The interaction leads to oxygen dissociation and the formation of carbon-oxygen single bonds on graphene, along with a band gap opening and a rounding of the Dirac cone. The change of the N 1 s core level signal indicates that graphitic nitrogen is involved in the observed mechanism: the adsorbed oxygen molecule is dissociated and the two O atoms chemisorb with epoxy bonds to the nearest carbon neighbours of the graphitic nitrogen. Our findings help resolve existing controversies and offer compelling new evidence of the ORR pathway.Carbon nanomaterials’ reactivity towards oxygen is very poor, limiting their potential applications. However, nitrogen doping is an established way to introduce active sites that facilitate interaction with gases. This boosts the materials’ reactivity for bio-/gas sensing and enhances their catalytic performance for the oxygen reduction reaction. Despite this interest, the role of differently bonded nitrogen dopants in the interaction with oxygen is obscured by experimental challenges and has so far resisted clear conclusions. We study the interaction of molecular oxygen with graphene doped via nitrogen plasma by  While pristine graphene and carbon nanotubes (CNTs) are chemically inert, the active sites induced by functionalization with nitrogen lower the energy barrier for O2 dissociation7, the first step of the ORR. This makes doping appealing also for applications other than heterogeneous catalysis, such as chemical or bio-sensing where the possibility to detect ppm levels of gases such as NO2, CO, H2 has been demonstrated, thus possibly avoiding the use of expensive metals9. The activationz barrier for oxygen dissociation on pristine graphene is higher than on CNTs 5 since the CNT curvature has a strong effect in lowering the required energy6.Due to their surprisingly high electrocatalytic activity, nitrogen-doped carbon nanomaterials have been proposed as low-cost candidates to substitute platinum catalysts for the oxygen reduction reaction (ORR)graphitic, a substitution with a vacancy as a neighbour is called pyridinic (both with sp² hybridization), or the N can be included in a pentagonal ring (either with sp³ or sp² hybridization) or in a local lattice distortion 10 to form a pyrrolic configuration. Theoretical models show different energy barriers for oxygen molecule adsorption and dissociation depending on the nitrogen configuration5. Since their five valence electrons are distributed differently, graphitic and pyridinic nitrogen atoms have different effects on the electronic properties of graphene: the graphitic nitrogen contributes with three σ bonds and two pz orbitals, with the extra electron available for conduction in a partially-occupied π* band; on the contrary, the pyridinic nitrogen only forms two σ bonds with the neighbouring carbon atoms, one electron occupies the pz orbital, and the other two form an electron lone pair. Since there is no occupation of the π* band in the pyridinic nitrogen, it does not behave as an electron dopant like graphitic N does (nor is it thought to be responsible for good ORR activity7). Furthermore, due to the proximity of the carbon vacancy, the pyridinic-vacancy complex has a hole doping effect. These predictions were recently experimentally confirmed by following the Dirac cone shift upon modification of the graphitic/pyridinic ratio by thermal treatments of graphene11 and by the intercalation of different metal atoms12.A nitrogen atom can be incorporated into the hexagonal carbon network in three main configurations: N simply substituting a single C atom is called 16. Three main issues make direct experimental observation very challenging: (i) low doping level, (ii) poor control over the bonding of the nitrogen dopants and (iii) the indirect nature of experimental studies reported so far, which often involve steps where the contamination of the sample cannot be controlled. To overcome limitations caused by commonly used N-doped graphene synthesis methods , or via wet chemistry), we used a post-synthesis plasma treatment17. This method is clean, it does not add undesired oxygen contamination, and it does not destroy the sp² character of the graphene network even while reaching a high concentration of N (on the order of 10 at.%)18. Further temperature treatments allow both the passivation of carbon defects and the conversion of pyridinic-N into graphitic-N, resulting in a larger density of states at the Fermi level with increasing graphitic-N level19. With our work, we build further steps to link the field of catalysis with fundamental surface science, making substantial progress in the comprehension of the role of differently bonded nitrogen dopants in the interaction with molecular oxygen.From an experimental point of view, the role of these active sites in the ORR is still not well understood and the mechanism for the enhanced catalytic activity is controversial2. The graphene synthesis and measurements were performed in situ in an ultra-high-vacuum (UHV) environment and the interaction with molecular oxygen was followed by the direct measurement of the core levels, valence band and unoccupied states of the system with synchrotron-based photoemission  and absorption (NEXAFS: near-edge absorption fine structure) techniques. The electronic structure of N-graphene was probed in a contamination-free environment before and after UHV exposure to oxygen. The experimental measurements are interpreted in the light of core level binding energy shifts calculated via density functional theory (DFT), leading to a detailed atomistic picture of the oxygen dissociation process.Using advanced synchrotron techniques, we investigated the response of nitrogen functionalities to the interaction with O2 gas. As evaluated by the survey XPS spectra, nitrogen concentrations of 7.3 and 7.8 at.