Hereby, we could monitor changes in the thermal conductance in response to varying degrees of disorder. We find that the measured conductance increases as the density of the physical disorder increases, but undergoes an abrupt modulation with increasing degrees of chemical modification, which decreases at first and then increases considerably. Moreover, we find that the conductance varies inverse proportionally to the average distance between the structural defects in the graphene, implying a strong in-plane influence of phonon kinetics on interfacial heat flow. We attribute the bimodal results to an interplay between the distinct effects on graphene’s vibrational modes exerted by graphene modification and by the scattering of modes.Thermal energy transport across the interfaces of physically and chemically modified graphene with two metals, Al and Cu, was investigated by measuring thermal conductance using the time-domain thermoreflectance method. Graphene was processed using a He Analysis of the probe–beam response obtained from the TDTR measurements yields a thermal conductance of G ≈ 21 MW m−2 K−1 for the junction containing pristine graphene grown on Cu foil. For pristine graphene grown on a different Cu foil, the measured conductance increases by ~50%  as shown in 24G < 30 MW m−2 K−1 for junctions such as Au/Ti/graphene/SiO2 and Al/graphene/SiO2 at room temperature12The temporal profiles of the ratio of the in-phase and out-of-phase signals G of the junction could be expressed as a sum-of-resistance seriesThermal transport across the Al/graphene/Cu junction is mediated by monolayer graphene sandwiched between two different metals, where the graphene is processed to contain a varying amount of structural or chemical disorder. In the sandwich structure, the thermal resistance 1/GAl-gr and Ggr-Cu are the thermal conductances of the Al–graphene and graphene–Cu interfaces, respectively. The internal thermal resistance per unit area of graphene can be regarded to be small compared with the resistance at the interfaces, which is ~10−8 m2 K W−1, because the internal resistance would be, at most, on the order of 10−9 m2 K W−1 12. We may therefore assume that interfacial heat transport mostly determines the total resistance 1/G across the graphene–metal junction and any change that may result from modification of the graphene lattice. We discuss below how thermal transport evolves with disorder in the graphene and what causes the change.where 2+ ion beam with a Gaussian distribution. The 1/e2 radius of the beam intensity was 3.2 mm and the peak ion dose was ~1015 cm−2 at a kinetic energy of 3.04 MeV. We then traced the defect signature using Raman spectroscopy. −1 and the ‘G’ band intensity at ~1580 cm−1, as obtained from the Raman measurements. The profile clearly follows a Gaussian distribution and 1/e2 distance of the distribution approximately matches the diameter of the ion beam. The seemingly asymmetric distribution is because the irradiation occurred closer to one edge of the sample. These results suggest that the density of the produced defects could be proportional to the bombarding He2+ beam intensity.To investigate the influence of structural disorder on thermal transport, we produced spatially varied defects in the graphene and carried out profiling measurements using both Raman spectra and thermal conductance along the bombarding ion beam. Spatial distribution of the structural disorder was produced in graphene by irradiation using a HeD and ‘G’ band intensity IG allows us to estimate the defect density. With the peak dose roughly equivalent to 1 × 1015 He2+ cm−2, we find that the defect density falls to a low-defect density regimeD is greater than 10 nm. This contrasts with Ar+ ion bombardment at a similar dose, which generates a much larger number of defects2+ compared with using heavier but lower energy ions D is simply proportional to the total number of defects probed by the Raman laser spot because the Raman signal becomes an independent sum of the contributions from each defect26D/IG) can then be related to the average inter-defect distance LD by an empirical formulaL is the wave length of the Raman laser beam at 514 nm. This leads to a rather simple relation D at the center position and edge of the beam profile can be estimated as 13.2 ± 3.6 nm and 34.5 ± 9.6 nm, respectively. Hence the defect density should be less than 0.1% even at the center position.Analysis of the ‘D’ band intensity IThe measured thermal conductance also changes with ion beam intensity, as shown in D/IG and G/Go follow Gaussian distributions, the use of a proportional relation between LD−2 and ID/IG allows us to extract a relation between LD and the thermal conductance. A Gaussian distribution can be expressed as w is the distribution width, x and xc are the measurement location and peak position of the distribution, respectively, and A is a constant. Assuming a dependence of thermal conductance on the distance LD as w is the width from the defect density distribution and w′ is the width of the Gaussian distribution fitted to G/Go − 1. From the data in D, whereas β ≈ 2 holds quite well for the Raman ‘D’ band profile .To gain insight into how the defects affect heat transport, we examine a correlation between the measured defect distribution and thermal conductance. Since both the profiles of ICvl states that the phonon mean-free-path l is longer than 100 nm in graphene, where C is the specific heat capacity and v is the speed of soundlP will determine the total MFP change and thus thermal conductivity. While the shortened distance between defects and accordingly reduced MFP should significantly impair in-plane heat transportThis result is evidence that a kinetic in-plane interaction between the defects strongly influences the cross-plane thermal transport occurring at the interface. The kinetic description of thermal conductivity Λ∼D/IG increases approximately linearly with exposure time after the initial exposure suggests an increase in oxidation coverage proportional to the processing time.For comparison, we investigate how heat flow responds to chemical modification of the graphene lattice by adsorbed foreign species. Graphene was processed by varying its exposure to a UV–ozone environment. This method utilizes UV rays to induce the chemical reaction of oxygen radical molecules with the graphene surface by generating ozone molecules from UV-activated oxygen (see Methods for details)192 junction as a result of oxidation using oxygen plasma, in which the enhanced conductance was attributed to an increased strength of the chemical bonding between the Al layer and the oxygen-adsorbed graphene. Despite the similar results, the reduced conductance observed in the earlier stages of oxidation, shown in The development of the Raman bands is similar to that of ion-bombarded graphene, but the trend in thermal conductance appears largely different and even opposite. In GAl-CuO/Cu ~100 MW m−2 K−1 is higher than that of the graphene-removed junction, but it is significantly lower than GAl-Cu ~4 GW m−2 K−1 for the Al–Cu interfaceG′ asTo appreciate the abrupt rise in thermal conductance and the measurement uncertainty following longer graphene processing times (over 6-hour), we measure the conductance of graphene-removed and graphene-free junctions. The graphene was intentionally removed using oxygen plasma to prepare the graphene-removed junction. The graphene-free junction was prepared by simply depositing an Al thin film on a Cu substrate. Measured values for both samples are plotted in f is the areal fraction of the remaining graphene and G. This relation gives us simple estimates of the fraction , implying that the conductance may vary considerably with a relatively small fraction of direct contact. The abruptly large change in the conductance can thus be accounted for by partial removal of the graphene after broad coverage of carbon–oxygen bonds. Indeed, as shown in the inset of where On the other hand, the reduction in thermal conductance observed in the earlier stages of UV–ozone processing is distinguished from previous results and remains to be explained. Since the elastic property of graphene changes with oxidation3032302 is known to be ~200 MW m−2 K−1, which is a factor of two greater than GAl-CuO/Cu. Yet these factors are insufficient when explaining the seemingly discrepant conclusions because the thermal transport pathway is likely to be similar. Although we exhibit the varying stages in thermal conductance in response to a range of oxidation, the cause of the discrepancy remains unclear.There are two more factors to consider regarding the oxidizing method and the material system. XPS measurements indicate that the signature of the carbon–oxygen bonds and a portion of the functional group with a higher bonding energy  are larger in our method than in the previous report19Cu) could change after ion irradiation and affect thermal transport across the junction. (2) We consider the possibility of forming a direct contact between the Al film and substrate through graphene pinholes that might be created by the ion bombardment, which would result in larger conductance. Bombarding the graphene on Cu substrate not only produces defects in the graphene, but also damages the substrate. Indeed, the expected damage reduced the thermal conductivity ΛCu by ~10% (see Cu). Additionally, the supposed thermal transport through pinholes would contribute to thermal conductance, as included in −2 K−1, which is negligible compared with the observed enhancement. Furthermore, the grain size of the Al thin film is a few tens of nanometers  The thermal conductivity of the Cu substrate  to obtain Raman spectra of the graphene. The laser wavelength for Raman excitement and beam spot size were 514 nm and ~3 μmThis process uses UV lights with wavelengths λ of 184 and 253 nm. Ozone molecules, which are generated by UV (λ = 184 nm) activation of oxygen, sequentially decompose to oxygen radicals that can oxidize graphene, which is assisted by UV rays at λ = 253 nm. These oxygen radicals are known to absorb on the graphene basal plane and chemically react with the carbon atoms to form functional groups . The Lorentz numbers for Al and Cu are slightly different because of the interaction of electrons with the lattice vibration−3).In thermal modeling, there is only one free parameter: the thermal conductance of the Al/Gr/Cu interfaces. The thermal conductivity of Al and Cu were obtained using the Wiedemann–Franz law .


