05), Figure Survival analysis of the TCGA patients showed no differences in the overall survival (OS) of patients with high IDH1/2 mutation, IDH1/2 mutation+1p19q Co-deletion, IDH1/2 wild-type) revealed a strong PD-L1 expression in the IDH1/2 wild-type samples  [PD-L1 promoter region, a hypermethylation in IDH1/2 mutated patients was found  . Analysis Figure . The met(G-CIMP) . Those ad Figure , possiblP Figure . SimilarPD-L1 was not directly correlated to the total mutation load of each patient (R=0.2 p>0.05). However, after performing a logistic regression analysis, NF1 mutation was identified as a predictor of increased PD-L1 expression. Other very frequently occurring mutations like PTEN (p>0.05), EGFR (p>0.05) or TP53 (p>0.05) did not correlate with PD-L1 expression. However, the effect of NF1 alteration on PD-L1 expression was not exclusively for NF1 mutation, also patients with NF1 deletions showed increased expression of PD-L1 (p<0.01 for homozygote and heterozygote deletions). These findings could be confirmed in a validation cohort (Freiburg) (p<0.01 heterozygote deletions) . This result is in line with the above-described PD-L1 expression in the IDH1/2 mutated subgroups.Whole-exome sequencing data of 264 patients derived from the TCGA database were analysed. 19 mutations with high occurrence were selected as shown in Figure ) Figure . IDH 1/2PD-L1 regulatory network at protein level and connected proteins were summarised in modules by WPNA  was identified as highly correlated to PD-L1 expression (R=0.32 p=2.0E-5). A network was calculated based on the intramodule connectivity of each protein in PM1  and phospho-S6 (p<0.05) were associated with NF1 loss-of-function  of the Freiburg cohort were performed to validate the coherence of MAPK-pathway activation and PD-L1 expression. A significant differences between patients with/without a NF1 loss-of-function. Additionally, p-MAPK14 (p-p38) as a maker of the MAPK-pathway activation was positively correlated  to PD-L1 expression . Permutation based pre-ranked GSEA was performed and revealed a significant enrichment of mesenchymal subgroup genes (pFWER<0.01) (data not shown). Expression module 2 . Further GSEA characterised this module as highly associated to immune response and INFγ-activation (pFWER<0.01)  Figure . A constPD-L1 expression. These functional alterations could be used in future diagnostics and help to identify patients who may profit from a PD-L1/PD-1 inhibition.Here we present an integrative analysis based on data of the TCGA database and glioblastoma multiforme samples of the Department of Neurosurgery, Medical Center Freiburg. The purpose of this study was to identify potential molecular or genetic alterations in glioblastoma multiforme influencing the PD-L1  resulted in an activation of MAPK pathway, which affected down-stream targets like MAPK, MEK and JNK  data were used for analysis. Data were downloaded at the UCSC Cancer Genome Browser. Expression analysis was performed based on Agilent array data (TCGA GBM G4502A) for high-grade glioma and RNA-seq data (TCGA LGG HiSeqV2 PANCAN) for low-grade glioma. Both datasets were normalized and log2 transformed. For mutation analysis the PANCAN AWG gene-level mutation data set was used for further analysis. Proteomic data were taken from TCGA database as normalized level 3 RPPA data.Public available Level 3 TCGA  during tumor resection. The tissue was snap-frozen in liquid nitrogen immediately after resection and processed for further genetic/metabolic analysis. Tissue samples were fixed using 4% phosphate buffered formaldehyde and paraffin-embedded with standard procedures. H&E staining was performed on 4 μm paraffin sections using standard protocols. Immunohistochemistry was applied using an autostainer (Dako) after heat-induced epitope retrieval in citrate buffer. IDH1 mutation was assessed by immunohistochemistry using an anti-IDH1-R123 antibody .Genome wide expression data of 48 patients (Histology: Glioblastoma multiforme WHO Grade IV)  was used for expression analysis in addition to TCGA data  of the TCGA database were processed as level III data. Co-mutation analysis was performed in R software and free-available implemented packages. Additional, copy-number variants from 577 patients (Level 3 TCGA data) were analysed. 45 Patients of the Freiburg validation cohort were analysed by Genome-Wide Human SNP Array 6.0 (Affymetrix) and pre-processed by the genome core facility in Freiburg .PD-L1 expression. The WGCNA analysis is a robust tool for integrative network analysis and was used in several recent studies [Expression analysis was performed on 585 patients (Array by AgilentG4502A). WGCNA uses the topological overlapping measurement to identify corresponding modules. These modules were analysed by their eigengene correlation to  studies –33. WGCN studies  for furt studies .http://www.broadinstitute.org/gsea) as input databases for this analysis. GSEA plots were visualised by limma R-package (barcodeplot function).Permutation based gene set enrichment analysis (GSEA) was performed for each module to find specifically enriched biological functions and related pathways . Pre-ranPD-L1 expression level. Additional networks were created based on intermodule connectivity in Cytoscape 2.0 as described above.Protein data of 215 patients were extracted from the TCGA database. WPNA was performed for proteome data of the TCGA database and PD-L1 expression. WPCNA was performed with the WCNA-package (R-software) as described above. Modules were built by topological overlapping measurement. The power for network analysis was adapted until scale-free topology was achieved. Derived modules were analysed by their eigengene correlation to PD-L1 promoter. Analysis of the correlation coefficient  between expression and methylation values of all CpG sides of the PD-L1 promoter was performed as described for MGMT promoter by Bady et al. 2016 [PD-L1 expression and used for further validation of PD-L1 promoter methylation  was used for an epigenetic analysis of al. 2016   was available for 45/48 patients . The Kaplan-Meier method was used to provide median point estimates and time-specific rates. The Hazard-Ratio (HR) was calculated by Cox-Regressions tests. Analysis was performed by survival package included in R-Software.The following antibodies were used in immunostaining analyses: phospho-JNK (Phospho-SAPK/JNK (Thr183/Tyr185), Cell signaling, 1:200), p-MAPK14 (p-p38 Antikörper (Thr 180/Tyr 182), Santa Cruz 1:200), PD-L1  and phospho-Akt (Phospho-Akt (Thr 308), Cell signaling, 1:200). Primary antibodies were used at the concentration indicated by the manufacturers. Anti-Mouse and anti-Rabbit Alexa488- or Alexa594-conjugated (Life Technologies) were used as secondary antibodies. Alexa594-conjugated antibodies were used at 1:200 dilution and Alexa488-conjugated antibodies were used at 1:100 dilution. Pictures were acquired using a fluorescent microscope . Image quantification was performed by ImageJ and analyzed by R-software.PD-L1 expression in glioblastoma multiforme. PD-L1 was highly associated with the mesenchymal subgroups and in line with mesenchymal-like genetic alterations like a loss-of-function in NF1. Patients with a NF1 mutations/deletion or RAS-MAPK pathway activation may profit from a targeted therapy with PD-1/PD-L1 inhibition.In conclusion, this study reported a comprehensive analysis of