% are reached in the two samples. The thermal stability and rearrangement of the nitrogen species were studied by temperature-programmed XPS: the two identically prepared samples were annealed with a controlled temperature ramp of 0.4 °C/s respectively up to 300 and 600 °C, while following the N 1 s core level signal evolution. Figure s core level spectra, recorded at room temperature, for the as-doped and after each annealing treatment are shown in Fig. 20 and with our previous findings on carbon nanomaterials doped via nitrogen plasma19. At the binding energy corresponding to pyrrolic nitrogen, many other defective components may be found, such as C-N single bonds, three-coordinated N atom in a Stone-Wales (SW) defects or nitrogen adatoms21. A fourth component at a higher binding energy  has been recently also assigned to graphitic nitrogen, with the N atom thought to occupy a substitutional position close to a crystallite edge22. Overlapping at a similar binding energy, nitrogen oxide could also contribute, which we can exclude here since no trace of oxygen was detected after the plasma exposure. A further component at a lower binding energy  is due to nitrogen atoms in the carbon network closer to the metallic substrate18, likely due to the pinning of graphene23. The 300 °C treatment slightly reduces the amount of nitrogen to 5.7 at.%. The main effect of annealing is the rising intensity of the high-binding energy side in the N 1 s spectra, corresponding to the graphitic components that pass from 1.5 to 2.1 at.% in absolute content at the expense of pyridinic and pyrrolic components, which decrease from 6.4 to 3.6 at.%. Judging from the slight loss in N content, these less stable functionalities partially desorb, and partially convert into graphitic-N. This is further evidenced after reaching 600 °C on the second sample, where the greatest amount (56.3%) of nitrogen present is in graphitic form. The overall nitrogen content decreased to 2.5 at.%, but the amount of graphitic is almost the same as for previous annealing, meaning that the loss is mainly due to pyridinic and pyrrolic functionalities, further confirming the high temperature stability of graphitic nitrogen18. The results of the fitting procedure  are summarized in Table We prepared two graphene/Ir111) samples that we exposed for 15 minutes to the downstream of a plasma source fed with N11 sample17. The C 1 s core level of graphene/Ir(111) is shown in Fig. 24, by a sharp asymmetric peak centred at 284.16 eV corresponding to C-C sp², plus a small shoulder at + 0.66 eV that can be attributed to defective carbon . Upon nitrogen plasma exposure, new components appear in the C 1 s spectrum at lower binding energies with respect to the main peak (283.34 eV and 283.80 eV), attributed to photoelectrons emitted by carbon atoms near vacancies25. A slightly higher binding energy component is likely due to second-nearest carbon neighbours of N atoms (284.53 eV), with additional contributions from sp² and sp³ C-N bonds 18.Annealing favours the passivation of defects and dangling bonds on the graphene surface, with the filling of carbon vacancies next to the N sites explaining the transformation of pyridinic into graphitic bondingn-type doping of graphene, deduced by the shift towards higher binding energy of the C-C sp² component in the C 1 s core level: this peak broadens and shifts with respect to the pristine C 1 s position. In particular, a shift of +0.08 eV was evaluated after the plasma exposure and a further shift of +0.22 eV after annealing, in both 300 and 600 °C cases. This behaviour highlights the n-doping action of graphitic nitrogen only, counterbalanced by the complex carbon vacancy-pyridinic nitrogen responsible for p-doping. In fact, although after annealing the total amount of nitrogen decreases, the shift towards higher binding energy of the C-C sp² peak due to n-doping increases, thanks to the change in the graphitic/pyridinic ratio11. As the annealing has a healing effect on the carbon network with the passivation of vacancies, the two pre-sp² peaks are quenched: in particular, the amount of vacancies decreases from 2.3 at.% after the plasma to 1.3 and 0.8 at.% in the two samples respectively annealed at 300 and 600 °C. Such a low amount of residual carbon vacancies testifies the highly crystalline quality of the nitrogen-doped graphene, also in agreement with the observed decrease in pyridinic nitrogen content. The effect of nitrogen implantation in the graphene network is also reflected in the Ir 4f7/2 core level . After exposure, there is an oxygen uptake corresponding to 2.0 at.% for the N-graphene annealed up to 300 °C and to 1.2 at.% for the one annealed up to 600 °C. In both cases, two O 1 s components are found at 530.6 and 532.3 eV, as shown in Fig. 26. In fact, the intensity of the C 1 s does not show any decrease (etching) during the process, indicating the integrity of the graphene flakes, as reported in Supplementary Fig. To study the interaction with molecular oxygen, we exposed both annealed N-graphene/Ir samples s features to a particular bonding, we consider the possibility for molecular oxygen of intercalating between the substrate and the graphene and to dissociate on the metal. Larciprete and co-workers27 found a complete intercalation of pristine graphene/Ir(111) for a substrate temperature of 250 °C and a pressure two order of magnitude higher than ours. Even though for defective graphene, this process can be activated at a lower gas dose27, we did not see any indication of oxygen intercalation. There are three typical XPS fingerprints of an effective penetration of O2 molecules under the graphene layer and their dissociation on the Ir substrate: (1) the appearance of an O 1 s peak at 529.8 eV; (2) the transformation of the C 1 s which broadens due to the formation of a second component at −0.5 eV by the co-existence of partially intercalated areas until it completely shifts to its new position; (3) the appearance of two new components on the Ir 4f7/2 core level spectra at the expense of the surface component due to iridium atoms bonding to one (at 60.57 eV) or two (at 61.08 eV) oxygen atoms28. The same elements were observed in the experiment of Grånäs and co-workers, where a partial graphene layer on Ir(111) was used29. In our case, the C 1 s shows a shift of −0.08 eV with respect to the