Results were compared with clinical parameters and outcome. Flow cytometry was exploited to determine the PD-L1 and PD-1 protein expression in conjunctival and cutaneous melanoma cell lines. PD-L1 expression was identified on tumor cells in five (19%) primary CM and on stromal cells (mainly CD68+CD163+ M2 macrophages) in 16 (59%) cases. PD-L1 expression on tumor cells was associated with the presence of distant metastases and a worse melanoma-related survival. PD-1 expression was seen in 17 (63%) cases, all of which were T2 stage tumors. Small tumors had a higher density of TILs than large tumors. The density of TILs was not correlated with survival, tumoral/stromal PD-L1 or PD-1 expression. In vitro results showed that most CM and cutaneous melanoma cell lines do not constitutively express PD-L1. However, expression could be upregulated after interferon gamma stimulation. Our findings suggest that blocking the PD-1/PD-L1 axis should be evaluated as a treatment for CM.Conjunctival melanoma (CM) is an infrequent but potentially lethal malignancy, with limited therapeutic options for metastases. Recent inhibitors of the interaction of programmed cell death protein 1 (PD-1) and its ligand PD-L1 are associated with good clinical responses in many malignancies. To investigate the therapeutic potential of targeting the PD-1/PD-L1 axis in CM, we analyzed the expression of PD-1 and PD-L1 and the density of various types of tumor-infiltrating lymphocytes (TILs) in primary CM ( Conjunctival melanoma (CM) is a rare ocular malignancy, accounting for 5% of all ocular melanoma . CM is aRecently, immunotherapies aiming at immune checkpoint pathways, such as cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), have been successfully exploited in the treatment of metastases of different malignancies and have led to long-lasting clinical responses . Both CTCM in many ways resembles cutaneous melanoma, suggesting that patients with CM metastases might also benefit from treatment with anti-PD-1/PD-L1 agents. PD-L1 expression determined by immunohistochemistry (IHC) on tumor cells is thought to be a potential biomarker predicting the sensitivity of anti-PD-1/PD-L1 treatment –13. Whetn = 20) is comprised of limbal (n = 16) and bulbar conjunctiva (n = 4). The non-epibulbar localization (n = 7) includes tarsal, forniceal and caruncular conjunctiva. The clinical TNM stage was T1 in 20 (74%) and T2 in 7 (26%) cases. Two (7%) of the patients underwent surgical excision alone as primary treatment, three (11%) excision with cryotherapy, one (4%) excision and mitomycin C, 16 (59%) excision and subsequent brachytherapy, one (4%) external beam radiation, and four (15%) were treated by exenteration. The median follow-up time was 46 months (range 3–247 months). Eleven (41%) cases developed local recurrences. At the end of the follow-up period, four patients had died from CM metastases, two from unknown diseases without any signs of metastases, and 21 patients were alive.We studied primary CM from 27 patients who had been treated at the LUMC between 1996 and 2014 , while positive PD-L1 staining in tumor areas was associated with the development of distant metastases (p = 0.01). Kaplan-Meier analysis and log rank testing similarly showed that PD-L1 positive staining in the tumor was associated with a worse melanoma-related survival  and 16 (59%) CM sections, respectively, as illustrated in Figure s Figure .p = 0.03). Absence of PD-1 tended to correlate with less local recurrence (p = 0.12). A prior study on cutaneous melanoma showed that those melanomas often harbor intrinsically PD-1-positive tumor cell subpopulations [PD-1 expression was localized on the membrane of T cells Figure , and wasulations ; however+CD8+, CD3+CD8-, CD3+CD8-Foxp3+ and CD3+CD8-Foxp3- T cells, and CD68 (macrophages) and CD68+CD163+ (M2 macrophages) within tumor areas of 26 primary CM sections. Figure +CD8- and CD3+CD8-Foxp3- positive cells than non-T2 tumors  and tumor-infiltrating M2 macrophages , CD3+CD8+ (p = 0.02), CD3+CD8- (p = 0.01), CD3+CD8-Foxp3- (p = 0.02) and CD3+CD8-Foxp3+(p = 0.03) T cells within their tumor areas than tumors with smaller basal diameters (Table p > 0.05) or with melanoma-related survival. IF staining of CD68 and CD68+CD163+ showed that the majority of macrophages belong to the M2 phenotype, suggesting a potential tumor-favorable environment created by macrophages in CM. As high cytotoxic T lymphocyte (CTL)/regulatory T cell (Treg) and high M1 (CD68+CD163-)/M2 macrophage ratios have been found to be associated with improved survival in breast cancer and cutaneous melanoma, respectively [p > 0.05). However, higher CTL/Treg ratio tended to correlate with local recurrence (p = 0.13). Correlation coefficients are shown in Table In order to see whether specific types of infiltrating leukocytes contributed to PD-L1 and PD-1 expression on tumor cells, we determined the presence of different subsets of T cells and myeloid cells in the same CM, by performing immunofluorescence (IF) staining according to previously described techniques : we meas T cells . The difectively , 18, we in vivo, we stimulated these cells with IFN-γ. As a control, we determined the upregulation of IFN-γ pathway by analyzing HLA Class I expression, using an anti-HLA class I antibody (Infiltration lymphocytes may be a source of interferon gamma (IFN-γ), which has been reported to enhance PD-L1 and PD-1 expression , 20. To antibody . HLA Claantibody .Immunotherapies that work through inhibiting the PD-L1/PD-1 axis have been successful in inducing clinical responses in patients with different malignancies, including cutaneous melanoma, non-small cell lung cancer, and bladder cancer –23. Howen = 5).PD-L1 expression is a potential biomarker for prognosis in different types of cancer –29. Expr+CD163+ M2 macrophages, similar to what has been described previously [In vitro experiment showed that all CM cell lines expressed low levels of membranous PD-L1, and a variable but clear increase of PD-L1 expression was seen in two out of three CM cell lines following IFN-γ stimulation. These findings suggest that in CM, initially PD-L1 negative or weakly positive tumors may display enhanced PD-L1 expression after exposure to IFN-γ produced by TILs.Not only expression of PD-L1 on tumor cells may be important, also PD-L1 expressed by myeloid cells in the tumor microenvironment may play an essential role in suppression of the host's immune response, even when the malignant cells lack PD-L1 , 35. Streviously . In vitr+CD8-), CD4 helper (CD3+CD8-Foxp3-) and Foxp3 (CD3+CD8-Foxp3+) cells than UM. However, the densities of CD8 (CD3+CD8+), CD68 and CD68CD163 cells were lower than those in UM. Compared to one study of cutaneous melanoma metastasis [Cancer exploits multiple mechanisms to avoid antitumor immune responses. Based on the “cancer immunogram” depicted by Blank, et al. , the gentastasis , the dentastasis , 39. We A major limitation of the present study is the small size of the cohort, coming from a single institute, due to the rarity of CM. We need more patients and tumor material, especially metastases, to carry out further studies and draw solid conclusions. In addition, we should be aware that CM samples are generally quite small, and that a representative section accounts for a small volume of tumor, and may not represent the PD-L1 expression of the whole tumor, as it is known that PD-L1 expression may be quite heterogeneous .In general, we provide a comprehensive view of PD-L1 and PD-1 protein expression, and immune infiltration status in CM. These findings deepen our understanding of the immunology of CM. We believe that these results support the rationale of PD-L1/PD-1 checkpoint immunotherapy for patients with metastatic CM and recommend to include these patients in future immunotherapy clinical trials inhibiting the PD-L1/PD-L1 pathway.th edition of the AJCC TNM cancer staging manual [Twenty-seven patients with histologically-proven primary CM were included in this study. All patients were seen at the Leiden University Medical Center, The Netherlands, and diagnosed with CM between 1996 and 2014. The medical files were reviewed for clinical and histopathological data. Information regarding the localisation and size of the primary tumors was obtained from the patient files, histology reports, and pre-excision color photographs. All tumors were evaluated by an experienced ophthalmic pathologist. Tumor stage was determined according to the 7g manual . TreatmeFormalin-fixed, paraffin-embedded blocks containing tumor material were cut in 4 μm sections, and mounted on slides. After deparaffinization with Xylene, rehydration with alcohol , and Tris-EDTA (pH 9.0) heat-based antigen retrieval, the tissues were incubated with primary antibodies at 4°C overnight. On the second day, after washing with phosphate-buffered saline (PBS), the samples were incubated with AlexaFluor  secondary antibodies for 1 hour at room temperature, followed by washing steps. The slides were counterstained and mounted with VECTASHIELD mounting medium with DAPI . Tonsil tissues were used as positive control. Incubation with 1% bovine serum albumin (BSA)/PBS instead of primary antibodies served as negative control. One tumor contained only enough material for PD-L1 and PD-1 staining, and not for additional staining. The primary antibodies are listed below: HMB45/Mart-1 , anti-PD-L1 , anti-PD1 , CD3 , anti-CD8 , anti-FoxP3 , anti-CD68  and anti-CD163 . The secondary antibodies are in 2) were evaluated using Leica Application Suite X or Zeiss Zen 2.1 software. Positive cells were counted manually by two observers, as previously described [2.The images of hematoxylin and eosin (HE) stained tumor sections were captured using Philips Image Management System 2.2. Images of IF staining were captured using either a Leica TCS SP8 X or Zeiss LSM 700 confocal laser scanning microscope. Depending on the tumor size, one to seven representative images at high power (250X) in different areas were randomly selected. Tumor areas were morphologically recognized by DAPI nuclear staining. Two investigators, without prior knowledge of clinicopathological data, scored membranous PD-L1 and PD1 expression. Expression of PD-L1 and PD-1 was designated as positive, when ≥ 5% of the tumor/stromal cells were positive , 41. Forescribed . ResultsThree conjunctival melanoma cell lines  , 43 and Cells were incubated with the previously determined optimal dilution of mouse monoclonal PD-L1 , PD-1  or HLA class I antibodies . Cells were collected (10000-20000 per live gate) using the FACSCalibur cytometer (Becton Dickinson), and results were analysed using FlowJo software .p ≤ 0.05. Pearson's chi square and Fisher's exact test were applied for categorical data; Mann Whitney U test was used for numerical data. Spearman's rank correlation analysis (two-tailed) was performed to compare correlations between different TILs and tumor size. Survival analysis was performed using Kaplan-Meier with log rank tests.Data were analysed with SPSS software version 23.0 . Data were considered statistically significant if


Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 pathway are expected to be a novel therapy for combating future increases in numbers of malignant pleural mesothelioma (MPM) patients. However, the PD-L1 expression, which is a predictor of the response to ICIs, is unclear in MPM. We studied the PD-L1 expression using four immunohistochemical assays  in 32 MPM patients. The PD-L1 expression in tumor cells and immune cells was evaluated to clarify the rate of PD-L1 expression and the concordance among the four assays in MPM. The positivity rate of PD-L1 expression was 53.1% for SP142, 28.1% for SP263, 53.1% for 28-8, and 56.3% for 22C3. Nine cases were positive and 10 were negative for all assays. Discordance among the four assays was found in 13 cases. The concordance rates between SP142 and 22C3 and between 28-8 and 22C3 were the highest (84.4%). The concordance rates between SP263 and the other three assays were low (71.9% to 75.0%). The PD-L1 expression in MPM was almost equivalent for three of the assays. Given the cut-off values set in our study, these findings suggested that these assays, except for SP263, can be used for accurate PD-L1 immunostaining in MPM. Malignant pleural mesothelioma (MPM) is a rare disease with no effective standardized systemic therapy. The median survival of untreated MPM is generally less than one year –3. MultiImmunotherapies targeting the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway are a standard treatment for various malignant tumors –12. ClinHowever, there are many PD-L1 immunohistochemistry (IHC) assays available for examining the PD-L1 expression of tumor cells (TCs) and immune cells (ICs), and selected assays vary among studies. In the Blueprint PD-L1 IHC Assay Comparison Project, the authors stained 39 cases of non-small cell lung cancer (NSCLC) using 4 PD-L1 IHC assays and reported that changing the assays and cut-off values used would lead to the misclassification of the PD-L1 status .Based on these findings, we evaluated the PD-L1 expression in different histological types of MPMs using four kinds of PD-L1 IHC assays and investigated the PD-L1 expression rate in MPM and the differences in the PD-L1 expression among the four assays. The aim of this study was to establish a highly reproducible standard assessment for each companion or complementary PD-L1 antibody in MPM and to elucidate the association between the expression of PD-L1 and the clinicopathological features.n = 19, 59.3%), biphasic type , and sarcomatous type . The study population included 27 male patients and five female patients . The TNM stage classifications were 1 as stage I, 4 as stage II, 17 as stage III, 8 as stage IV, and 2 as unknown because they were from biopsied cases [The patients’ characteristics are shown in Table e n = 6, .8%. The Figure Table The heat map in Figure p = 0.49, SP263: p = 0.06, 28–8: p = 0.43, 22C3: p = 0.62). Sarcomatous type had the highest mean percentage of positive cells in all assays except for 22C3, and epithelial type had the lowest mean percentage of positive cells in all assays.Regarding the histological types, the positive rate of epithelial type was low in all assays, while the positive rate of sarcomatous type was high. In the analysis of the relationship between the PD-L1 expression and histological types , there was no significant correlation between the PD-L1 expression and the histological type in any of the assays .In the analysis of the overall survival (OS) by the PD-L1 expression in each assay, there were no significant differences Figure . We evalMPM is a rare malignant tumor arising from mesothelial cells of the pleura and accounting for less than 0.3% of all cancers . There iImmunotherapies targeting the PD-1/PD-L1 pathway are now a standard treatment in various malignant tumors, such as melanoma, NSCLC, renal cell carcinoma, Hodgkin’s lymphoma, carcinoma of head and neck, and gastric carcinoma –12. PD-Ln = 53) and a median progression free survival (PFS) of 17.1 weeks [n = 25), a median PFS of 5.4 months, and a median OS of 18 months [n = 34) was reported at the 2016 World Conference on Lung Cancer [Several clinical trials have been conducted to investigate the effect of immunotherapy using an ICI targeting the PD-1/PD-L1 pathway in MPM patients. The phase JAVELIN trial using avelumab (regardless of PD-L1 expression) , the pha.1 weeks . In the 8 months . In the g Cancer . Based oMany PD-L1 IHC assays have been developed to examine the PD-L1 expression of TCs, and the assays used vary among studies and ICIs. In the Blueprint PD-L1 IHC Assay Comparison Project, the authors stained 39 cases of NSCLC using four PD-L1 IHC assays. They reported that changing the assays and cut-off values used would lead to the misclassification of PD-L1 status . Given tet al. and Mansfield et al. reported values of 41.7% and 40% with the same cut-off [The PD-L1-positive rate of MPM differs wildly among reports, ranging from around 20% to about 70% –24. In t cut-off , 26. GivThe cut-off values in the present study were the same as those used in the Blueprint project . SP263 iRegarding the PD-L1 expression by histological types of MPM, some reports have found that PD-L1-positive cases were more frequently non-epithelial type  than epithelial type , 23, 25,et al. reported that slides stained within 90 days had a slightly higher prevalence of PD-L1 positivity in comparison to specimens that were stored for ≥90 days [Among the four assays used in this study, SP142 is reported to be less likely to stain TCs in NSCLC or MPM than the other three assays , 23, 33.≥90 days . The den≥90 days , 39, to In IC staining, no significant correlations were observed among the four assays. Tumor-infiltrating lymphocytes (TILs) are measured morphologically, and there is currently no established threshold for TILs . HoweverTo the best of our knowledge, this is the first validation trial for MPM using the four companion diagnostic assays. In addition, most of the samples were surgical specimens, and the results from quality-controlled samples were verified by staining with IHC antibodies specific for MPM. We believe that this research gives MPM patients very realistic information that will be useful in the clinical administration of ICIs.Finally, as a future issue of immunostaining for MPM, it is important to determine whether or not the four PD-L1 IHC assays have specific staining properties for any TCs, as some MPMs have a heterogeneous tissue morphology within the tumor –46. TherIn conclusion, the positive rate of PD-L1 expression in MPM was over 50% and almost equivalent among the SP142, 28-8, and 22C3 assays. The relatively low rate of PD-L1-positive cases with the SP263 assay may have been due to the cut-off value used. Our results suggest that ICIs might be effective for MPM as novel therapeutic agents, and the three assays with the cut-off values used in our study may all be suitable for accurate PD-L1 immunostaining in MPM.Thirty-two MPM tissue specimens were obtained by surgical excision (including two thoracoscopic biopsy specimens) from January 1992 to December 2016 at Nagoya City University Hospital. All cases were microscopically reviewed and diagnosed by two expert pathologists (TM and HI). All specimens were stained with IHC antibodies specific for MPM such as calretinin, cytokeratin 5/6, and vimentin , 39, andAn appropriate formalin-fixed and paraffin-embedded (FFPE) block containing the tumor in each case was selected by reviewing H&E-stained specimens, and each FFPE block was sliced into 3-μm-thick tissue sections. The tissue sections were deparaffinized and subjected to immunostaining with the following for anti-PD-L1 antibodies: clone SP142 , clone SP263 , clone 28-8 , and clone 22C3 (Dako). SP142 and SP263 immunostaining was carried out with a Bond-Max autoimmunostainer  and a Bond polymer-refine detection kit (Leica Microsystems). 28-8 and 22C3 immunostaining was carried out with a Dako autostainer Link48 system (Dako) and a PD-L1 PharmDx kit (Dako).The positivity of TCs and ICs was assessed by two expert pathologists (TM and HI). TCs in which the membrane was immunostained at any intensity were considered to be positive for PD-L1. The ratios of PD-L1-positive TCs were evaluated by microscopic observation. Meanwhile, ICs in which the membrane or cytoplasm was immunostained at any intensity were considered to be positive for PD-L1 because the stained membrane and cytoplasm could not be distinguished in lymphocytes due to their small size . Most PDp value < 0.05. We performed all analyses using the JMP software program .The analysis was performed using a Fisher’s exact test for qualitative variables. Survival curves were generated using the Kaplan-Meier method, and the log-rank test was used to assess the statistical significance of the differences between groups. The two-sided significance level was at


Fonsecaea comprises black yeast-like fungi of clinical relevance, including etiologic agents of chromoblastomycosis and cerebral phaeohyphomycosis. Presence of melanin and assimilation of monoaromatic hydrocarbons and alkylbenzenes have been proposed as virulence factors. Multicopper oxidase (MCO) is a family of enzymes including laccases, ferroxidases and ascorbate oxidases which are able to catalyze the oxidation of various aromatic organic compounds with the reduction of molecular oxygen to water. Additionally, laccases are required for the production of fungal melanins, a cell-wall black pigment recognized as a key polymer for pathogenicity and extremotolerance in black yeast-like fungi. Although the activity of laccase enzymes has previously been reported in many wood-rotting fungi, the diversity of laccase genes in Fonsecaea has not yet been assessed. In this study, we identified and characterized laccase-coding genes and determined their genomic location in five clinical and environmental Fonsecaea species. The identification of laccases sensu stricto will provide insights into carbon acquisition strategies as well as melanin production in Fonsecaea.The genus  Fonsecaea is a melanized fungal genus defined by sympodial conidiogenesis with conidia arranged in short chains, and in absence of budding cells. The genus affiliates to the ascomycetes order Chaetothyriales, comprising proven causative agents of human chromoblastomycosis and cerebral phaeohyphomycosis  as C-terminus, which has not yet been described in basidiomycetes. It was suggested that incorrect processing of the C-terminus leads to lack of enzyme activity. The deletion of DSG[LIV] in 2 centre , 38. The removed . In thisFonsecaea laccases possess between 1 and 9 putative N-glycosylation sites, together with Asn-X-Ser/Thr sequons : subfamily I with zero introns, subfamily II with 2 introns and subfamily III containing 6 introns. Two other subfamilies, which had genes with 1 or 3 introns, were restricted to F. multimorphosa and F. erecta  and F. pedrosoi, F. monophora and F. multimorphosa   .Fonsecaea laccase genes, particularly in the T1 region, could increase the diversity of this enzyme impacting its oxidative ability.These introns were found distributed similarly within subfamilies. Interestingly, we observed a prevalence of gain or loss of introns in the internal region of enzymes belonging to subfamilies II and III . InternaFonsecaea species, F. pedrosoi, F. monophora and F. nubica, agents of chromoblastomycosis each have two extracellular and two intracellular laccases, while the saprobic species F. erecta has three extracellular and one intracellular laccases and both F. multimorphosa strains three extracellular and two intracellular laccases Click here for additional data file.