Immune checkpoint blockade therapeutics, notably antibodies targeting the programmed death 1 (PD-1) receptor and its PD-L1 and PD-L2 ligands, are currently revolutionizing the treatment of cancer. For a sizeable fraction of patients with melanoma, lung, kidney and several other solid cancers, monoclonal antibodies that neutralize the interactions of the PD-1/PD-L1 complex allow the reconstitution of long-lasting antitumor immunity. In hematological malignancies this novel therapeutic strategy is far less documented, although promising clinical responses have been seen in refractory and relapsed Hodgkin lymphoma patients. This review describes our current knowledge of PD-1 and PD-L1 expression, as reported by immunohistochemical staining in both non-Hodgkin lymphoma cells and their surrounding immune cells. Here, we discuss the multiple intrinsic and extrinsic mechanisms by which both T and B cell lymphomas up-regulate the PD-1/PD-L1 axis, and review current knowledge about the prognostic significance of its immunohistochemical detection. This body of literature establishes the cell surface expression of PD-1/PD-L1 as a critical determinant for the identification of non-Hodgkin lymphoma patients eligible for immune checkpoint blockade therapies. In order to develop within immunocompetent hosts, it is imperative that tumors evolve several immune escape strategies, such as mutations causing antigen loss or alteration of the antigen processing and presentation machinery , 2. OtheHere, we review the literature on PD-1 and PD-L1 protein expression levels in NHL and the mechanisms of their up-regulation, as well as the prognostic relevance of these proteins in NHL patients.The main physiological role of PD-1 is in limiting autoimmunity in an inflammatory context (e.g. in response to infection) by restricting the activity of T cells in peripheral tissues , 21. PD-In lymphoma, PD-1 is frequently upregulated in tumor cells themselves. For example, its expression is regularly reported in peripheral T cell lymphoma (PTCL) derived from follicular helper T cells (TFH) such as angioimmunoblastic T cell lymphoma, follicular T cell lymphoma and nodal peripheral T cell lymphoma with TFH phenotype. Likewise, some neoplastic B cells (prolymphocytes/paraimmunoblasts) in chronic lymphocytic leukemia (CLL) frequently up-regulate PD-1 –31. Its e.g. through CD79A/B mutations) and present with other genetic alterations such as BCL2 amplifications or INK4-ARF deletions. The chronic BCR signaling pathway is a well-known target for therapeutic interventions  but activating mutations  occasionally hamper drug efficacy [Diffuse large B cell lymphoma (DLBCL) are the most common type of lymphoma in adults. The prognosis for DLBCL patients is heterogeneous and remains poor in 40% of cases despite the introduction of therapy combining rituximab with cyclophosphamide-doxorubicin-oncovin-prednisone (CHOP) –46. The efficacy . Howeverefficacy –52. In aefficacy .+ cells in both the tumor and non-tumor compartments [n=132/1253 DLBCL samples; using a cut-off of 30%), while it was expressed in 15.3% of ME cells, which were essentially composed of macrophages . This study also confirmed the predominant expression of PD-L1 in non-GCB subtypes of DLBCL NOS.PD-L1 is expressed by both DLBCL tumor B cells and by non-malignant cells from their immune microenvironment, such as macrophages , 54. In 2 examined [+ TILs was higher in GCB DLBCL and was inversely correlated with the number of PD-L1+ tumor and ME cells, although these conclusions remain controversial [In contrast to PD-L1, PD-1 expression has almost exclusively been detected in the ME cells of DLBCL, with a varying number of cells per mmexamined , 55, 56.examined , 55–57, examined , 58. Kiyexamined  also repoversial , 60.+ plasmablastic lymphoma (PL) (where 20% of tumor cells are PD-L1+) [+ post-transplant lymphoproliferative disorders (PTLD) (where 60% of tumor cells are PD-L1+) [+ DLBCL of the elderly (where 100% of tumor cells are PD-L1+) [+ DLBCL subtype [+ lymphoma cells, and the inhibition of EBV-induced lymphomagenesis following PD-1/PD-L1 blockade in a mouse model [+ lymphoma patients, suggesting that their expression underlies the tolerogenic immune response induced by EBV [- PTLD [- PL [PD-L1 expression is also considered to be a hallmark of EBV-associated lymphoproliferative disorders. These include EBV PD-L1+) , EBV+ po PD-L1+) , EBV+ DL PD-L1+)  and the  subtype  found in subtype . This frse model , suggestd by EBV , 61. How [- PTLD  and EBV-LD [- PL , 58.IGH genes that lead to PD-L1 overexpression have also been reported [et al. [+ DLBCL since antiviral and inflammatory cytokine responses also activate the JAK/STAT pathway. In addition, the EBV-encoded latent membrane protein (LMP)-1 activates AP-1 (via cJUN/JUN-B components) and the JAK/STAT signaling pathways which, respectively, activate the PD-L1 enhancer and promoter [Nearly 20% of DLBCL NOS are reported to carry genetic anomalies and chromosomal alterations that lead to PD-L1/2 overexpression . Specifireported . Recentl [et al.  reported [et al. , 42, 43.promoter .+ tumor cells [Beside DLBCL NOS, primary central nervous system large B cell lymphoma (PCNSL) and primitive testicular lymphoma (PTL) are extranodal DLBCLs that arise at sites considered to be immune sanctuaries , 65. PCNor cells .PD-L1/2 locus, involving either the CIITA or the IGH loci [The expression of PD-L1 by tumor cells in primary mediastinal B cell lymphoma (PMBL) has also been investigated by a number of studies and is reported in 36% to 100% of cases , 56, 68.IGH loci , 69.+ DLBCL had inferior overall survival rates than PD-L1- DLBCL patients. Moreover, patients with PD-L1+ tumor cells but low PD-1+ TIL counts had poorer prognoses than patients with PD-L1- DLBCL and high PD-1+ TIL counts [+ T cells showed an aggressive clinical course [As depicted in Figure L counts . ElevateL counts , 72. MorL counts , 12, 54.L counts , 59, 70.l course .+ cells are abundant in the ME of FL [+ cells include not only TILs but also follicular helper T cells (TFH) from lymphomatous follicles or residual germinal centers [In contrast to DLBCL, most follicular lymphoma (FL) tumor cells do not express PD-L1 or PD-L2 , 68, 74,ME of FL . In FL,  centers , 76  and progression-free survival (PFS), although these results remain controversial [+ lymphocytes tested . The prognostic value of PD-1 staining in immune cells may also rely on the pattern of infiltration in FL samples, since high rates of intra-follicular PD-1+ cells have been correlated with a good prognosis while PD-1+ inter-follicular and diffuse infiltrates have poorer outcomes [+ PD-1bright and CD4+ PD-1dim T cell subtypes that were associated with different outcomes [Conflicting data have been reported regarding the prognostic impact of PD-1/PD-L1 expression in FL. As depicted in Figure to DLBCL . Carreraoversial . These doutcomes , 79–81. outcomes . In contoutcomes .+ cells among the circulating blood cells of CLL patients [The PD-1/PD-L1 axis also remains poorly documented in other small B cell lymphoma. From the few available studies it appears that LPL, MCL and MZL tumor cells are usually negative for PD-L1 IHC staining , 56, 68.patients , 84, andFH. This also applies to other lymphoma with a TFH phenotype according to the new WHO 2016 classification [FH subtypes, with no PD-L1+ tumors from 4 cases reported in one study [+ [The PD-1/PD-L1 axis is involved in both the development and the immune escape of some PTCL malignancies. From a diagnostic point of view, PD-1 expression is usually observed at the surface of angioimmunoblastic T cell lymphoma (AITL) tumor cells derived from normal Tfication  includinne study  and 1/21ne study . PD-L1 ene study  to 17% one study , howeverstudy [+ .et al. [+ ALCL) cell lines. This report was confirmed in biopsies from ALK+ ALCL tumors, with frequencies of PD-L1+ cases varying from 34 to 100% of the analyzed cases [+ tumor cells. Moreover, the same group showed that NPM-ALK induces the activation of the IL-10 and TGF-β cytokines. Since IL-10 can also activate JAK/STAT signaling via STAT3, and thus induce the up-regulation of PD-L1 [via STAT3 or IL-10.In contrast, Marzec et al.  reporteded cases , 85, 87.ed cases  showed tof PD-L1 , one may+ T cells have been found to be PD-1+ [+ tumor cells, together with the lack of PD-L1+ ME cells, has been correlated with a poor prognosis in ATLL patients [The expression of PD-1 and/or PD-L1 has also been reported in some cutaneous lymphoproliferative disorders. For instance, some mycosis fungoïdes (MF) and Sezary syndrome (SS) tumor cells have been found to express PD-1 at their surface. Similarly, some rare cutaneous disorders characterized by small- and average-sized CD4be PD-1+ –90. In abe PD-1+ . Likewisbe PD-1+ , 91, 92,+ tumor cells in 67% of cases. This was presumably secondary to EBV infection via LMP1-mediated upregulation of PD-L1 or via interferon (IFN) signaling [Finally, studies on extranodal NK/T cell lymphoma-nasal type have reported PD-L1ignaling , 43.