value measured after the annealing of the N-graphene, moving from 284.38 eV to 284.30 eV. This shift is not compatible with oxygen intercalation, since that mechanism does not implicate a gradual progressive shift in the binding energy of the C 1 s as in the case of doping. Instead, intercalation causes a peak broadening due to the appearance of a second component arising from O-(2 × 1) intercalated regions27. We thus attribute this shift to reduced n-doping by the graphitic nitrogen due to absorbed oxygen atoms, as we will show later by core level simulations. Our C 1 s lineshape only slightly varies, as reported in Supplementary Fig. Before assigning the O 1 f7/2 core level does not show any further peaks indicative of an interaction between the metal and the gas molecules . Epoxy is the most dominant species formed while oxidizing graphene31. Ether is usually found at the edges and defects of graphene, and therefore we would expect a decrease of this component when the annealing is performed to a higher temperature . In fact, its relative amount is slightly different in the two samples: ether is 43% for the N-graphene previously annealed to 300 °C, while it is 30% on the one annealed to 600 °C. Together with ethers, some carbonyl groups (C=O) may also form32, but their contribution to the O 1 s spectrum cannot be disentangled from epoxies because they appear at the same binding energy and their amount is expected to be very low. In fact, DFT calculations33 and experimental results30 showed that the dominant species is epoxy, followed by ether and finally carbonyl.The above evidence indicates that OK-edge  do actually provide partially occupied π* anti-bonding orbitals, thus acting as active site, contrary to pyridinic-N5. In terms of energetics, it is known that O2 adsorption on the C-N bridge site is not favourable, and instead takes place on a C-C site close to a N site6.All these observations cross-confirm our hypothesis of oxygen dissociation on N-doped graphene resulting in the creation of C-O epoxy and ether bonds, as we have excluded the possibility of Os lineshape39, as we show in Supplementary Fig. s line are not very informative. Furthermore, the available computational method is known to give different absolute energies for the core levels of different elements40, making it doubtful that such a large and expensive simulation would offer additional insights in the present context. Thus, starting with a relaxed 6 × 6 supercell of graphene in vacuum, we created and relaxed the structure of the graphitic N site, then added between one and three O atoms near their expected absorption sites and relaxed the structures further. For comparison, we also calculated pristine graphene, and added epoxide oxygen. Images of the structures are shown in Fig. To simulate the atomic structures involved and to predict the XPS signatures of differently bonded and oxygenated N sites, we turned to density functional theory (see Methods). While it would be feasible to simulate an entire 524-atom graphene/Ir(111) system to model the C 1 s core level binding energies, we calculated total energy differences between the ground state and the first core-excited state, as described in the Methods. There are two factors that complicate a direct interpretation of calculated BE values. One is the system: we simulate a single layer of graphene in vacuum, whereas the experimental sample lies on the Ir(111) substrate. This clearly has some effect on the BEs, since the experimental C 1 s is at 284.16 eV instead of the expected value near graphite’s 284.42 eV41. However, it is not clear whether all other binding energies are similarly affected, or is the bulk value more sensitive since it is more influenced by the long-range metallic screening that is perturbed by the moiré. The other factor is the known underestimation of the values42, which yield a pristine graphene C 1 s at 283.827 eV for our simulation parameters, about 0.6 eV (0.02%) lower than the experimental graphite value. For ease of comparison to the experimental data, we have aligned the calculated BEs to the relevant experimental references  in Table To obtain C, N and O 1 s level downshifts by −0.48 eV for a single epoxide O, or −0.94 eV for two epoxide O. The O 1 s of epoxide atoms bonded at the N-neighboring C likewise downshift, by −0.67 eV for a single O or by −0.52 eV for two O, in good agreement with our experimental BE that is −0.6 eV downshifted from what is expected on pristine graphene30.Upon oxidation, the graphitic N 1 s core level spectra before and after the oxygen exposure, shown in Fig. s response also changes in important ways. In particular, we see a decrease of the graphitic-N signal at 400.9 eV and the pyrrolic at 400.0 eV, which is a clear indication that the oxygen atoms are adsorbed on graphene in a way that perturbs the chemical environment of those particular nitrogen sites. Furthermore, the absence of nitrogen oxide components, expected at binding energies higher than 402 eV44, indicates that the O2 dissociation, although catalysed by nitrogen, does not lead to chemisorption of oxygen atoms directly with nitrogen. In the fit of the N 1 s spectra in Fig. 2, a further component  is needed to properly fit the spectra after exposure to oxygen. This sixth component is found in both cases at −1.2 eV from the graphitic peak (red), in good agreement with the shift caused by the presence of two oxygen atoms surrounding a graphitic-N site  and Supplementary Fig. s), show that the oxygen dissociation is an almost reversible process. The faster desorption of the oxygen component at 530.6 eV with respect to the one at 532.3 eV, which remains almost constant, is in agreement with thermal desorption observed on graphene oxide46 and graphite30. These results further confirm our assignment to epoxy , there is a decrease of the N6 component, in agreement with the desorption of epoxy-oxygen and a partial restoring of the original graphitic-N. The decrease of the N1 component may be due to a reorganization of the graphene flakes after annealing, resulting in reduced contact with the underlying Ir. During this heating step the nitrogen amount further decreases from 5.1 to 4.6 at.