The Method of Levels (MOL) is a transdiagnostic cognitive therapy that promotes contentment, wellbeing, and goal achievement through the resolution of internal conflicts underlying psychological distress. MOL, based on Perceptual Control Theory (PCT), was developed in routine clinical practice and has been used effectively across different health services by different practitioners. Access to MOL-style questions through a smartphone app could, potentially, help both the general public maintain robust mental health, and also be a useful adjunct to therapy for clinical populations. The app is called MindSurf because of its focus on helping people explore their thinking. Prior to developing the app and using it with different populations it was necessary to determine whether such an idea would be usable for and acceptable to potential app users. Therefore, a pilot study was conducted with a non-clinical sample to assess the usability and acceptability of the app including monitoring whether the questions delivered in this way were associated with any adverse events.A pilot study using quantitative as well as qualitative methods and incorporating a repeated measures, A-B design was conducted.The 23 participants were healthy adult volunteers who were all either undergraduate students, postgraduate students, or staff of the University of Manchester. They received MOL-style questions on their mobile phones over a 1-week period. Qualitative results were encouraging and indicated that the format and style of questioning were acceptable to participants and did not lead to increased worry or concern. A one-way, repeated measures ANOVA indicated that there was a nonsignificant decrease in scores on the 21-item Depression, Anxiety, and Stress Scale (DASS21) over a 2 week period.The results of the pilot study justified development of MindSurf and further testing once it is available for use. A power analysis indicated that the pilot study was underpowered to detect significant effects but provided important information regarding the appropriate sample size for future research. The pilot study also indicated that future research should investigate the effects of receiving more than three questions per day.Results of the pilot study indicate that MindSurf will be a usable and acceptable app. Its benefits should be further explored through longer studies with larger sample of both the general population as well as clinical populations. Mental health problems are a growing international concern. Approximately half of all ill-health in UK residents under the age of 65 is due to mental illness  accountiThe rise of the smartphone has been astonishing with an estimated 1.82 billion smartphones in active use worldwide by the end of 2013 . ImportaSmartphones have been described as revolutionizing approaches to wellbeing investment . Links bDespite the potential benefits of smartphone applications there is a concerning lack of regulation and an absence of evidence for many of these applications . AlthougThe Method of Levels (MOL) –16 is a Apart from the demonstrated effectiveness of MOL , 19, oneIt was planned to develop a mobile phone app that would push MOL-style questions at random times throughout the day to the user’s phone. The number of questions would be set by the user as would the time period within which the questions were delivered. The user would be prompted to either type an answer or to say an answer out loud, because in MOL it is considered that the external expression of internal cognitions is a useful way to promote greater depth of processing .Given the importance of ensuring that the app is evidence-based, a systematic program of research was developed  .The first research question required a qualitative methodology to answer whereas the second question used quantitative methods. For RQ2 an hypothesis was generated:For RQ1, semi-structured interviews were used. The usability and acceptability of the app was determined by participants’ responses in the interviews. Also, completing the week of responding to three text messages a day was used as a behavioral indicator of the usability of the app.http://intqhc.oxfordjournals.org/content/19/6/349.long).With regard to the methods of the study, this paper conforms to COREQ guidelines . The volunteering database is a website where all research projects for the University of Manchester that require research participants are listed. Volunteers are then able to select studies in which they are interested in participating and establish contact with the researcher for more information. Of the 23 people who volunteered, three were male and all were over the age of 18. A large proportion of female participants is not unusual in these types of studies  and is lInformation about the rationale for the study and what would be required of participants was displayed on posters around the University of Manchester. This information was also available at the online database where people could indicate their willingness to participate in the research. Furthermore, all participants were provided with a standardised Participant Information Sheet prior to consenting to participate in the research. Given that the focus of the study was usability and acceptability, other potentially useful measures such as assessing the roles of expectancy or credibility were not used but may be included in future research. Participants were aware, however, that they would be asked to participate in an interview at the end of the study which would explore their experience of receiving the questions. A small monetary incentive of £5 was offered to participants upon completion of the 2-week data collection period.As with other studies assessing the usability and acceptability of smartphone apps, inclusion and exclusion criteria were minimal . IndividThe DASS-21 , a 21-itThe DASS-21  requiresParticipants were provided with two paper-copy diary booklets for completion, one for each week of data collection.The diary booklet for week one consisted of two 10-point Likert scales to be completed once daily; one indicating the perceived levels of wellbeing and the other indicating perceived levels of distress. For the perceived wellbeing scale, a score of zero indicated an extremely low level of wellbeing and a score of ten indicated an extremely high level of wellbeing. For the perceived distress scale, a score of zero indicated “not distressed at all” and a score of ten indicated “extremely distressed”.The diary booklet for week two prompted participants to write the responses to the questions they were sent via text message in the text boxes provided (three per day). As in week one, participants were also requested to record once daily their perceived levels of wellbeing and distress . Participants were asked to record these ratings after having received all the questions for the day.Interviews were recorded using a digital dictaphone device. Both SPSS (Version 20) and NVivo (Version 10) were used in the analysis of the data.“What’s going through your mind just at the minute?”; “What’s your perspective on things right now? What’s the thought that stands out the most?”; “What goal are you heading for right now?”; “What is going through your mind at this very minute? What do you make of that?”; and “Where’s your attention just now?”A bank of 52 questions was developed by the first author (TAC) based on the types of questions asked in MOL. The questions are designed to help people increase their awareness of their current cognitive activity. Some of the questions include a pair of questions to draw people’s attention to various cognitions and then have them reflect on that. The question bank includes questions such as: The intervention for this study was the app described in the introduction. The essential feature of this app is that it pushes to a user’s phone questions, in the form of text messages, which encourage metacognitive thinking. These questions have been found to be therapeutically useful in clinical situations. The concept of the app is innovative in that it pushes questions at random times throughout the day. Without any evidence of usability or acceptability it was considered premature to develop an actual app so the researcher functioned as the app and sent three text messages to the participants’ phones at random times throughout the day over a 1-week period. If the results of this study indicate that the development of an app should proceed, the number of questions (between 0 and 10) will be set by the user as will the time period each day within which the questions will be sent .Ethics approval was obtained from the University of Manchester Research Ethics Committee prior to starting the study. Participants were informed of the nature of the study and the content of the questions they would receive  before consent was obtained. Participants were advised to only provide that amount of information in response to the questions which they felt comfortable sharing.Table The semi-structured interviews were recorded and then transcribed. A topic guide consisting of five core questions to explore usability and acceptability was used to structure the interviews. The questions were: What was your experience of receiving the questions daily via text message?; What did you think of the questions themselves?; Did you find the questions helpful at all? Why?; Did the questions help you realise anything? What was this?; Would you have preferred more or fewer questions each day? Why is this? From these questions, additional probes were used to examine responses in greater detail.Thematic Analysis (TA) was used to identify recurring themes in the data obtained from the transcripts. The initial stage of TA was familiarisation with the data including reading the interview transcripts several times in order to increase sensitivity to emerging themes. The data were systematically coded with attention focused on recurrent content and content relevant to MOL and PCT. These codes were then grouped and organised into subthemes which, when reviewed and analysed, were regrouped under broader, conceptual themes. All subthemes and themes were reviewed in an iterative process and subthemes that did not have sufficient recurrent, representative data were discarded.A repeated measures, A-B design was used for this study. Data were collected over a 2 month period because participants completed their 2 week engagement in the study at different times depending on their availability and time of recruitment.The quantitative data and analysis, for the purposes of this study, indicated potentially adverse effects and should be regarded as an addition to the qualitative data which explores the usability and acceptability of the app. Scores from the DASS-21 at the three time-points of baseline, pre app, and post app were analysed using a one-way, repeated measures analysis of variance (ANOVA). Repeated-measures ANOVAs were also conducted on daily ratings of perceived wellbeing and distress which were obtained over a 14 day period.F = 3.10, p = 0.06. This trend was encouraging and suggests that adverse events do not seem to be indicated.It was hypothesised that the MOL-style questions would result in significant decreases in scores on the DASS-21. The means and standard deviations for the DASS-21 at three different time points  are presented in Table Daily ratings of wellbeing and distress were obtained from participants over 14 days (7 days prior to receiving the questions and 7 days while receiving the questions). The mean ratings for Day 1, Day 8, and Day 14 are provided in Table η2 = 0.20 respectively, the power for this study was 0.52. To increase power to 0.95 a sample size of 66 is required.Given that the direction of change for the DASS-21 was in the desired direction, a power analysis was conducted to determine if the failure to achieve statistical significance was due to the study being underpowered. With a sample size of 23, the alpha level set at 0.05, and an effect size for the DASS-21 of Of the 23 participants in the study, 11 consented to participate in semi-structured interviews. The most common reason participants gave for not participating in the interview was a lack of time available in their busy schedules. Table Analysis of the transcripts revealed that all participants had varying opinions regarding the app and differential experiences of receiving the questions. All interviewed participants recognised the potential benefits of receiving the questions and engaging in a psychotherapeutic intervention via technological modalities, although there were differing views of how relevant such interventions were to their everyday experiences. Interviewees clearly expressed their perceptions of engaging in interventions via mobile phone technology and their degree of willingness to engage in such interventions via technological modalities.