+ tumor cells were those who benefitted most from PD-1/PD-L1 immune checkpoint blockade treatments [- patients [+ T cell infiltration and PD-L1 expression (with a 1% cut-off) represent predictive factors for immunotherapeutic response [+ lymphoma such as EBV+ DLBCL or NKTCL [+ or EBV- PTLD frequently show PD-L1-expressing tumor and ME cells. Thus, in these subtypes, PD-L1 may be used as a biomarker to identify patients who may benefit from anti-PD-1/PD-L1 inhibitors. Thus, the clinical impact of PD-L1 status in EBV+ lymphoma and PTLD has not yet been firmly established. Nevertheless, a significant association between PD-L1 expression and poor outcomes has been detected in early-diagnosed NKTCL patients treated with chemotherapy containing asparaginase [+ ALCL patients may also be eligible for treatment by PD-1/PD-L1 inhibitors since their NPM-ALK rearrangement up-regulates PD-L1 expression. Nevertheless, correlating this expression with clinical outcome needs to be formally validated in ALK+ ALCL patients. Ongoing preclinical trials and translational research into the PD-1/PD-L1 axis in lymphoma will assess the effectiveness of PD-1/PD-L1 inhibitors in PD-1/PD-L1-positive r/r B cell and T cell lymphoma. Thus, the validation of standardized procedures for assessing PD-1/PD-L1 expression is paramount for establishing a reliable predictive marker of response, which is currently missing from our therapeutic armament for NHL patients.Preliminary data from clinical trials in solid tumors and HL indicate that patients with PD-L1eatments , 93–94. patients . This obpatients  with difpatients . In factresponse . So far,response . For exaresponse . Pembrolresponse . In contresponse , 100. Inor NKTCL , 58, 102raginase , 102. ALEncouraged by the promising results in melanoma  and mult+ and PD-L1/L2+ cells. This encompasses both PD-1-expressing TILs as well as PD-L1/2-expressing macrophages and tumor cells in some NHL subtypes. Although PD-1 expression by TILs is the mere hallmark of their physiological activation, PD-L1/2 expression by malignant cells results from several tumor-intrinsic (genetic or oncogenic) and -extrinsic immune escape mechanisms selected by pressure from antitumor immunity. Hence PD-L1 overexpression is associated with the poorest prognosis in several types of aggressive NHLs. Thus, monoclonal antibodies selectively blocking the PD-1/PD-L1 axis could preserve TILs from exhaustion and promote antitumor immunity as an effective therapeutic strategy for NHL.In conclusion, tumors from most NHLs carry PD-1


The calculated results indicate that graphene intercalation can improve dielectric reliability of HfO2 dielectric even for the interfaces having interfacial oxygen vacancy or a small amount carbon vacancy. Moreover, the calculated results indicate that, inserting graphene into Ni/HfO2 interface induces the EWF’s to decline, and controlling interfacial oxygen or carbon vacancy can effectively tune the EWF of Ni/Gr/HfO2 interface. Our work strongly suggests that the use of graphene synthesized into Ni/HfO2 interface is a very effective way to improve the interface quality, and controlling interfacial oxygen or carbon vacancy is also an attractive and promising way for modulating the EWF of Ni/Gr/HfO2 interfaces.We have investigated the effects of graphene intercalation on dielectric reliability of HfO Having appropriate effective work function (EWF) and good dielectric reliability are crucial requirements for desired metal gate and gate dielectric, respectively. However, during the actual sample deposition and integration procedures, metal gate inevitably reacts with gate dielectric substrate. This reaction between them can induce metal contamination in thin gate dielectric5 and fermi level pinning6 which makes the EWF of gate electrode to deviate from the desired value. As a result, reliability and performance of device has been degraded. Therefore, improving dielectric properties of thin gate dielectric and effective tuning work function are essential for reliable integration of MOS devices. Yet, as well known, proper tuning the EWF is still difficult because of its extreme sensitivity to many interfacial factors8. It has been reported that processing conditions of device may strongly affect the EWF10. And then in the actual experimental process, there may exist many defects in the interface of metal/oxide structure. For example, with the help of the oxidation of underlying silicon, oxygen vacancies in HfO2 can be easily generated12. These interface defects would alter the interface structure and then interface dipole. As a result, the EWF is changed.Metal gate and high dielectric constant “high-14. But, it is too complex to be commonly used. Because of graphene’s two-dimensional sheet with monoatomic thickness, the exceptionally high conductivity and high thermal stability, it has a great potential for application in both low-dimensional sciences and nano-scale electronic devices. For example, it has been used as a gate stack electrode in MOS technology16. Recently, the experimental research17 has shown that the use of graphene (Gr) as a gate electrode instead of metal on a high-k gate dielectric improves the gate dielectric quality. Furthermore, several experimental studies on the work function tuning of metal-graphene stack electrode also have been done for metal-graphene-oxide structure18. Among them, Misra et al.18 explored multilayer graphene as metal gate electrode by inserting it between SiO2 dielectric and TiN metal. They found that incorporation of graphene between SiO2 dielectric and TiN metal gave rise to significantly improved dielectric reliability and an EWF tuning of gate electrode up to 0.5 eV by controlling the number of graphene layers. And Song et al. found the work function for graphene-metal electrode varied depending on the metal species, and it was either pinned to the work function of metal or pinned to a particular value regardless of the work function of metal16. They also found that varying the number of graphene layers can tune the work function of graphene/metal from 4.3 eV to 5.1 eV15. It has been experimentally investigated that the effects of incorporation of graphene on gate dielectric reliability and work function tuning for metal-oxide interface. Unfortunately, so far, there is little theoretical research on this subject. This motivates us to investigate the impacts of intercalation of graphene into metal/oxide structure.Replacement gate process which avoids high temperature processing after metal gate deposition could address some concerns induced by the reaction between metal gate and gate dielectric substrate2) has high dielectric constant and excellent thermal stability and satisfies various technical requirements etc, it has emerged as one of the most preferred gate oxide in metal/high-k oxide7. Furthermore, due to its high work function, thermal stability and good compatibility with high-k oxide, Ni has become a more ideal gate metal material. In this paper, we used first-principles calculations based on density functional theory (DFT) to investigate the following two aspects: (1) the effects of graphene intercalation on dielectric reliability of HfO2. (2) the effects of graphene intercalation and interfacial oxygen or carbon vacancy on the EWF of Ni/Gr/HfO2 interface.Currently, since hafnium oxide . Since the non-local dispersive interaction of graphene with metal Ni surface is very important and must be considered according to previous researchers28, then in our concerned Gr/Ni, Gr/HfO2 and Ni/Gr/HfO2 interfaces without or with atom vacancy, van der Waals contributions should also be considered. And here we employed optB88 exchange functional to describe van der Waals forces. The more details are presented in the back section. Spin polarization was included, and the plane-wave basis cutoff was set at 400 eV which shows a good convergence. A dipole correction was applied to avoid spurious interactions between periodic images of the slab. A 4 × 4 × 1 Monkhorst-Pack k-mesh was adopted for the calculations. The atoms were fully relaxed through the conjugate-gradient algorithm until the residual force on each atom was less than 0.03 eV/Å. Our calculated lattice constants for bulk cubic-HfO2 and fcc-Ni are 5.03 Å and 3.51 Å, respectively, which are slightly lower than the experimental values of 5.08 Å29 and 3.52 Å30. Ni(111) and HfO2(111) surfaces were chosen as the building blocks to build Ni/HfO2 interface, because both of them were the