%.Concerning the N 1 ex-situ grown graphene on copper foil to compare with an inert substrate. In the case of graphene on copper, we also found an O uptake of ~2.0 at. % after exposure. The N 1 s core level spectra before and after O2 interaction are shown in Supplementary Fig. s lineshape in N-graphene/Cu differs from N-graphene/Ir(111) because of its different morphology . However, interaction with O2 shows the same behaviour as in Fig. Since iridium itself is catalytically active, even though we have excluded oxygen intercalation and thus its obvious role in the dissociation, we repeated the experiment using 47 of the Elettra synchrotron  on a freshly grown graphene/Ir(111) sample. According to the experimental geometry and the use of linearly polarized light, the anisotropy of the two branches of the Dirac cone is enhanced48. In Fig. p and s polarization. In our analysis, we concentrated on the Dirac cone region along the Γ–K–M high symmetry direction of the Brillouin zone, as sketched in the inset of Fig. p-doping. In addition to the π band of graphene, we can also identify iridium surface states (indicated with S1 and S2) and minigaps (at 1.1 eV on the Γ–K branch and at 0.7 eV on the K–M), as already reported in literature49 and confirming the high quality of the present sample. The FWHM of the π band provides an indication of the disorder in the system. In particular, the FWHM of the MDC  calculated at 0.3 eV BE for the pristine sample is 0.045 Å−1, slightly higher than the value (0.035 Å−1)49 reported for graphene grown on single-crystal Ir(111). This difference can be ascribed to the lower growth temperature used in the case of Ir(111) films on YSZ.The interaction of graphene with oxygen has important effects also on its valence band. Graphene on Ir(111) is an optimum sample since it grows in large domains in register with the hexagonal symmetry of the substrate and can be easily studied by angle-resolved photoemission spectroscopy (ARPES). We repeated the same procedure, with the same plasma source, at the BaDElPh beamlinen-doping action of graphitic nitrogen. The π band maintains its linear behaviour after doping and the surface states of iridium are still visible in the spectra of Fig. −1. This broadening is explained by the higher scattering rate induced by the nitrogen defects, but it is much lower than the one found in a similar experiment of graphene on copper11 (0.19 Å−1). This difference can be ascribed to the different morphology of the graphene layer caused by the two substrates: the moiré modulation present in graphene/Ir(111) and absent in graphene/Cu may play a role in hampering the graphene interaction with the metallic substrate. Indeed, the sharp long-range ordered super-symmetric over-layer due to the mismatch between graphene and Ir(111) is maintained after exposure to nitrogen plasma, as testified by the extra spots surrounding each diffraction spot in the hexagonal pattern of the low energy electron diffraction images (LEED) shown on the bottom panels of Fig. 51.After nitrogen plasma exposure and subsequent annealing at 600 °C, a relatively large downshift of the Dirac cone takes place, with the Dirac point now located at 0.44 ± 0.02 eV and consistent with the significant in situ synchrotron spectroscopy experiments. The creation of single C-O bonds was highlighted by XPS, NEXAFS and ARPES measurements. Supported by first principles calculations, the modification of the N 1 s lineshape reveals a change in the chemical environment of graphitic nitrogen upon oxidation that is consistent with the adsorption of two oxygen atoms in epoxy position on the nearest carbon atom neighbours of the graphitic nitrogen, as predicted theoretically. A possible participation in this mechanism of three-fold coordinated nitrogen in a Stone-Wales defect can not be excluded. We ruled out any possible role played by the catalytically active iridium substrate in the oxygen dissociation by carefully comparing with previous studies on oxygen intercalated graphene/Ir(111). To further exclude its involvement in this mechanism, we compared our results with graphene on copper foil, a catalytically inert substrate, finding very similar results. Our work thus offers the first direct experimental description of the first step in the oxygen reduction reaction, providing detailed atomic-level understanding of the role of the nitrogen active sites in the interaction with molecular oxygen. Such knowledge will be crucial in understanding and improving the use of this novel metal-free material in gas-sensing devices and fuel cells.We have studied the adsorption and dissociation of molecular oxygen on nitrogen-doped graphene directly with multiple in situ by the chemical vapour deposition of ethylene in ultra-high vacuum  on iridium(111) films evaporated on top of an epitaxial yttria-stabilized zirconia (YSZ) film, following an established recipe that allows to achieve a long-range ordered layer52. Prior to graphene growth, the Ir(111) substrate was cleaned in UHV via cycles of Ar+ ion sputtering and annealing at 940 °C. The quality of the sample was confirmed by XPS, ARPES and LEED before each experimental step. Nitrogen was incorporated in graphene/Ir(111) by exposing the sample to the downstream of a µ-wave plasma source fed with N2 gas in the 10−5 mbar range. Two distinct samples with very similar initial nitrogen content were grown and then annealed at either 300 or 600 °C. Exposure to oxygen was performed in the same chamber, with a pressure of 5 × 10−5 mbar of pure molecular oxygen (O2) while keeping the sample at 200 °C for 30 min and then cooled down in O2 atmosphere until reaching T < 100 °C.Graphene was grown f, C 1 s, N 1 s and O 1 s core levels. Photoelectrons were collected at normal emission by a Phoibos electron energy analyser equipped with a homemade delay line detection system. Photoemission intensities were normalized to the beam intensity and number of scans and the binding energy (BE) scale was calibrated using the Fermi edge of the Ir substrate. The error bar on the atomic concentration is on the order of ±0.5 at.