‘Yeah, I thought it was good … text messages suited me fine … it’s good to text you, like, at random times to catch you in the moment.’ [103]‘… it was quite good to be honest because most people, y’know, keep their phone with them at all times so, like it was something I’d do right there and then.’ [109]When questioned regarding the acceptability of using mobile phones for such interventions, the majority of participants thought that mobile phones were acceptable and convenient. Participants reported that they were able to integrate a mobile phone-based intervention into their everyday lives relatively easily due to mobile phones being integral to daily routines:‘… some of them, like, actually made me just think more about the bigger picture so that was quite positive.’ [113]Participants recognised the potential benefits of the app and were able to identify ways in which it could be personally helpful:‘I think it depends on the situation you're in at the time as well - because if you're in a situation that's either stressful or quite demanding - the way you're thinking at that time and the question you receive whether it's helpful or not I think can have a big difference on…how it would make you cope later on … the questions I think mostly would all be really helpful just depending on what kind of circumstances you’re under at that time.’ [104]Also, participants identified particular circumstances in which the questions would be helpful such as when they feel their wellbeing is compromised or their distress levels are increasing.‘… it took up quite a bit of time … well, it didn’t take up time it was just, erm, hard to answer them when I got them…’ [102]‘… at times they were a bit inconvenient I think just because of obviously what I was doing at the time …’ [104]Participants emphasised how important it was to be able to incorporate the app into their everyday lives in terms of the usability and acceptability of the approach.‘… some of them, most of them, were quite straightforward and quite easy to answer but there were some of them that were quite hard to answer…because you weren’t quite sure what it was actually asking you about.’ [102]‘I just thought it was a bit, it was a bit too vague … so I didn’t really know … what the questions were asking.’ [105]Some participants expressed difficulty at understanding what particular questions were asking about or at generating answers to questions. Three participants felt that this was due to the vague nature of the questions, but two participants expressed difficulty in generating an answer to the questions which they deemed sufficient.All participants identified and acknowledged that the questions received via text messages enhanced their self-awareness to varying degrees. Participants reported individually that the MOL-style questions prompted them to enhance their awareness of issues such as their thought patterns, their personal goals, and their behavioural responses. The overarching theme of enhanced awareness could be clearly separated into the subthemes of: present-moment thinking, self-reflection, and barriers to self-reflection.‘[The questions] made me more aware in that moment of the way I think …’ [101]‘I think doing the questions … made me think about – how to be more aware of my thoughts … it sort of … made me think about thinking about my thinking if that makes sense?’ [103]Participants made reference to the fact that the questions prompted them to consider their present-moment thoughts and thought patterns, potentially allowing them to adopt a more mindful approach to their emotions and cognitions. For example:‘… it enables you to reflect on what you’re thinking and then … kind of check yourself and be like ‘I shouldn’t be really thinking that’ so that was quite good I think …’[101]‘… it was nice to sort of think about what I’m doing and whether it makes sense or not.’ [105]Participants reported that receiving the questions prompted reflection. This reflection appears to have often been inwardly directed, thus resulting in participants becoming increasingly self-aware and reflecting on patterns of thought and behaviour which may play a general role in their wellbeing.‘… if you’re busy on a particular, sort of, job or task or whatever where you’re preoccupied by things where you’re … preoccupied by the task and not preoccupied by other thoughts.’ [103]‘… a lot of the time it would just be like, well I’m not really thinking anything right now. I was just like not really doing anything and my mind was pretty blank.’ [108]Despite participants reporting that the questions and the app as a whole enabled them to be more self-reflective and more aware of patterns of thoughts, behaviours, and emotions, participants also reported sometimes experiencing difficulty engaging in the activity of self-reflection. This emerged as a separate issue to the comprehension of the questions, but instead appeared to revolve around having difficulty capturing and analysing current thoughts.Given the increasing burden of mental health problems and the difficulty in accessing effective services in a timely manner, a pilot study, preceded by a proof-of-concept exercise, was conducted to investigate the usability and acceptability of a smartphone app. The app is based on MOL, a transdiagnostic cognitive therapy. The proof-of-concept exercise indicated that the questions were received favourably across a 7 day period and that some useful realisations were achieved. The focus of this paper, the pilot study, was similar to other projects investigating the use of apps in that it had a small, self-selected convenience sample, was conducted over a relatively short time-frame, and focussed on usability and acceptability , 12. TheFrom the qualitative data in the pilot study the themes of: “perceptions of the app” ; “convenience”; “comprehension”; and “enhanced awareness”  were generated. Participants indicated that, generally, they found the questions helpful in the way that was intended. That is, they became more aware of their thought processes and more reflective. Participants also reported finding some of the questions difficult to answer, but found the modality of a mobile phone convenient. It could be that, when the app is used an adjunct to MOL therapy, or even over a longer time frame, the questions would not be vague because people would be familiar with them. The qualitative data, therefore, indicate that the app is both usable and acceptable.n = 10), convenience sample with no inclusion criteria to investigate the usability and acceptability of an app to promote wellbeing in a clinical population [Although there is a paucity of research about health related apps , the respulation . As withThe quantitative data did not demonstrate a significant reduction in psychological distress although the focus of this pilot study was on assessing the usability and acceptability of the app and the function of the quantitative data was to indicate the possibility of adverse events. These pilot study data indicate that adverse events seem unlikely. In fact, given the way in which receiving the questions was experienced by the participants, we can have some confidence when developing the app that the questions are unlikely to excessively irritate or distress the app users. To investigate reductions in psychological distress it would seem that the lessons from this pilot study are that a clinical sample would need to be used in a research program over a longer period of time and perhaps receiving more than three questions per day.It was encouraging that even over the period of 7 days, receiving just three questions per day, the participants reported increases in awareness and self-reflection. If the app was developed to allow users to schedule more questions per day and to receive the questions over longer periods of time, then greater benefits may be observed. Using larger samples with varying levels of wellbeing and distress will provide more information on the benefits of the app for different populations.Even though small sample sizes are appropriate for pilot studies, the sample size does limit the conclusions that can be drawn. Moreover, the power analysis indicated that the pilot study was underpowered to detect the effect of the app. Also, the impact of the questioning may have been restricted given that only three questions per day were sent to participants’ phones, and this occurred only over a 7-day period. Sending three text messages per day to 23 people can be an onerous task when done manually, however, when the app is developed, the app users will be able to specify the number of questions to receive each day providing the flexibility that allows different users to suit their individual needs.Another limitation may be that only approximately half of the sample participated agreed to be interviewed. This may have introduced a selection bias whereby it was those participants who experienced the questions positively who agreed to be interviewed. People who did not participate in the interviews most commonly gave the explanation of being too busy and did not indicate that they had experienced the questions negatively, however, this remains an empirical question that could be clarified in future research.The results of the pilot study have justified development of the app. Because of the focus on exploring the contents of one’s mind, the app will be called MindSurf. Once MindSurf has been developed it will be possible to conduct further studies with large sample sizes over longer periods of time and with both non-clinical and clinical populations. As indicated in Table From the qualitative analysis in this study it was evident that some participants found one or more of the questions difficult to understand. In future research it will be possible to explore this lack of understanding in greater detail and to revise and improve the questions where necessary. It could be that if people use the app over a longer period of time with a greater range of questions that the questions in general will become more comprehensible. Participants themselves may also be able to offer questions that could be included in the question bank.The post hoc power analysis that was conducted has provided useful information regarding the sample size required in future research (n = 66). It will also be possible in future research to investigate the impact of question frequency on effect size. It may be that, with an increase in questions per day from 3 to, perhaps 10, that a larger effect size would be obtained.Given the number of mobile phone apps available now that address different aspects of mental health, it would also be possible to use research designs that incorporated comparison groups. As well as comparing MindSurf to other apps, it will also be of interest to ascertain whether MindSurf enhances the effects of therapy by comparing a therapy as usual group with a therapy plus MindSurf group. Despite the availability of wellbeing apps, there is a startling lack of evidence for most of them  and the The results of this pilot study indicate that MindSurf will be an acceptable and usable app that does not appear to generate adverse events. Further research is warranted to investigate the use of the app as a way of promoting wellbeing in the general public and also helping to reduce psychological distress in clinical populations. The unique feature of generating questions randomly throughout the day to enable the examination of a person’s cognitive processes “in the moment” may prove to be an important factor in experiencing sustained and robust contentment and satisfaction and lasting reductions in psychological distress. The systematic development and evaluation of MindSurf is providing an evidence-based addition to the large number of health and wellbeing apps already available and helping to address the urgent priority of providing timely access to effective mental health support.