close-packed and most stable facets31. The 3 × 3 Ni(111) primitive surface unit cell has very good lattice match to 2 × 2 HfO2(111) primitive surface unit cell with a lattice mismatch about 5%. The Ni(111)/HfO2(111) structure used to mimic the Ni/HfO2 interface is similar to that previously reported by Li et al.32. The Ni(111)/HfO2(111) interface supercell was constructed by stacking 7 Ni layers on HfO2(111) slab (5 Hf layers and 10 O layers) and a 15Å-vacuum which is large enough to avoid any spurious interaction between the periodic replica. The supercell of the interface is shown in Fig. 2 base considered in this study, the lateral lattice parameters were fixed to be the value of HfO2(111) and two Hf bottom layers and four O bottom layers were frozen to act as HfO2 bulk. The Ni(111) surface was then fully relaxed. It may correspond to the features of Ni experimentally deposited on HfO2 substrate.All calculations were carried out using Vienna ab initio simulation package (VASP) with projector augmented wave approachS is the cross-sectional area of supercell. With this definition, a negative 2(111) interface was −0.027 eV/Å2. It implies that metal Ni(111) surface would energetically favorable to combined with HfO2(111) to form Ni(111)/HfO2(111) interface.The stability of interface was examined by calculation of the binding energy 2 and the EWF of Ni/HfO2 interface, we need to obtain a stable structure for Ni(111)/Gr/HfO2(111) interface. In order to get the stable Ni(111)/Gr/HfO2(111) interface structure, firstly, we built interface structure of graphene absorbed on HfO2(111) and then found the appropriate structure for Gr/HfO2(111) system. Generally, there are three different terminations: O-term, Hf-term and OO-term (terminated by two O layers) for HfO2(111) surface. According to refs34, the O-term surface was found to be energetically stable under O-rich condition. Therefore, to simplify, the graphene was considered to combine onto O-term surface of HfO2. Gr/HfO2(111) was modeled by a slab model that (3 × 3) graphene was deposited on (2 × 2) HfO2(111) surface with five Hf-O trilayers, since they had very good lattice match with a lattice mismatch about 4%. And a 15 Å-vacuum region was also inserted along the direction perpendicular to graphene. In view of previous studies36, we considered four binding configurations of graphene on HfO2(111) substrate. In these configurations, a C atom is adsorbed directly above the center of a Hf-O bond (Bridge), above the hollow site at the center of the Hf-O hexagonal ring (Hollow), on the top of a Hf atom (top-Hf), and on the top of an O atom (top-O), meanwhile other C atoms are adsorbed above HfO2(111) surface according to the structure of graphene. The binding energies of Gr/HfO2(111) systems for all binding configurations were calculated according to Eq. (2(111) substrate. More, we find that graphene is adsorbed on the HfO2 substrate with the average separation between graphene and topmost O layer about 2.91–2.96 Å which is very close to the reported values of 3.05 Å in ref.35 and 3.03. Å in ref.36. For simplicity, in subsequent study for Ni(111)/Gr/HfO2(111) interface, we used the beforehand generated Gr/HfO2(111) structure with top-O configuration to build Ni(111)/Gr/HfO2(111) interface structure further.To study the effects of graphene intercalation on dielectric properties of HfOre for Ni11/Gr/HfO2(111) system has been acquired. Nextly, interfacial bonding between graphene and Ni(111) was considered. The graphene/metal systems have been extensively studied39. Several authors have investigated them with standard DFT approaches where the van der Waals (vdW) contributions are neglected38. Yet as we all know, it fails to reproduce non-local dispersive interaction which is important in weak binding between graphene and metal surface. Recent studies28 have evidenced that the optB88-vdW functional has been found to be an effective solution to describe vdW contributions, which plays an important role in binding between graphene and metal surface. Therefore, to reasonably consider the interaction between metal slab and graphene, we employed the vdW density functional in form of the optB88-vdW. Moreover, the three typical symmetric positions of Ni(111) surface which are located above Ni atoms in the first layer, the second layer and third layer are named as top, hcp and fcc position40, respectively, as shown in Fig. 28. Therefore, in subsequent study for Ni(111)/Gr/HfO2(111) interfaces, we employed the optB88 exchange functional to describe van der Waals forces. In addition, from Fig. The appropriate structure for Gr/HfO2(111) interface was built by combining Ni(111) with the previously acquired Gr/HfO2(111) structure. Assume that Ni and graphene being deposited on a prefabricated HfO2 substrate to form an interface. So in structure relaxation, the lateral lattice parameters were fixed to be the value of c-HfO2(111) and the Ni(111) surface and graphene were then fully relaxed in finding the equilibrium structure of interface. The schematic for the supercell of the Ni(111)/Gr/HfO2(111) supercell is shown in Fig. 2 and −0.230 eV/Å2 for top-hcp and top-fcc, respectively. Noted that the binding energies for the two binding configurations are all negative and their difference is little. Thus it is reasonable to infer that Ni(111) energetically prefers to combine with Gr/HfO2(111). Moreover, it is found that for both binding configurations, Ni(111) slab is bound with Gr/HfO2(111) interface in a typical physical adsorption distance. The average separations between graphene and the nearest neighbor Ni layer are about 3.20 Å and 3.27 Å for top-fcc and top-hcp, respectively.The Ni(111)/Gr/HfO432, respectively. As is well-known, DFT underestimates the oxide gap. And our calculation of HfO2 is 3.70 eV which is close to the result of 3.95eV45, indicating that our calculations are reliable. As shown in Fig. p-type Schottky-barrier height (SBH) V is the difference between the macroscopic average potentials residing in Ni and HfO2 bulk-like-regions respectively46. The first term for equation /Gr/HfO2(111) interface, when the graphene is inserted into Ni(111)/HfO2(111) interface, it would only change interfacial structure but Ni and HfO2 bulk electronic structures were not affected. Thus, according to equation  interface and Ni(111)/Gr/HfO2(111) interface with Ni combining onto Gr/HfO2 in top-hcp configuration. It can be evidently seen from Fig. V about 0.7 eV. That is, the EWF fells by about 0.7 eV. Both calculated results of ΔV for Ni(111)/Gr/HfO2(111) interfaces with Ni combining on Gr/HfO2 in the top-fcc and top-hcp configurations are the same. Furthermore, the calculated EWF of Ni(111)/Gr/HfO2(111) interface is about 5.3 eV being close to the PMOS’ requirement, which deviates the calculated work function value and other theoretical value47 of graphene (~4.5 eV), and is close to the value of Ni (~5.0 eV). This is very similar to the previous experimental report for Ni/graphene/SiO216.The interface EWF can be estimated using potential-line-up method, as schematically shown in Fig. entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaV reflects the change of interface dipole barrier. And the change of interface dipole barrier originates in interfacial charge redistribution. To see the charge transfer effect, the plane averaged electron density difference of the valence electron 47The change of Δ2(111) and Ni(111)/Gr/HfO2(111) interfaces, respectively. Blue and yellow denote electron accumulation and electron depletion, respectively. As shown in Fig. 2(111) interface. The interface dipole is formed because of the electron accumulation near O layer and the electron depletion near interfacial Ni layer. And the electronic perturbations associated with the formation of interface extend into HfO2 and Ni side deeply. However, for Ni(111)/Gr/HfO2(111) interface, as shown in Fig. 2 side, also just goes slightly deep into Ni side. The total electron transfer is obviously diminished compared to that for Ni(111)/HfO2(111) interface. This might be attributed to graphene’s unique aspects of charge screening by its relativistic low energy carriers, which can be clearly seen in Fig. 2(111) interface. There are electron accumulation near graphene layer and electron depletion near interfacial O and