%. NEXAFS measurements across the C K-edges were taken in Auger yield mode, with  linearly polarized radiation and with the electric field vector parallel to the surface plane  by appropriately rotating the sample. ARPES measurements were performed at the BaDElPh beamline47 with a photon energy of 34 eV for both p and s polarization to highlight both branches of the Dirac cone (K point at 1.7 Å−1) along the high-symmetry direction Γ–K–M of the Brillouin zone. The overall energy and angular resolution was set to 50 meV and 0.3°. At both beamlines, the full experiment  was performed in situ without breaking UHV conditions. XPS spectra were fitted using a commercial software (CasaXPS), with a Doniach-Sunjich line shape convoluted with a Gaussian used for the C 1 s (C-C sp²) and Ir 4 f core levels, while the other peaks had Voigtian line shapes. The FWHM of the N 1 s components reported in Table All measurements were performed at the Elettra synchrotron radiation facility in Trieste . XPS and NEXAFS were performed at the SuperESCA beamline, with photon energies of 200, 400, 500 and 650 eV used respectively for the Ir 4 53. For each of the studied structures, we used a 6 × 6 supercell of graphene with 10 Å of vacuum, the Perdew-Burke-Erzerhof (PBE) functional, and a Monkhorst-Pack k-point mesh of 5 × 5 × 1 (these parameters are sufficient for core level binding energies converged to within 100 meV)42. After introducing a N site, we relaxed the structure until maximum forces were <0.02 eV/Å. For calculations of the C, N and O 1 s core level binding energies, we utilized a standard delta Kohn–Sham (ΔKS) calculation with a projector-augmented wave dataset including an explicit core-hole on an atom of interest54.For atomistic simulations, we used density functional theory modelling implemented in the GPAW simulation package42, we corrected the calculated values by aligning the C 1 s energy of a C atom far away from the N site to the experimental C 1 s energy of 284.16 eV. However, since the error depends on the core level in question40, to facilitate direct comparison with the experiment, we also corrected the N 1 s to match the experimental graphitic N and the O 1 s to match the known value for epoxide on pristine graphene. Note that since this affects all 1 s levels of each element identically, the alignment does not affect the shifts upon oxidation or our conclusions.Since the simulation method slightly underestimates the binding energiesSupplementary Information


P < 0.05). In addition, the two methods were analyzed for the quantitative detection of PD-L1 expression. The results from the estimation of transcriptional RNA expression and quantitative protein immunohistochemistry were consistent (r = 0.745). Furthermore, we also analyzed PD-L1 expression level in different types of TETs from TCGA database and observed that higher PD-L1 expression was in thymic carcinoma than in thymoma. Therefore, it could be concluded that PD-L1 expression in TET cells was correlated with the degree of malignancy, whereas the estimation of PD-L1 expression was potentially applicable in the clinical staging of TETs.The incidence of thymic epithelial tumors (TETs) in the Chinese population was much higher than that in the North American population. In clinical treatment, the prognosis of benign tumors after surgical resection was significantly better than that of malignant tumors. Currently, the commonly used clinical indicators for TET staging included Masaoka staging and WHO (2015) pathological criteria; however, the distinction between the benign and malignant tumors and diagnosis is yet to be explored. The current study demonstrated that the expression of PD-L1 in tumor cells was correlated with the degree of TET malignancy. The quantitative analysis of PD-L1 expression in 70 cases of TET tumor samples revealed that the positive rate of PD-L1 expression in types A, AB, B1, and B2 of thymoma (40 cases) was 37.5% (15/40), which was significantly lower than that in type B3 thymoma and thymic carcinoma  as demonstrated by chi-square test ( Thymic epithelial tumors (TETs) referred to tumors originating from thymic epithelial cells or those differentiating into the thymic epithelium, including thymoma and thymic carcinoma . The incA total of 70 cases of pathologically diagnosed TETs by surgical resection or needle biopsy were collected from 2012 to 2017 at the Department of Pathology in Weifang People's Hospital and Wenzhou People's Hospital, China. The cohort consisted of 50 thymoma and 20 thymic carcinoma cases. According to the WHO (2015) diagnostic criteria, two pathologists reviewed the section, confirmed the diagnosis, and deduced that there were sufficient tumor cells for subsequent immunohistochemistry (IHC) and genetic testing. Also, the clinicopathological data, including age, gender, with/without myasthenia gravis, with/without preoperative and postoperative radiotherapy and chemotherapy, tumor size, pathological classification, Masaoka staging, with/without lymph node, and distant metastasis were collected.A 4 × 4 array of acceptor wax blocks was made using a tissue chip instrument. The diameter of each micropore was 0.9 mm, the interval about 1 mm, and the depth about 3 mm. The IHC staining was performed on a Roche BenchMark XT fully automated IHC instrument. PD-L1  and PD-1  staining was performed according to the manufacturers' instructions. Phosphate buffer saline (PBS) was used as a negative control instead of the primary antibody. The placental tissue and tonsil tissue were used as positive control for PD-L1 and PD-1, respectively. PD-L1 was primarily located in the cell membrane; the positive color was manifested as yellow to brown linear staining on the tumor cell membrane. PD-1 was localized in the cytoplasm of the interstitial lymphocytes. The interpretation of PD-L1 results was divided into two parts: the percentage of positive cells (A) and staining intensity (B). The percentage of positive cells (A): enumeration of the percentage of stained tumor cells (0–100%), percentage of positive cells = number of positive cells/total cells × 100%. The standard of staining intensity score for positive cells: 0 point, negative; 1 point, weakly positive (light yellow); 2 points, moderately positive (brownish yellow); 3 points, strongly positive (tan). The expression of PD-L1 was evaluated by the product of positive cell percentage and the staining intensity, and the positive expression was defined when the above formula-based estimation was ≥3% . The intμm. Firstly, the total tissue RNA was extracted from the FFPE samples by RNA isolation and extraction kit , followed by reverse transcription into cDNA and qPCR. The qPCR reaction system consisted of 2 μL cDNA template, 1 μL PD-L1 primers and probes , 7 μL ribozyme-free water, and 10 μL Master Mix. The specific reaction program was as follows: Uracil-DNA Glycosylase incubation at 50°C for 2 min, polymerase activation at 95°C for 10 min, and PCR reaction at 95°C for 15 s and 60°C for 1 min for a total of 40 cycles. The gene mRNA expression level was detected based on the difference of Ct values (ΔCt) of the patient's target gene and the housekeeping gene as compared to the corresponding ΔCt database , and the normal distribution frequency of the patient's ΔCt in the population was obtained. The relative expression level of the genes in this study was defined as follows: <0.25 was low expression; 0.25–0.75 was moderate expression; and >0.75 was high expression.The wax blocks containing corresponding tumor tissues were serially sectioned into 5–8 slices, each with a thickness of 5 P values ≤ 0.05 were considered to be statistically significant.105 TET cases were selected for analysis. All cases had detailed clinical information and RNA-seq results. According to the clinical information of TET patients, all patients were divided into two categories, including thymic carcinoma and thymoma. Then we analyzed PD-L1 expression level of patients with thymic carcinoma or thymoma, respectively, and the difference of PD-L1 expression level in different categories of TETs was calculated by test.  PD-L1 mRNA expression and PD-L1 protein and between PD-L1 protein expression and clinicopathological features were conducted by chi-square or Fisher's exact test, as appropriate. The comparisons between the PD-L1 protein and PD-1 protein expression were conducted by chi-square or Fisher's exact test, as appropriate. The correlation between the variables was analyzed by Spearman correlation analysis. Two-sided P values ≤ 0.05 were considered to be statistically significant.Statistical analysis was performed using SPSS 21.0 . The comparisons between theA total of 70 TET patients  aged 29–77  years were included in the study. The diameters of the primary tumors were 1.2–11.74 cm. All the cases were classified according to the World Health Organization (WHO) (2015) histological criteria of thymic tumor. The cohort consisted of 11, 13, 9, 7, 10, and 20 cases of type A, type AB, type B1, type B2, type B3, and thymic carcinoma, respectively. All the thymic carcinoma cases were squamous cell carcinomas. According to the Masaoka clinical staging of thymoma, the cases of stages I, II, III, and IV were 32, 4, 20, and 14, respectively. Eleven patients exhibited comorbidity with myasthenia gravis, 37 patients underwent radiotherapy after surgery or biopsy diagnosis, and 21 patients were given chemotherapy after surgery or biopsy diagnosis; cervical lymph node metastasis, classified as type B3, was detected in 1 case of thymoma, and lymph node or other organ metastasis was observed in 13 thymic carcinoma patients.χ2 = 2.786, P > 0.05). Considering that the biological behaviors of type B3 thymoma were similar to that of thymic carcinoma, it was classified as thymus carcinoma for analysis. The positive rate of PD-L1 in type B3 thymoma and thymic carcinoma  was significantly higher than that in other types of thymomas  . In 20 cases of thymic carcinoma tissues, the rate of PD-1 was positive in 13 cases of tumor-infiltrating lymphocytes (TILs) with a positive rate of 65%, and in 14 cases of tumor cells with a positive rate of 70%. In addition, PD-1 was positively correlated with PD-L1 expression in tumor cells .PD-L1 protein was expressed on the cell membrane of tumor cells that appeared as light yellow, brownish yellow, and tan depending on the expression level Figures 1. PD-1 prP < 0.05)  . PD-L1 mRNA was highly expressed in 17 cases, with positive PD-L1 protein detection and positive coincidence rate of 100%. On the other hand, PD-L1 mRNA was moderately expressed in 22 cases, of which the PD-L1 protein was positive in 15 cases with a positive coincidence rate of 68.18%. The PD-L1 mRNA was lowly expressed in 17 cases, in which the PD-L1 protein was positive in only 1 case, with a negative coincidence rate of 94.12%. The concentration and purity of RNA extracted from 14 samples were infinitesimally low, and, hence, these were regarded as unqualified samples and not included in the statistical analysis. Five cases of PD-L1 protein were detected as positive. The coincidence rate of the two detection methods was 85.71%. Spearman correlation analysis showed that PD-L1 mRNA expression was positively correlated with PD-L1 protein expression  could be simply divided into two categories, including thymoma (n = 84) and thymic carcinoma (n = 21). We found that the median PD-L1 expression in patients with thymoma was 5.68, and the median PD-L1 expression in patients with thymic carcinoma was 9.39 (P = 0.0419). The result further suggests that high PD-L1 expression is substantially correlated with malignancy of TET tumors.We obtained 105 TETs cases with detailed clinical information and RNA-seq results from TCGA database. Based on the clinical characteristics, all TET patients (was 9.39 . The difThe study of the PD-1/PD-L1 pathway in many tumors has been under intensive focus. However, only a few reports about PD-1/PD-L1 in TETs are available in recent years. In this study, we analyzed the expression of PD-L1 protein in 70 