We study the mode and dispersion properties of graphene-dielectric nonlinear plasmonic waveguide considering the dual nonlinearity of dielectric and graphene. For TM polarization, the mode distribution, the permittivity distribution, and dispersion relation were obtained by numerically solving the Maxwell equations. Compared with the case considering only the nonlinearity of dielectric, the initial field intensity to excite plasmon modes reduces obviously when introducing the dual nonlinearity. In addition, the influence of dual nonlinearity on dispersion relation is discussed, and we find that the graphene’s nonlinearity affects strongly the dispersion properties. The introduction of dual nonlinearity leads to the decrease of the initial field intensity, which has potential application in all-optical switches with low threshold. The graphene plasmonics have attracted widespread attention –4 due toConsiderable efforts have been devoted to investigating the optical properties of graphene-dielectric nonlinear composite structures –12 for tBased on the graphene’s strong nonlinearity, several nonlinear optical effects have been predicted –17. Nestσg is placed at x = ± d/2, where the dielectric is a kerr-type medium with a permittivity ε = εL + α|E|2. In our analysis, the graphene is treated as a boundary due to its thickness in one atom scale. Considering a transverse-magnetic(TM) SPPs that propagate along z direction with a propagation constant β and exponentially decays along the x direction into the air and nonlinear medium, respectively.The graphene-nonlinear dielectric plasmonic waveguide is schematically illustrated in Fig. Ex, Ez, and Hy. The magnetic field H = Hyy and electric field E = Exx + Ezz satisfy the equationsFor the TM polarization, we know that there are three field components ε0 and μ0 are the electric permittivity and magnetic permeability of vacuum. From Eq.  at low frequency (ℏω ≤ μc). For the strong field condition, the nonlinear part of the conductivity must be considered and the total conductivity of graphene reads [where ne reads 12\documeEτ is the tangential component of the electric field and σNL denotes nonlinear conductivity [where uctivity 13\documeνF = 0.95 × 108cm/s is the Fermi velocity.where λ = 10 μm. Other parameters are fixed to the values ε1 = 1, εL = 2.25, α = 5 × 10− 16(m/v)2 [EF = 0.27 ev, τ = 1.5 ps. It is well known that there are two modes in graphene-dielectric-graphene linear structures, which are symmetric and antisymmetric modes, respectively. In the following, we will discuss the influence of nonlinearity on modes distribution in the graphene- dielectric composite structures.For the graphene, only in THz and far-infrared frequency range can its surface conductivity be simplified to a simple Drude type; therefore, we choose the incident wavelength as 16(m/v)2  EF = 0.H0 as the initial magnetic field component at incident interface, by solving Eqs. and different chemical potential and nonlinear coefficients of dielectric. In Fig. α = 0, σNL = 0), the nonlinear structure degenerate into a linear structure. In Fig. Next, we turn our attention to discuss the influence of nonlinearity of dielectric and graphene on dispersion relation. Figure α = 5 × 10− 16(m/V)2. The results are shown in Fig. μc = 0.27 eV to μc = 0.16 eV, as shown in Fig. μc = 0.10eV), as shown in Fig. H0 and chemical potential the dispersion relation only shows a symmetric mode with a closed loop.In the following, we introduce simultaneously the nonlinearity of dielectric and graphene, and discuss the influence of nonlinearity of graphene on dispersion relation with a fixed nonlinear coefficient of dielectric In summary, we have investigated the mode and dispersion properties of graphene-dielectric nonlinear plasmonic waveguide. The mode distribution, permittivity, and dispersion relations were obtained by numerically solving Maxwell equation for TM polarization. Compared with the case considering only the dielectric’s nonlinearity, the initial field intensity reduced apparently when considering the nonlinearity of dielectric and graphene simultaneously. In addition, the dual nonlinearity affects the dispersion properties of the waveguide significantly. Especially, as the graphene’s nonlinearity increases, the antisymmetric and asymmetric modes merge into one and gradually disappear. Therefore, only the symmetric mode can be found in the case of strong nonlinearity.


Positive tumor PD-L1 expression in KTs from GCs was associated with poor prognosis. In contrast, positive tumor PD-L1 expression in KTs from CRCs was associated with an improved prognosis. We analyzed copy number variations of the PD-L1 gene in KTs. PD-L1 expression was higher in cases with copy number gains. The T cell densities within KTs and their corresponding primary tumors were compared. The densities of CD8+ T cells correlated significantly between the primary tumors and KTs from the same case. Taken together, the research further highlighted targets for immune-based therapy in KTs from GCs and CRCs.Krukenberg tumor (KT) is an uncommon ovarian metastatic signet-ring cell adenocarcinoma that mostly metastasizes from gastrointestinal carcinoma. Optimal treatment options for KTs are limited. Programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors have shown remarkable activity in clinical trials for metastatic tumors. Here, we evaluated PD-L1 expression and T cell infiltration in KTs and their corresponding primary tumors. Positive tumor PD-L1 expression was detected in 9 (25.7%) KTs from gastric carcinomas (GCs) and in 20 (66.7%) KTs from colorectal carcinomas (CRCs). Patient survival was assessed according to the PD-L1 status and CD8 Ovarian metastatic tumors that contain a component of signet-ring cells are known as Krukenberg tumors (KTs) and originate mainly from the stomach (76%), intestines 11%), breast 4%), and other organs 1%, breas–4. Most %, and otImmunotherapy has emerged as a promising measure for cancer treatment . AccumulPD-L1 gene in KTs from GCs were analyzed. The immune microenvironment of KTs was also assessed and compared with that of the primary tumor from the same case.We investigated PD-L1 expression in KTs from GCs and CRCs, examined the correlation between PD-L1 expression and T cell infiltration, and evaluated the impact of PD-L1 expression on prognosis. Copy number variations in the We reviewed a retrospective cohort study of 65 cases. A tissue microarray was constructed for paraffin sample tissues and included 35 KTs with 23 matched primary GCs and 30 KTs with 28 matched primary CRCs collected at Fudan University Affiliated Obstetrics and Gynecology Hospital and cooperative hospitals between 2000 and 2015. The overall survival (OS) time was defined as the interval between the ovarian metastasectomy operation and death or survival. Exclusion criteria included (a) the absence of surgery of KT and (b) the validation of an ovarian nonadenocarcinoma metastasis. Samples and medical records were approved by the research ethics committee of Fudan University Affiliated Obstetrics and Gynecology Hospital and cooperative hospitals.The primary antibodies used were as follows: anti-PD-L1 for immunohistochemistry , anti-PD-L1 for multiplex IHC , anti-PD-1 , anti-FOXP3 , anti-CD3 , anti-CD8 , and anti-CD8 for multiplex IHC . Positive staining was visualized with DAB substrate liquid , and counterstaining was performed with hematoxylin. Scoring was performed by two senior pathologists.+, FOXP3+, CD3+, and CD8+ cell densities (cells/mm2) were quantified using digital image analysis. The CD8+ T cell densities were dichotomized into “high” and “low” groups according to the median.Tumor PD-L1 expression was determined by the presence of membrane staining in tumor cells as previously reported . StromalThe tissue sections were deparaffinized, rehydrated, and incubated with 0.3% hydrogen peroxide, and the antigen was unmasked in 10 mM sodium citrate buffer using a microwave. After the sections were incubated with the primary antibody for 45 min at RT, slides were incubated with anti-rabbit secondary antibody  or anti-mouse secondary antibody  for 30 min at RT. Tyramide (TSA) plus fluorescein  or TSA plus Cyanine 5  was added to the slides at a 1 : 50 dilution, and the slides were incubated for 10 min at RT. A microwave was used to remove the combined antibody. TSA plus fluorescein or TSA plus Cyanine 5 was added to slides at a 1 : 50 dilution. Cell nuclei were counterstained with DAPI. The slides were imaged using Vectra imaging software.PD-L1 copy number in 13 paraffin samples was analyzed using the OncoScan formalin-fixed and paraffin-embedded (FFPE) Assay Kit . Total FFPE DNA was extracted using the QIAamp DNA Mini Kit . Results were analyzed using ChAS  software. Raw data was submitted to ArrayExpress, and the data accession number is E-MTAB-6277.The P values < 0.05 were considered to be statistically significant.Statistical analyses were conducted using Statistical Product and Service Solutions 16.0 software . Chi-square tests in cross tables were performed to examine the correlation between the PD-L1 expression levels and clinical parameters or lymphocyte variables. Correlations of T cell infiltration between the primary tumor and metastases were calculated with the Spearman correlation test. OS was estimated using the Kaplan-Meier method, and differences were evaluated using the log-rank test. Graphic representations were analyzed with GraphPad Prism software . A retrospective cohort study of 65 patients, including 35 KTs from GCs and CRCs, was conducted. The clinicopathological characteristics of the patients are detailed . In the CRC patients, an inverse relationship between the tumor PD-L1 expression and the extent of signet-ring cells was observed (P = 0.009). No significant relationship was evident between the stromal PD-L1 expression and any clinicopathological feature  and menopause status (HR = 2.860 (95% CI: 1.057–7.736), P = 0.038) were analyzed to be risk factors for OS.Characteristics including PD-L1 expression in ovarian metastases were analyzed using COX proportion hazard regression models to assess the prognostic values . In GC pP = 0.007) and high ratio of signet cells (HR = 4.655 (95% CI: 1.366–15.863), P = 0.014) were identified as potential prognostic factors ,  factors . The negPD-L1 gene copy number gains in 2 cases (15.4%), PD-L1 gene copy number losses in 2 cases (15.4%), and a normal copy number status in 9 cases (69.2%). The PD-L1 expression was higher in cases with copy number gains than in normal cases (P = 0.0209) or cases with copy number losses (P = 0.0101)  .+, CD8+, FOXP3+, or PD-1+ T cell infiltration . The densities of CD8+, CD3+, and FOXP3+ cells were significantly higher in the primary GCs than in the KTs . Similarly, the densities of CD8+, CD3+, and FOXP3+ cells were significantly higher in the primary CRCs than in the KTs  expression is upregulated in multiple human cancers and attenuates the antitumor immune response –24. Two e system , 23, 25.e system .Genome abnormalities might affect the tumor PD-L1 expression . A previPD-L1 gene amplification was identified in only 2 of 13 samples, and this evidence was insufficient to explain the overexpression of PD-L1. Previous studies have reported a significant relationship between tumor PD-L1 expression and the CD8+ cell density [+ regulatory T cell inhibitory pathways might serve as a negative feedback mechanism that follows CD8+ T cell infiltration [+ cell density [+, CD8+, or FOXP3+ T cell density, which may be due to differences in the study populations.Although DNA copy number gains play important roles in gene overexpression,  density , 28. IDO density . However+ cells. Consistently, previous studies have demonstrated that the immune contexture in the primary tumor results in “educated” immune cells that are recalled at the metastatic sites [We further evaluated the immune contexture of the primary tumors and KTs. From the primary tumors to the metastases, the GCs and CRCs had significant correlation patterns between CD8ic sites . Tumor cic sites –32. Our Our study has some limitations. First, most of the KTs were over 5 cm in size and most were associated with no normal tissues in the margins. Thus, we were not able to assess the tumor and stromal PD-L1 expressions and the immune cell infiltration within the tumor-invasive margin. Second, the study included a small sample size because (i) Krukenberg tumor is uncommon, only accounting for 1% to 2% of all ovarian tumors [In conclusion, positive tumor PD-L1 expression implies that there might be implications for targeting the PD-L1/PD-1 axis in KTs. The prognostic value and immune pattern in the metastases might be useful for guiding treatment.+ cells that were correlated between the primary tumors and KTs from the same case, indicating that the primary tumor exerted an influence on the immune environment in the metastases. It further highlighted targets for immune-based therapy in KTs from GCs and CRCs.Our results characterized the role of PD-L1 and the immune microenvironment in KTs. Tumor PD-L1 expression predicted diverse prognoses in KTs with different corresponding origins. In KTs from GCs, positive tumor PD-L1 expression was associated with poor prognosis. In contrast, in KTs from CRCs, the positive tumor PD-L1 expression was associated with an improved prognosis compared to the negative PD-L1 expression. The GCs and CRCs had patterns of CD8