Ni layers. Consequently, the total interface dipole comprises two parts: one is pointing from graphene layer to O layer and the other is pointing from graphene layer to Ni layer. Both parts are opposite and may be counteracted. Consequently, the whole dipole decreases. On the whole, the reduction of charge-transfer along with mutual competition between the two parts of interface dipole has led to the EWF to decrease.Here, z denotes the direction which is parallel to interface normal. EF), the EWF can be modulated. To better observe the variation behavior of Fermi level induced by graphene intercalating into Ni/HfO2 interface, we have plotted the density of states (DOS) for Ni(111)/HfO2(111) interfaces without and with graphene intercalation. Figure 2(111) and Ni(111)/Gr/HfO2(111), respectively. Graphene intercalation into Ni/HfO2 interface shifts the Fermi level towards conduction band minimum (CBM) of HfO2. As a result, graphene intercalation acts as a donor dopant (n-type doping). Thus, it gives a well qualitative explanation for the EWF’s variation behavior. Moreover, as shown in Fig. 2(111). However, comparing the PDOS of interfacial Hf and O atoms in Fig. 2 with that of surfacial Hf and O atoms for HfO2 in Fig. 2 without graphene intercalation. Especially, interfacial Hf and O atoms lost their dielectric properties but exhibited metallic properties. Thus interfacial interactions between Ni and HfO2 have induced contamination in HfO2 gate dielectrics and then degrade the dielectric reliability. However, for Ni(111)/Gr/HfO2(111) interface shown in Fig. 2 gate dielectrics can be greatly improved by inserting a graphene layer between Ni and HfO2. This agrees with the experimental results reported by Misra et al.18 that inserting graphene between SiO2 and TiN has significantly improved dielectric reliability. We think that this improvement brought by graphene intercalation may be due to graphene’s excellent charge screening role on both Ni and HfO2 sides.Substantially, by controlling the relative position of Fermi level  in graphene layer, and O vacancy with a higher vacancy content of 1/4 ML in O atom layer closest to graphene layer, called Ni(111)/Cvac-Gr/HfO2(111) and Ni(111)/Gr/Ovac-HfO2(111) interfaces, respectively. The calculated DOS results for Ni(111)/Cvac-Gr/HfO2(111) shown in Fig. 2(111) without C vacancy, graphene exhibits metallic properties. This may be attributed to chemical bonding between C and Ni atoms near C atomic vacancy. The bond length is about 1.847 Å which is shown in illustrations of Fig. vac-Gr/HfO2(111) interface, and it is found that C vacancy brought about an ascent of 0.3 eV compared to non-defective Ni(111)/Gr/HfO2(111). The calculated result of ΔV for Ni(111)/Cvac-Gr/HfO2(111) is about 1.7 eV. According to Fig. 2(111), C vacancy acts as an acceptor dopant (p-type doping). This decline of EWF might be due to the change of charge transfer derived from C vacancy. Figure vac-Gr/HfO2(111) interface. It can be clearly seen that electron transfer decreases compared with that for Ni(111)/Gr/HfO2(111) shown in Fig. High dielectric reliability of oxide and effective work function tuning of metal-oxide interface are two important factors in determining the CMOS’s overall performance. In fact, experimentally, interfacial atom vacancy can be easy to form in interfacial layer for metal-oxide interface, and these defects should affect many properties of interface. In order to research the impacts of the interfacial atom vacancy, we study Ni/Gr/HfOvac-HfO2(111), it can be easy to find that interfacial Hf and O atoms also remain their dielectric properties. Interfacial O vacancy does hardly change graphene’s properties. It is worthwhile noted that, compared with the case of non-defective Ni(111)/Gr/HfO2(111), interfacial O vacancies only bring about an EWF’s decline of 1.0 eV. The calculated result of ΔV for Ni(111)/Gr/Ovac-HfO2(111) is about 3.0 eV. In other words, interfacial O vacancy can effectively tune the EWF from 5.3 eV for non-defective Ni/Gr/HfO2 to 4.3 eV (being close to the NMOS’ requirement) without sacrificing dielectric reliability of gate dielectric, which is fascinating and desirable. The EWF’s big reduction is associated with interfacial charge redistribution. Seen in Fig. inter-d in Fig. 2, obtained by Chiu et al.36. Corresponding to the relative movement behavior for the fermi energy, contrary to the case for C vacancy, interfacial O vacancy acts as a donor dopant (n-type doping).As seen in Fig. 2, and the effects of graphene intercalation and interfacial oxygen or carbon vacancy on the effective work function of Ni/Gr/HfO2 interface using first-principle calculation. It is found that the graphene intercalation reduces metallic contamination in HfO2 dielectric even for those interfaces with interfacial O vacancy and a small amount C vacancy. Consequently, the graphene intercalation improves the dielectric reliability of HfO2 and the interface quality. Our work strongly indicates that the use of graphene synthesized into Ni/HfO2 interface can significantly improve dielectric reliability of gate dielectric and then to effectively reduce the physical thickness of gate dielectric. In addition, controlling interfacial oxygen or carbon vacancy is a very effective way for modulating the EWF of Ni/Gr/HfO2 interface. By inserting graphene into Ni/HfO2 interface, the EWF could drop about 0.7 eV. Furthermore, only the removal of 1/18 ML carbon atom in graphene layer could induce a considerable EWF increment of 0.3 eV, while the removal of 1/4 ML interfacial oxygen atom could give rise to a great EWF decrement of 1.0 eV. These are very important for metal gate technology and application where the EWF of metal/oxide interface needs to be modulated. And these results also have practical significance for the realization of high-performance graphene-based devices.In summary, we have investigated the effects of graphene intercalation on dielectric reliability of HfO


PD-L1 DNA methylation (mPD-L1) in prostate tissue samples and to evaluate its potential as a biomarker in prostate cancer (PCa).The rapid development of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors has generated an urgent need for biomarkers assisting the selection of patients eligible for therapy. The use of PD-L1 immunohistochemistry, which has been suggested as a predictive biomarker, however, is confounded by multiple unresolved issues. The aim of this study therefore was to quantify mPD-L1 compared to tumor tissue. High mPD-L1 in PCa was associated with biochemical recurrence (BCR) in univariate Cox proportional hazards (hazard ratio (HR)=2.60 [95%CI: 1.50-4.51], p=0.001) and Kaplan-Meier analyses (p<0.001). These results were corroborated in an independent validation cohort in univariate Cox  and Kaplan-Meier analyses (p=0.029). Although mPD-L1 and PD-L1 protein expression did not correlate in the validation cohort, both parameters added significant prognostic information in bivariate Cox analysis .In the training cohort, normal tissue showed significantly lower levels of mPD-L1 was analyzed in a training cohort from The Cancer Genome Atlas (n=498) and was subsequently measured in an independent validation cohort (n=299) by quantitative methylation-specific real-time PCR. All patients had undergone radical prostatectomy.mPD-L1 is a promising biomarker for the risk stratification of PCa patients and might offer additional relevant prognostic information to the implemented clinical parameters, particularly in the setting of immune checkpoint inhibition. We further analyzed the prognosis of PCa patients stratified according to their mPD-L1 status (high vs. low) in combination with the dichotomized PD-L1 protein expression status. Figure mPD-L1 present with shorter BCR-free intervals compared to patients with simultaneous low PD-L1 protein expression and low mPD-L1. Patients with either high PD-L1 protein expression and low mPD-L1 or low PD-L1 protein expression and high mPD-L1 showed an intermediate BCR-free interval. The finding that PD-L1 protein expression and methylation seem to be unrelated in our cohort prompted us to investigate additional mechanisms of post-transcriptional regulation of PD-L1.High PD-L1 protein expression determined semi-quantitatively mPD-L1, suggesting that mPD-L1 was associated not