cases of TETs. The results showed that the positive expression rate of PD-L1 protein in TETs was 54.29%. Previous studies , 13–18 d PD-L1 mRNA expression in TETs, which also provided a credible basis for the estimation of PD-L1 protein. The results showed that the expression of PD-L1 mRNA in high- and low-expression groups was in agreement with that by IHC detection; however, the difference primarily occurred in the moderate-expression group, in which the concordance rate with protein was 68.18%. These differential phenomena might be attributed to the following: real-time fluorescence qPCR detected the mRNA expression level in all cells, including tumor and interstitial cells; however, the IHC interpretation of PD-L1 protein was confined to the tumor cells, and the analysis did not include the nontumor lymphocytes that expressed the PD-L1 protein. Furthermore, TETs were different from other solid tumors, especially type B1 and B2 thymoma. The TETs were similar to the normal thymus, with relatively abundant nontumor immune cells. A total of 14 cases of patients with tissues expressing PD-L1 mRNA did not reveal any unqualified RNA. However, IHC showed positive PD-L1 expression in 5 cases. Although the sensitivity of mRNA detection was higher than that of IHC, it could not clearly identify the same in tumor cells, and the specificity was poor. On the other hand, the RNA was degraded due to the method of sample fixation, the length of time, and the long shelf-life, which led to failed obtaining results. Although a certain degree of subjectivity was noted in the selection of IHC antibody and the judgment of positive intensity and positive threshold, the percentage of false-positive and false-negative results would appear with advantages of simple operation, mature technology, low cost, and improvement in the drawbacks by quality control. Therefore, the IHC method can be used as a detection indicator for PD-L1 in TETs with its unique advantages of simple operation and cost-efficiency.Moreover, this study, for the first time, used real-time fluorescence qPCR to detect theStudies have found that the positive expression of the PD-L1 protein was an adverse prognostic factor , 20. TheA majority of the TETs were composed of a mixture of neoplastic epithelial and nonneoplastic lymphocytes. Moreover, the detection of PD-L1 alone cannot comprehensively determine the prognosis of TETs. PD-1 is primarily expressed on activated T lymphocytes. Studies have shown that an increase of PD-1-positive TILs suggested a poor prognosis , 24. ThyThe present study also found that PD-L1 protein expression was associated with radiotherapy and chemotherapy; however, the underlying mechanism was yet unknown. Katsuya et al.  reportedOverall, the current findings indicated that the expressions of PD-L1 protein and mRNA differed in thymoma and thymic carcinomas, and PD-L1 may serve as a potential marker of invasiveness and prognosis. In addition, the high expression of PD-L1 and PD-1 in TETs has also made it possible to clinically adopt the immunotherapy for targeting PD-1/PD-L1.


Gender inequity in science and academia, especially in senior positions, is a recognised problem. The reasons are poorly understood, but include the persistence of historical gender ratios, discrimination and other factors, including gender-based behavioural differences. We studied participation in a professional context by observing question-asking behaviour at a large international conference with a clear equality code of conduct that prohibited any form of discrimination. Accounting for audience gender ratio, male attendees asked 1.8 questions for each question asked by a female attendee. Amongst only younger researchers, male attendees also asked 1.8 questions per female question, suggesting the pattern cannot be attributed to the temporary problem of demographic inertia. We link our findings to the ‘chilly’ climate for women in STEM, including wider experiences of discrimination likely encountered by women throughout their education and careers. We call for a broader and coordinated approach to understanding and addressing the barriers to women and other under-represented groups. We encourage the scientific community to recognise the context in which these gender differences occur, and evaluate and develop methods to support full participation from all attendees. Gender imbalances in science, technology, engineering and mathematics (STEM) careers have been widely recognised, with women particularly underrepresented at senior levels ; 2. Thishttp://www.academia-net.org/), and codes of conduct for professional meetings  forms of discrimination, including the introduction of double blind peer review , lists ogs (e.g. ). These gs , supporThis pattern of lower participation from women can also be considered within this theoretical framework. Scientific reputation is obviously important although it is clearly not one dimensional, for example individuals may be considered especially strong in generating ideas, experimental practice, analysis or in making penetrating comments. High scientific reputation, as with other positive feedback, may enhance personal wellbeing as well as potentially leading to offers to participate in meetings, or suggestions to collaborate. As an example of its importance, Göktepe-Hulten and Mahagaonkar  showed tHowever, as demonstrated in the Reputation model, scientific reputation may be poorly related to actual contributions to the field, with some obtaining high credit due to the promotion by themselves or followers . In addiWithin the context of this framework, here we hypothesise that women are less likely to participate in the question sessions at a large scientific conference, due to behavioural differences linked to external factors. We aim to address the question of whether women ask fewer questions than men in this context and to investigate the influence of factors other than gender-based behavioural differences, such as demographic inertia and discrimination by session chairs. Finally, we aim to discuss whether the likely causes of gender