We further demonstrate near-100% position-controlled particle trapping at voltages as low as 0.45 V with nanodiamonds, nanobeads, and DNA from bulk solution within seconds. This trapping scheme can be seamlessly integrated with sensors utilizing graphene as well as other two-dimensional materials.The many unique properties of graphene, such as the tunable optical, electrical, and plasmonic response make it ideally suited for applications such as biosensing. As with other surface-based biosensors, however, the performance is limited by the diffusive transport of target molecules to the surface. Here we show that atomically sharp edges of monolayer graphene can generate singular electrical field gradients for trapping biomolecules via dielectrophoresis. Graphene-edge dielectrophoresis pushes the physical limit of gradient-force-based trapping by creating atomically sharp tweezers. We have fabricated locally backgated devices with an 8-nm-thick HfO The capability of positioning target molecules onto the edges of patterned graphene nanostructures is highly desirable. Here, the authors demonstrate that the atomically sharp edges of graphene can be used as dielectrophoretic tweezers for gradient-force-based trapping applications. Unlike noble metals, such as gold or silver, the carrier concentration in graphene can be tuned, hence enabling the possibility of electrically reconfigurable biosensing11. Nanopatterned graphene such as ribbons11, nanochannels12, or nanopores have all been shown to offer benefits for use in sensing applications. However, biomolecules in viscous media is generally governed by diffusive transport, and the placement of target molecules at the region of highest sensitivity is a key prerequisite to biosensing. Currently, graphene-based biosensors mostly employ DNA or protein immobilization across the entire graphene surface or rely on the random adsorption of biomolecules to the most sensitive regions‒the edges or pores in graphene13. The ability to precisely position and concentrate target molecules onto the edge of patterned graphene nanostructures is highly desirable yet is not extensively studied. Besides biosensing, such capability can also benefit nanophotonic applications for integrating quantum emitters and plasmonic antennas with tunable optoelectronic properties of graphene15.Graphene18, optoelectronic tweezers20, flow-through nanopores22, electrokinetics23, dielectrophoresis (DEP)24, or photothermal methods25 can be used for the aforementioned purposes. However, it is not trivial to integrate such schemes on patterned graphene chips. We show that the atomic-scale thickness of graphene enables ultra-strong DEP forces for trapping nanoscale objects and molecules along patterned edges of graphene. DEP is a widely used method to manipulate biomolecules or polarizable nanoscale objects by using gradient electrical forces obtained from sharp conducting tips, edges, or small gaps between electrodes24. The time-averaged DEP force on a particle of radius R and in a medium of permittivity 2426. However, in this case as our model system is based on nanoscale particles, we assumed a dipole approximation.Various on-chip manipulation techniques, such as optical trappingFT, given byTR is the room temperature and The DEP force depends on the particle volume that goes down with the particle size. However, the thermal Brownian motion becomes very important as we reduce the particle size to sub-micron length scale. To trap a particle, it is required to overcome the thermal force, E|2, it is a scalable particle trapping method, i.e., reducing the radius of curvature of electrode features  or electrode-to-electrode separation28 can significantly boost its magnitude. Patterned metallic nanoelectrodes29 or bottom-up structures such as isolated carbon nanotubes (CNTs)31 have been used to shrink the critical dimension of DEP electrodes into single-digit nanometer scale. Extrapolating this idea further toward the ultimate limit, we conceive a design to turn Angstrom-scale-thickness monolayer graphene into an electrode that provides the sharpest possible edge over millimeter length scales, which cannot be created using lithographically patterned metal nanostructures or even CNTs. Although single-walled CNT electrodes can provide sharp nanometer-scale edges, these devices often suffer from inability to control the gap between CNT electrode and the gate, as well as difficulty to form high-density regular arrays of CNTs. Previously, Xie et al.32 used an atomic force microscopy probe to cut graphene into small-area interdigitated electrodes and showed DEP trapping of CNTs over these structures.Since the DEP force is proportional to the field gradient term ∇|33.In this work, we demonstrate a novel scheme to turn exposed edges of graphene into strong DEP trapping sites. This was done by constructing a robust large-scale graphene-edge DEP platform and trapping nanoparticles and biomolecules at the edge of this monolayer graphene atop an 8-nm-thick insulator with low bias voltages. Such capability can offer graphene-based sensors a unique solution to diffusion-limited mass transport problems2)). A photograph of the chip is shown in Fig. 2 on it. Next, the entire wafer surface, including the Pd electrode, was coated with 8-nm-thick HfO2 deposited by atomic layer deposition (ALD). Then, single-layer graphene grown by chemical vapor deposition (CVD) was transferred onto the wafer and etched into rectangular patterns. In addition to the sharpness of the graphene edge, the nanoscale gap between graphene and the Pd gate electrode (defined by the thickness of the HfO2 layer) further enhances the electric field. Finally, photolithography was employed to pattern Cr/Au contact electrodes, which formed Ohmic contacts to the graphene. The gate and the contact electrodes were arranged in an interdigitated fashion to minimize series resistance and to produce more exposed edges efficiently trap the nanoparticles and molecules. Details of the fabrication process are elaborated in Methods section.Being a gradient-field dependent phenomenon, DEP is greatly enhanced by reducing the radius of curvature of the electrode, with graphene edge providing the sharpest tip Fig. . Reducin2. This density can be further enhanced by reducing the pitch of the gate fingers as well as the graphene segments to 1 µm, which is realistic using i-line optical lithography. In such a case, the trapping site density could be increased to 4 sites per 1 μm2, a nearly 1000× improvement over the current devices. Furthermore, using advanced optical lithography or electron-beam lithography to form the gate fingers and segment the graphene, the trapping density could be further increased by another 1–2 orders of magnitude.The key feature of our processing scheme is the wafer-scale throughput and scalability of trap arrays. In the layout we employed, the gate finger spacing is 15 μm with two graphene segments on either side, and these segments have a pitch of 60 μm. The current design provides a density of four trapping sites per 900 µmE|2), which is responsible for DEP trapping, for a semi-infinite graphene “electrode”, electrically biased by a metal gate electrode, as shown in Fig. 2 layer with a dielectric constant of 13 is assumed between the electrodes. As the experiments were all performed in water, the rest of the simulation domain is assumed to be water with a dielectric constant of 80. The bottom electrode is treated as an ideal metal and graphene is modeled as a layer with zero thickness. An illustration of the simulation domain is shown in Fig. We calculated the gradient of the electric field intensity  factor  for 190 nm polystyrene beads. This is the voltage at which the DEP force is enough to overcome the Brownian motion of the nanoparticles due to their thermal energy. The amplitude of the applied voltage was increased from 400 to 700 mV at a step interval of 50 mV. At each voltage, fluorescence images were collected for 50 s. More beads were trapped at the trapping sites as the voltage amplitude was increased  particles with nitrogen-vacancy (NV) centers  at the trapping sites on graphene  collected after turning on the bias (amplitude 2 V and frequency 100 kHz) show ND particles localized at the trapping sites on a two-dimensional grid defined by orthogonal edges of graphene and buried metal lines.Besides potential applications in biosensing, graphene-based DEP can be utilized for on-chip assembly and array formation of nanoscale quantum emitters such as nanodiamond particles, semiconductor nanocrystals, or plasmonic metal nanoparticles. Rapid on-chip integration of these elements with patterned graphene can be highly desirable for applications in nanosensingene Fig. . The ND Cions) present in the solution should be much greater than the total number of charged sites present across all DNA molecules, as expressed byCDNA is the concentration of DNA and Cions/CDNA (=106) is much higher than 37. The polarizability values were estimated from previously reported empirical findings that correspond to ~3.48 × 10−31 Fm2 and ~1.00 × 10−31 Fm2 for the 10 kbp and 500 bp DNA, respectively37. Next, we calculated the threshold Rg) of 10 kbp DNA with persistence length 50 nm, calculated using a worm-like chain model39, is 238 nm that corresponds to an FT value of 14.4 fN , the threshold 18V2m-3.To demonstrate the utility of the graphene electrodes in capturing biomolecules, we used 10 kbp and 500 bp DNA molecules tagged with YOYO-1 dye  of the system, which is given bySo far, we showed DEP manipulation using high frequency AC fields (1−10 MHz). Reducing the frequency to 100 kHz, results in an interesting localization of the DNA molecules near the graphene edge but the molecules are not tightly trapped at the edge Fig. . This phFor a solution of conductivity 12 μS/cm , the charge relaxation frequency is 270 kHz . As we used low voltages and low-conductivity solutions in our system, the expected temperature rise is minimal. Even for the case where we expect highest Stability of biomolecules is dependent on the temperature of the surrounding environment. We investigated the expected temperature rise in our system based on Joule heating from the relation 41 or tapered nanotips8 wherein field hotspots are located along the edges, enabling tunable mid-IR spectroscopy of ultralow-concentration molecules using graphene plasmons. Our wafer-scale chip platform can also be used for nano-positioning quantum emitters to build nanophotonic circuits