only with decreased mRNA expression but also with lower levels of mRNA destabilizing miR-197 and three of the miR-200 family members miR-200a-c . In particular miR-197, miR-200, miR-570, miR-34a, and miR-513 have been shown to play a key role in the regulation of PD-L1 expression (reviewed by ). MiRs aTargeting the immune checkpoint pathway PD-1/PD-1 has emerged as a promising therapy providing significant clinical activity and durable benefits with minimal toxicities in several solid tumors . The rapPD-L1 promoter methylation in PCa. The aim of this study therefore was to quantify PD-L1 DNA methylation in prostate tissue samples and to evaluate its potential as a biomarker in PCa.Aberrant DNA methylation of certain loci has been reported to play a major role in carcinogenesis . Such epDNA methylation of promoter regions is often associated with epigenetic silencing . In normPD-L1 gene were analyzed. In this training cohort, mPD-L1 levels were increased significantly in PCa compared to normal tissue, indicating that PD-L1 might be differentially methylated in prostatic malignancies. Further investigations revealed that two of the five examined beads (cg19724470 and cg15837913) showed a significant correlation of mPD-L1 with BCR in univariate Cox proportional hazards analysis and Kaplan-Meier analysis. Interestingly, differential methylation was found in the border regions and not the centre of the CpG-dense promoter area. One of the two beads (cg19724470) further showed an inverse correlation with PD-L1 mRNA expression and an association with prognostic clinicopathological variables including the ISUP grading group, pT, pre-surgical prostate-specific antigen (PSA), resection margin, and ERG status.In the present study, five Illumina Infinium HumanMethylation450 BeadChip beads taken from the TCGA dataset and located in the promoter region of the PD-L1 DNA methylation, analyzed as a continuous variable and as a dichtomized variable using an optimized cut-off, was significantly associated with an increased risk for BCR in patients in univariate Cox proportional hazards analysis and its prognostic value was further confirmed by Kaplan-Meier analysis.In order to validate these findings with an additional technology in an independent cohort, a quantitative methylation qPCR assay was designed within the region of the best performing bead (cg19724470). In this validation cohort, high PD-L1 promoter methylation and protein expression in PCa. This may point at a complex post-transcriptional and post-translational regulation of PD-L1. According to our findings, mPD-L1 is inversely correlated with mRNA transcription and associated with miR as cellular component, which modify the downstream processing of PD-L1 mRNA. Differential expression of miR may therefore potentially interfere with the linear translation of PD-L1 mRNA into PD-L1 protein. Thus, further studies are warranted to unravel the complex interplay between DNA methylation and protein expression in PCa. In addition, post-translational modifications which affect the stability of the protein (e.g. glycolization) and in turn alter the affinity of the anti-PD-L1 antibody to membranous PD-L1 might further be causative for ostensibly conflicting results =100%*2ΔΔCTThe DNA methylation of PD-L1 methylation, expression, and clinicopathological variables were analyzed using the Spearman's rank correlation, Kruskal-Wallis (≥ 3 groups) or Mann-Whitney-U (2 groups) tests. BCR-free survival analyses were conducted by Kaplan-Meier and Cox proportional hazards regression analyses. P-values < 0.05 were considered statistically significant.The statistical analyses were performed using SPSS, version 22 . Associations/correlations between


Furthermore, the chirality of the QHE edge channels can be changed by a top gate. These results demonstrate that basic QHE physics are experimentally accessible in graphene for a fraction of the price of conventional setups using superconducting magnets, which greatly increases the potential of the QHE in graphene for research and applications.We have observed the well-kown quantum Hall effect (QHE) in epitaxial graphene grown on silicon carbide (SiC) by using, for the first time, only commercial NdFeB permanent magnets at low temperature. The relatively large and homogeneous magnetic field generated by the magnets, together with the high quality of the epitaxial graphene films, enables the formation of well-developed quantum Hall states at Landau level filling factors  Experimentally demonstrated for the first time in 1980This very peculiar state of matter has been the object of an extremely vast corpus of fundamental research on various materials. Indeed, quantum Hall systems host a great variety of unique states and phenomena, such as fractal quantum Hall states345Experimentally, the QHE is obtained in materials such as gallium arsenide heterostructures, under a strong magnetic field (of at least a few Tesla) and at low temperatures. These constraints, which require the use of cryogenic refrigerators containing superconducting magnets to generate the magnetic field, stem from the small energy gap between Landau levels at low magnetic fieldsGraphene can circumvent those constraints because, owing to its unique band structure, the energy gap is more than one order of magnitude larger than in gallium arsenide heterostructures at typical magnetic fields of a few Tesla.Indeed, shortly after the first experimental observation of the QHE in graphene10 cm−2 can be attained, making the QHE at the Landau level filling factor v = 2 reachable for magnetic fields lower than 1 T13152Fe14B alloy, which have remanence fields of up to 1.4 T. Replacing the expensive, cumbersome and fragile superconducting solenoid magnets with affordable permanent ones would considerably expand efforts to probe and exploit the properties of the QHE.The ability to tune the density of carriers, and even their type, in graphene by the field effect implies that the constraint of high magnetic fields could also be lifted for clean enough samples, in which carrier densities lower than 5 × 10In this paper, we show that the QHE can be induced in a graphene sample using NdFeB permanent magnets instead of the usual superconducting magnets.v = 2 QHE plateau with respect to both gate voltage and magnetic field2021222425Several fabrication techniques yield clean enough graphene samples so as to reach the QHE at sub-tesla magnetic fields. While boron nitride-encapsulated graphene12 cm−2), the top gate dielectric, made of hydrogen silsesquioxane , decreases the carrier density to around 5 × 1011 cm−2 at zero top gate voltage Vgate, leading to high mobilities (up to 16000 cm2/Vs at Vgate = 0 V). This, combined with the ability to further decrease the density by changing Vgate, allows us to reach the QHE regime at magnetic fields as low as 0.5 T, which is easily attainable with permanent magnets. This is clearly shown in H) and longitudinal (Rxx) resistances under the magnetic field generated only by permanent magnets as a function of Vgate at the base temperature of our He3 fridge (300 mK).We used this setup to perform QHE measurements in an epitaxial graphene Hall bar. An optical micrograph of the sample is shown in H (red squares) at the value 0.5 × h/e2, accompanied by a zero value of Rxx (black circles), for Vgate between −33 and −39 V. This is the hallmark of the QHE at filling factor v = 2. As Vgate is further swept towards negative values, the v = 2 plateau vanishes and the charge neutrality point (CNP) is reached at Vgate ≈−45 V. This is indicated by the local maximum in Rxx and the change in the sign of RH. For Vgate between −50 and −55 V, Rxx again reaches zero while RH becomes quantized at −0.5 × h/e2, characteristic of the QHE at filling factor v = -2. This change in sign, corresponding to a change in the chirality of charge carriers, is a signature of the ambipolarity of charge transport in graphene.The results plotted in v = ±2 in epitaxial graphene, we now focus on the robustness of the QHE phase against temperature. H (red squares) and Rxx (black circles) on the v = ±2 plateau at up to T = 2 K. A sevenfold increase in the temperature only leads to a deviation smaller than one-thousandth of the expected values for RH. Note that the low-temperature value of RH is slightly