or single-photon source arrays42. While we used straight edges of graphene in this work, DEP can also be performed with conductive nanopores44, presenting new opportunities for single DNA translocation and analysis48. Beyond graphene, atomically sharp edges of other 2D materials49 can also be utilized for ultra-strong DEP, providing a practical route to building tunable biosensors.Monolayer graphene can generate ultra-strong dielectrophoresis forces and outperform conventional metal electrodes that are orders of magnitude thicker. We have presented the concept of graphene-edge DEP and constructed a practical device platform to realize it. In our scheme, the critical dimension of graphene electrodes for DEP is controlled by its natural thickness and the insulator deposited by ALD, thus our approach is scalable and highly reproducible at the wafer scale. Using arrays of electrodes, we can selectively trap and position nanoparticles and DNA molecules precisely along the atomically sharp edges of graphene. This could facilitate performing of biological assays at low concentrations as well as exploring molecular interactions and conformational changes in confined space. We also demonstrated trapping of DNA molecules from 10 pM solution and on-chip assembly and precise positioning of nanodiamonds. Notably all of these rapid DEP manipulation steps were performed using monolayer graphene around or below 1 V, which makes the low-power graphene-edge DEP attractive for many applications. For example, edge trapping of biomolecules can be readily integrated with graphene nanoresonators2 (300 nm) thermally grown on Si substrate. The gate electrode patterns were exposed and developed using photolithography, followed by a combination of reactive ion etching and wet etching on the SiO2 layer. A metallization layer of Ti/Pd (10/40 nm) was then deposited by electron-beam evaporation and lifted off. Atomic layer deposition of HfO2 at 300 °C was applied to form a dielectric layer (8 nm), which was annealed in nitrogen at 400 °C for 5 minutes. A via layer was patterned using photolithography, and then the HfO2 in localized regions above the Pd metal was reactive-ion etched in SF6 for 30–60 s. The via window through the dielectric layer is intended to provide the ability to subsequently contact the gate electrode with additional contact metallization. Single-layer graphene grown by chemical vapor deposition (CVD) was transferred onto the dielectric layer using a wet transfer process, patterned by photolithography, and then etched using an O2 plasma. Ohmic contacts were patterned next using photolithography, and Cr/Au (10/80 nm) was deposited using electron-beam evaporation and lifted off. Finally, for the convenience of the device measurement, a thick metal layer of Cr/Al (10/300 nm) was patterned and deposited to form low-resistance electrical leads from the gate and Ohmic contacts to the contact pads for electrical probing.Our graphene-edge DEP chip was fabricated starting from a thick layer of SiOA solution volume of ~10 µl was placed on top of graphene electrodes, confined within a reservoir made in adhesive tape. A cover slip was placed to avoid any unwanted evaporation. A probe station was used to apply an AC bias across the contact pads by a function generator (HP 33120 A). The peak amplitude of the voltage used was in the range of 1−3 V. Depending on the particle, the frequency of the AC bias was optimized for polystyrene, DNA molecules, and nanodiamonds. For the polystyrene and ND particles, water was used as a surrounding medium (conductivity ~4 µS/cm), whereas 10 µM KCl solution (conductivity ~12 µS/cm) was used for DNA.Fluorescent nanoparticles used in this work are 190 nm diameter carboxylate-modified polystyrene beads (The data that supports the finding of this study are available from the corresponding author upon request.Peer Review FileAdditional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


Our phenomenological model for contact noise because of current crowding in purely two-dimensional conductors confirms that the contacts dominate the measured resistance noise in all graphene field-effect transistors in the two-probe or invasive four-probe configurations, and surprisingly, also in nearly noninvasive four-probe  configuration in the high-mobility devices. The microscopic origin of contact noise is directly linked to the fluctuating electrostatic environment of the metal–channel interface, which could be generic to two-dimensional material-based electronic devices.The impact of the intrinsic time-dependent fluctuations in the electrical resistance at the graphene–metal interface or the contact noise, on the performance of graphene field-effect transistors, can be as adverse as the contact resistance itself, but remains largely unexplored. Here we have investigated the contact noise in graphene field-effect transistors of varying device geometry and contact configuration, with carrier mobility ranging from 5,000 to 80,000 cm The performance of graphene field effect transistors is adversely affected by fluctuations in the electrical resistance at the graphene/metal interface. Here, the authors unveil the microscopic origin of such contact noise, highlighting the role of current crowding. Over the past few years, there has been tremendous progress in creating high-mobility, atomically thin FETs through a combination of low-resistance ohmic contacts13f noise, which is known to degrade the performance of amplifiers, or introduce phase noise/jitter in high-frequency oscillators and converters6The wide spectrum of layered two-dimensional 2D) materials provides the opportunity to create ultimately thin devices with functionalities that cannot be achieved with standard semiconductors. The simplest of such devices is the field-effect transistor (FET). There are several factors that determine the performance of an FET, the key among them being the dielectric environment, quality of the metal–semiconductor contact and the level of low-frequency 1/D materiaf noise. Conflicting claims exist, where some studies attribute the 1/f noise in graphene transistors primarily to noise generated within the channel region78101178910111213141516171819202223242526f noise.Even for the widely studied graphene FET, it is still unclear what the dominant contribution is to the 1/LT22293032LT∼0.1–1 μm  and black phosphorus FETs26In this work we study a series of graphene FETs with different mobilities, substrates and contacting configurations to demonstrate that electrical noise at the metal–graphene junction can be the dominant source of 1/2/p++–Si substrate, etched into a Hall bar shape with surface-contacted Au (99.999%) leads (45VBG). We measure the two-probe  and four-probe  resistance and noise as a function of VBG between the leads 2 and 3  and current  leads). The VBG dependence of R2P and R4P is shown in R4P shows a slightly asymmetric transfer characteristic, known to occur for asymmetric contact dopingVBG≈6 V. R2P, however, shows a second Dirac point at VBG≈31 V because of the combination of hole doping and weak pinning by Au at the contact region3438n−p and R−VBG characteristics confirms the structural integrity and gate tunability of the Fermi level of the graphene channel underneath the contact.We first focus on a single-layer graphene channel on conventional (300 nm) SiO%) leads . Here we%) leads VBG. We mRc, we follow the Landauer approach where the net transmission probability T across the contact is determined by the interplay of the number of propagating modes in the channel and metal regions  and that calculated assuming the Dirac-like dispersion and level broadening ≈80 meV underneath the contact and ≈57 meV in the channel , indicates that the contact resistance is primarily composed of the resistance RT of the graphene layer over the charge transfer length (LT) underneath the contacts. Owing to mismatch between the resistivities of the metal and graphene, LT is significantly smaller than the geometric width Lc (∼1–1.5 μm) of the metal lead, resulting in the current-crowding effect.The shift in the local chemical potential by metal contacts results in a change in resistance that contributes to contact resistance, and determines the extent of current crowding at the gold–graphene interface. To compute the contact resistance  regions 38. FigurρT . Taking RT as the experimentally observed contact resistance Rc  for the second Dirac point at +31 V where the mismatch between the resistivity of the metal and that of the graphene layer underneath is maximum.where μm2 ref.  is the stance Rc , we calcρT=RTW/LT, it is evident that the contact noise is essentially the resistance fluctuations in the graphene layer underneath the contact, that is, nT are the phenomenological Hooge parameter and carrier density in the charge transfer region, respectively. nT and is determined by the kinetics of local disorder induced by trapped charges, chemical modifications and changes in the band dispersion due to hybridization. Assuming a diffusive transport in the charge transfer region with density-independent mobilitySince  refs , 24 and Rc is ≈1 for interface-type contacts or ≈3 for constriction-type contactsnT is the only gate-tunable parameter3438and implies a scaling relation R2P and R4P at all VBG consists of random time-dependent fluctuations with power spectral density f noise due to many independent fluctuators with wide distribution of characteristic switching rates. However, to estimate and compare the total noise magnitude, we have evaluated the ‘variance' SR(f) numerically over the experimental bandwidth.Noise in both density  , where  VBG dependence of R2P and Rc  is ∼10 times larger than that at the main Dirac peak, indicating the significantly larger noise where the current crowding is most severe and contact resistance is the largest. Surprisingly, P and Rc . The oriRc in VBG collapses on a single trace, and varies as n in 12 cm−2), that is, p–p′ regime where Rc reduces to ≲1 kΩ, we observed −3  for both R2P and R4P  and Rg are the metal–graphene interface resistance and graphene channel resistance, respectively. Owing to the inseparability of RT(=Rc) and Rg within this contacting scheme, we plot R4P in R4P≲150–200 Ω, where Rg is small because of heavy electrostatic doping of the channel. However, for R4P≳200 Ω, the deviation from the Rg that causes R4P to overestimate the true Rc. We have observed an R4 scaling of noise for high-mobility suspended graphene devices as well (see The effect of contact noise becomes more severe for invasive surface contacts (leads extending to the current flow path), as demonstrated with a device that has graphene on BN . The tra V−1 s−1  inset. Se of VBG , suggestwell see .I–V characteristics (not shown) and temperature independence of Rc  than the corresponding mobility  of the part on BN, as well as strong substrate-induced doping, both of which can be readily understood by the proximity to charge traps at the SiO2 surface. Although 2 substrate is up to a factor of 10 larger than that on BN, similar to that reported recently192 surface).To verify this, we have fabricated an invasively Cr/Au-contacted device where a single graphene channel was placed partially on BN (thickness ∼10 nm), thus physically separating the channel from the oxide traps2 . The fou2  confirm c points , it is ecently19  over thrRcW, from different classes of devices that were studied in this work. We identify two key factors that have an impact on the contact noise: first, as can be clearly seen in 2, lower on devices with graphene on BN and lowest for suspended graphene devices where all SiO2 has been etched away from under the graphene channel as well as partially from below the contact region  tape. The heterostructures were assembled using a method similar to that described in ref. 3 and O2 plasma, to be 23±2 nm per 60 s .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-5, Supplementary Notes 1-3 and Supplementary References.