smaller than 0.5 × h/e2, which is due to an error in the estimated gain of the room-temperature amplifiers of about 0.04%.Having shown that our setup enables us to reach the QHE regime at gate dependence of Rxx is plotted for several values of temperature up to 30 K. Note that fluctuations arise in Rxx around the CNP below 7 K, indicating the presence of residual electron/hole puddles at the CNP. In the explored temperature range, the minimum longitudinal conductivity σxx = Rxx /(Rxx2 + RH2), plotted in the main panel of VRH ∝1/T × exp[(TM/T)1/3]. This model is similar to the one presented in ref. xx in epitaxial graphene samples immersed in magnetic fields generated by a superconducting magnet.The deviations become larger as the temperature is increased, as shown in the inset of e.g. one cannot reverse in-situ the direction of the magnetic field) can be mitigated by implementing a mechanical displacement of the magnets. In addition, it is possible to combine it with a small coil to induce small changes in the applied magnetic field30In summary, we have combined the low carrier densities attainable in epitaxial graphene with the relatively large magnetic fields generated by NdFeB permanent magnets to obtain a robust quantum Hall phase without the use of either superconducting or normal electro-magnet. In those conditions, the QHE could be reached in various types of graphene samples. For fundamental physics, using this setup with encapsulated monolayer and bilayer graphene2 insulating layers, allows us to tune the density and the type of the charge carriers. At zero gate voltage, the sample has n-type carriers with a density of about 5 × 1011 cm2, due to the doping from the SiC substrate and the HSQ.The epitaxial graphene wafers were prepared by thermal decomposition of a 6H-SiC(0001) substrate, annealed at around 1800 °C in Ar at a pressure of less than 100 Torr. Metallic contacts are made of Cr/Au. An Au top gate, separated from the graphene film by a 100-nm-thick hydrogen silsesquioxane (HSQ) and a 60-nm-thick SiOThe measurements were performed using standard lock-in techniques at roughly 30 Hz with NF LI-75A room-temperature amplifiers.How to cite this article: Parmentier, F. D. et al. Quantum Hall effect in epitaxial graphene with permanent magnets. Sci. Rep.6, 38393; doi: 10.1038/srep38393 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


PD‐1/PD‐L1 immunotherapy is viewed as having clinical benefits in advanced cancers but is effective in only a few patients, suggesting that an efficient combination approach is needed to improve efficacy. Immunohistochemistry analysis indicated that PD‐L1 expression was correlated with the E6 expression in tumors from 122 lung cancer patients. The poorest survival occurred in PD‐L1‐positive/E6‐positive tumor. PD‐L1 expression was increased by the expression of E6, but not the E7, oncoprotein in lung and cervical cancer cells. PD‐L1 expression was responsible for E6‐mediated colony formation and soft agar growth. Therefore, PD‐L1 secreted from tumor cells may directly promote tumor progression, particularly in E6‐positive tumors. Immune deficiency nude mice were used to test the possibility that combining anti‐PD‐L1 mAb with Lm‐LLO‐E6 vaccine could have a higher antitumor activity compared with anti‐PD‐L1 mAb or Lm‐LLO‐E6 vaccine alone. A greater antitumor activity was obtained with anti‐PD‐L1 mAb + Lm‐LLO‐E6 vaccine than with anti‐PD‐L1 mAb or Lm‐LLO‐E6 alone in subcutaneous and metastatic tumors induced by TL‐1 and SiHa cells. The longest survival time for nude mice was observed in the anti‐PD‐L1 mAb + Lm‐LLO‐E6 vaccine group. In conclusion, an anti‐PD‐L1 mAb + Lm‐LLO‐E6 vaccine may be an efficient treatment for suppression of tumor growth and metastasis induced by HPV‐infected cells. Program death ligand‐1 (PD‐L1) acts as an inhibitor of human T‐cell responses by binding to its receptor PD‐1 to create the tumor microenvironment. This, in turn, results in tumor progression due to tumor immune surveillance Poor prognosis in NSCLC is associated with the epithelial‐mesenchymal transition (EMT), a key process that drives cancer metastasis Human papillomavirus (HPV) 16/18 infection is associated with lung cancer development in Taiwan Antibody‐mediated blockade of PD‐L1 can induce a durable tumor regression and prolonged stabilization of disease in patients with NSCLC Lung tumor specimens were collected from 122 patients who underwent primary NSCLC surgical resection at the Department of Thoracic Surgery, Taichung Veterans General Hospital  between 1998 and 2004. Patients were asked to submit written informed consent; the study was approved by the Institutional Review Board (TMUH No. 201301051). The tumor type and stage of each collected specimen were histologically determined in accordance with the World Health Organization classification system. Cancer relapse data were obtained from chart review and confirmed by thoracic surgeons. Clinical parameters and OS and relapse‐free survival (RFS) data were collected from chart review and the Taiwan Cancer Registry, Department of Health, Executive Yuan, ROC.TL‐1 and TL‐4 cells were cultured from the plural effusions from lung adenocarcinoma patients μm sections, mounted on glass, and dried overnight at 37°C. All sections were then deparaffinized in xylene, rehydrated through alcohol, and washed in phosphate‐buffered saline. This buffer was used for all subsequent washes. Sections were heated in a microwave oven twice for 5 min in citrate buffer (pH 6.0) and then incubated for 60 min at room temperature following a conventional streptavidin peroxidase method . Signals were developed with 3, 3′‐diaminobenzidine for 5 min and the sections were counterstained with hematoxylin. Negative controls were obtained by leaving out the primary antibody. The signal intensities were evaluated independently by three observers. Immunostaining scores were defined as the cell staining intensity  multiplied by the percentage of labeled cells (0–100%), leading to scores from 0 to 300. A score greater than or equal to 150 was defined as “high” immunostaining, whereas a score <150 was defined as “low”.Immunohistochemistry was performed to evaluate PD‐L1 protein expression. PD‐L1 antibody was purchased from Genetex . HPV E6 antibody was purchased from Santa Cruz Biotechnology . Formalin‐fixed and paraffin‐embedded specimens were cut into 3 The HPV 16 E6 cDNA plasmid and HPV 16 E6/E7 small hairpin (sh)RNA were described previously 2. Colonies were visualized and quantified with a microscope after 14 days of cultivation, and the colonies larger than 100 μm in diameter were counted.Anchorage‐independent growth was assayed as the ability of cells to form colonies on soft agar plates. The bottom agar consisted of growth medium containing 10% fetal bovine serum and 0.5% agarose in six‐well plates. A total of 300 cells were resuspended in growth medium containing 10% fetal bovine serum and 0.4% agarose and placed on top of the bottom agar. The cells were grown at 37°C in a humidified incubator in 5% CO6 CFU/mouse. The anti‐PD‐L1 mAb obtained from BioLegend  was injected intravenously at a dose of 25 μg/mouse. The aim of the experiment was to determine the tumor growth changes induced by different treatments when compared with the control group. Briefly, mice were subcutaneously implanted with 500,000 TL‐1 cells/mouse in the right flank on day 0. On day 7 (tumor size ~3–4 mm in diameter), the mice were injected with Lm‐LLO‐E6 or Lm‐LLO‐E7 intraperitoneally and/or with anti‐PD‐L1 mAb intravenously. Mice were treated with anti‐PD‐L1 mAb an additional three times, on days 14, 21, and 28 after tumor implantation. The control group of mice was only injected with TL‐1 or SiHa cells. Tumors were measured every 3–4 days using digital calipers, and tumor volume was calculated using the formula V = (W2 × L)/2, where V is volume, L is length (longer diameter), and W is width (shorter diameter). A flow chart is presented in Figure Six‐ to eight‐week‐old female BALB/c nude mice and C57Bl/6 mice were purchased from National Laboratory Animal Center . Mice were cared for under protocols approved by the NLAC Animal Care and Use Committee. Lm‐LLO‐E6 (E6) vaccine and Lm‐LLO‐E7 (E7) vaccine were injected intraperitoneally at doses of 5 × 106 CFU/mouse. The anti‐PD‐L1 mAb was injected intravenously at a dose of 25 μg/mouse. The aim of the experiment was to determine the changes in tumor growth and survival induced by the different treatments. Briefly, mice were injected with 100,000 TL‐1 or SiHa cells/mouse by tail‐vein injection on day 0. On day 14, the mice were injected with Lm‐LLO‐E6 or Lm‐LLO‐E7 vaccine intraperitoneally and/or with anti‐PD‐L1 mAb intravenously. Mice were treated with anti‐PD‐L1 mAb an additional three times, on days 21, 28, 35 after tumor cell injections. Mice continued to eat normally until death. A flow chart is presented in Figure Lm‐LLO‐E6 (E6) and Lm‐LLO‐E7 (E7) vaccines were injected intraperitoneally at a dose of 5 × 10P < 0.050 was considered statistically significant.All statistical analyses were conducted using the SPSS statistical software program, as described previously  P = 0.039; Table P = 0.042; Table We collected 122 surgically resected lung tumors from NSCLC patients to examine whether E6 expression could be associated with PD‐L1 expression. The data for E6 expression were obtained from previous studies P = 0.001 for OS, P = 0.003 for RFS; Fig. P = 0.017 for OS, P = 0.121 for RFS; Figure P = 0.026 for OS; HR, 1.55, 95% CI, 0.97–2.48, P = 0.045 for RFS; Table The possible association between E6 and PD‐L1 expression, singly and combined, and overall survival (OS) and relapse‐free survival (RFS) was examined in NSCLC patients. Kaplan–Meier analysis showed that shorter OS and RFS were observed in patients with positive PD‐L1 tumors than with negative PD‐L1 tumors RNA and for comparison with HPV16‐negative TL‐4 cells transfected with the E6 or E7 expression vector. This comparison would verify whether the E6, but not the E7 oncoprotein, is responsible for PD‐L1 expression in lung cancer cells Fig. A. WesterWe next examined whether manipulation of PD‐L1 expression by E6 could attenuate the abilities for colony formation and soft agar growth in lung cancer cells. The representative colony growth in agar plates and soft agar growth colonies in soft agar plates are shown in Figure The anti‐PD‐L1 mAb may provide durable tumor suppression and a clinical benefit in advanced NSCLC We also used a tail‐vein animal model to verify whether the anti‐PD‐L1 mAb + Lm‐LLO‐E6 combination can suppress metastatic lung tumor nodule formation induced by TL‐1 cells in nude mice. The experiments were halted at 77 days. The representative metastatic lung tumor nodules induced by TL‐1 cells in each group were compared with those induced by SiHa cells. Tumors were confirmed by H & E staining Fig. A. RepresP = 0.004 for anti‐PD‐L1 mAb + Lm‐LLO‐E7 vaccine, P = 0.037 for Lm‐LLO‐E6 vaccine, P = 0.043 for anti‐PD‐L1 mAb, Fig. The body weights of the control mice injected with TL‐1 cells were significantly lower than those of mice injected with anti‐PD‐L1 mAb, Lm‐LLO‐E6 vaccine, anti‐PD‐L1 mAb + Lm‐LLO‐E6 vaccine, and anti‐PD‐L1 mAb + Lm‐LLO‐E7 vaccine, but they did not differ from those that received Lm‐LLO‐E7 vaccine treatment Fig. . SimilarThe prevalence of HPV16/18 DNA and E6 oncoprotein expression were approximately 55% and 28% κB inhibitor BAY11‐7082, but unchanged by PI3K/AKT inhibitor wortamannin .Figure S4. Changes in body weight in mice injected with TL‐1 or SiHa cells and treated with PD‐L1 mAb, Lm‐LLO‐E6 vaccine, or Lm‐LLO‐E7 vaccine.Figure S5. Tyrosine Kinase inhibitor (Gefitinib), MEK/ERK inhibitor (AZD6244), PI3K/AKT inhibitor (Wortamannin), and NF‐κB inhibitor (BAY 11‐7082) were treated with TL1 and SiHa cells for 48h.Figure S6. Lm‐LLO‐E7 vaccine alone and the combination of anti‐PD‐L1 mAb+Lm‐LLO ‐E7 vaccine markedly suppress tumor growth induced by TC‐1 cells in C57B/L6 mice compared with other treatments. The mice were subcutaneously injected with HPV16‐infected mouse TC‐1 cells.Figure S7. Mature infiltrating T lymphocytes in tumor of each group of nude mice were not detected in nude mice with different treatments.Click here for additional data file.


Understanding the mechanism of humidity transduction calls for experimental data and a theory to interpret the data and design new experiments. A comprehensive theory of humidity transduction must start with agreement on what humidity parameters are measured by hygroreceptors and processed by the brain. Hygroreceptors have been found in cuticular sensilla of a broad range of insect species. Their structural features are far from uniform. Nevertheless, these sensilla always contain an antagonistic pair of a moist cell and a dry cell combined with a thermoreceptive cold cell. The strategy behind this arrangement remains unclear. Three main models of humidity transduction have been proposed. Hygroreceptors could operate as mechanical hygrometers, psychrometers or evaporation detectors. Each mode of action measures a different humidity parameter. Mechanical hygrometers measure the relative humidity, psychrometers indicate the wet-bulb temperature, and evaporimeters refer to the saturation deficit of the air. Here we assess the validity of the different functions by testing specific predictions drawn from each of the models. The effect of air temperature on the responses to humidity stimulation rules out the mechanical hygrometer function, but it supports the psychrometer function and highlights the action as evaporation rate detector. We suggest testing the effect of the flow rate of the air stream used for humidity stimulation. As the wind speed strongly affects the power of evaporation, experiments with changing saturation deficit at different flow rates would improve our knowledge on humidity transduction. Insects are continually challenged to achieve and maintain optimal functions as ambient humidity changes. In addition to metabolic adaptations, most insects improve their survival abilities by humidity preference and avoidance responses. These behaviors make the existence of hygroreceptors very likely. The structural and the electrophysiological properties of hygroreceptors have been studied in only a few insect species. These hygroreceptors are associated in antagonistic pairs of a moist and a dry cell in the same sensillum with a thermoreceptive cold cell. The mechanism by which humidity stimulates the moist and dry cells remains controversial. Three main models of humidity transduction have been proposed, namely in which hygroreceptors operate as mechanical hygrometers, psychrometers or evaporation detectors  of water evaporating from its surface. Rising air temperature (sensible heat) increases both the wet-bulb and dry-bulb temperatures Figures , but theThe loss of water by evaporation is directly proportional to the saturation deficit of the air. Rising air temperature increases the saturation deficit Figure  when the2 surface area; Apodaca, Thus, transduction may involve chemical or mechanical mechanisms. Nonetheless, an evaporation controller that relies on concentration sensors might be unstable because chemical concentration is affected by multiple factors. Evaporation acting by fluid shear stress, pressure across, or tension within the dendritic membranes may create mechanical forces that deform membranes—as has been suggested for the mechanical hygrometer model. By contrast, the mechanosensory transduction stimulated by evaporation requires fine-tuning of the transducing ion channels. Because the moist cell and the dry cell discharge continuously during upward and downward changes in the saturation deficit, a receptor current must continuously flow when the evaporation rate is in equilibrium with ambient humidity. The current will be modulated between maximum and minimum values, and the sign will be opposite in the moist and dry cell. Conductivity of both antagonists must account for this symmetric change in receptor current as well as for its maintenance. A simple mechanism described for mechanosensitive channels is based on variation of the membrane capacitance (Petrov and Usherwood, Electrophysiological studies on the cockroach revealed a dependence of the responses of the moist and the dry cell to slowly fluctuating changes in vapor pressure on the temperature level at which the humidity changes are carried out Figures . The posAll authors contributed to data analysis, interpretation of results, writing the manuscript and designing the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.