differences in behaviour in these settings are likely to be addressed by current efforts to tackle the gender imbalance in STEM fields. This is the first study of this scale to compare participatory behaviour of different genders at an international conference. Significant differences in behaviour would highlight that there are behavioural patterns aligned with gender (but not restricted to specific genders), that may be a symptom of and/or a contributing factor to, the continuing drop out of women at progressively higher levels of seniority in science.We observed sessions of talks at the International Congress for Conservation Biology and European Congress for Conservation Biology in Montpellier, France in August 2015. At four randomly selected times during the week we monitored up to 10 synchronised parallel sessions. Monitoring was conducted with a team of 14 voluntary observers for 34 of these 40 talk sessions. Each observer recorded the number of men and women in the audience (excluding themselves), the number of men and women sitting in the front half of the room, and the genders of the session chair(s), each speaker, and audience members selected to ask questions. For 20 sessions, the age of those asking questions was recorded in two categories: younger than 50, or 50 or older . See All work was carried out with the approval of the University of Kent’s School of Anthropology and Conservation Ethics Committee. We obtained permission from the conference organisers in advance, but delegates were not informed about the study, to ensure that results reflected the typical behaviour of chairs and conference participants. We ensured that all data from sessions were aggregated; no personal or identifying information was collected about any individual.We analysed question data from 31 sessions (three sessions were removed for incomplete data or the chair being aware of this study). Across the 31 sessions, we analysed 270 questions.genquest; 1 = male; 0 = female), so there was one data point for each question asked. We used a logistic regression model [p, the probability that a question is asked by a male audience member. As the gender ratio of the audience determines the numbers of each gender that are available to ask questions, the logit-transform of the proportion of the audience that was male (nm/(nm+nf)) was included as an offset:To test whether men asked significantly more questions than women, we used a binomial Generalized Estimating Equation (GEE)  with seson model  to estimβ0) to be zero on the logit scale, which implies an equal rate of questioning by men and women after accounting with the offset for the proportion of men and women in the room. We formulated the data such that 1 = male question and 0 = female question, therefore a positive intercept suggests that men ask more questions than women and a negative intercept suggests the opposite.If men and women asked questions at the same rate, we would expect the intercept of the binomial model (We included gender of the session chair and the speaker as covariates and used "quasi-likelihood under the independence model criterion” (QIC)  to distip).Previously it has been hypothesised that men ask more questions because there are currently more men in senior positions and experienced people have greater confidence and often a wider knowledge. To test this hypothesis, we repeated the question analysis above, using only those questions asked by people in the younger age group category (less than 50). Only some observers recorded the age of the questioners, therefore this analysis was conducted with data from 20 sessions and included 123 questions.We did not have information of the gender balance of young people in the audience as we considered achieving this would make our activities too conspicuous, therefore we used two separate approaches to estimating the gender balance of young people in the audiences. Firstly, we assumed the proportion of men and women was the same in the younger age group as all age groups. However if younger age groups are likely to have a greater proportion of women than older age groups, this will over-estimate the number of young men and under-estimate the number of young women. Secondly, we assumed that in each room 10% of participants were at least 50 years old and we assumed that 75% of those over 50 were male .In total across all 31 sessions analysed for questions, there were an estimated 1487 female audience members and 1116 male audience members. There were 270 questions: 152 from men and 118 from women. Therefore, on average there were 0.08 questions asked by each female audience member and 0.14 questions asked by each male audience member.After accounting for the gender balance in the audiences, men asked more questions than women, demonstrated by a positive intercept in the GEE model . Transforming this intercept back to the real scale, the estimated values suggest that in a hypothetical room with an even balance of men and women, 64% of questions would be asked by men . This point estimate suggests that men ask nearly two thirds of the questions and therefore for each question asked by a woman, each man asks on average 0.64/0.36 = 1.8 questions (95% CI 1.4–2.2) . There wEven amongst the younger researchers, male participants asked significantly more questions than female participants. Using the first estimate of gender ratio, the model estimated that, in a room with an even gender balance, young men would ask 61% of the questions asked by younger researchers and that therefore younger male researchers ask 1.5 questions (95% CI: 1.1–2.2) for every question asked by younger female researchers. However, we expect that this model will produce a negatively biased estimate of the ratio of male:female questions, given the biased offset used. Using the second adjusted estimate of gender ratio, the model estimated that with an even gender balance, young men would ask 66% of the questions asked by younger researchers and that therefore younger male researchers ask 1.8 questions (95% CI: 1.2–2.7) for every question asked by younger female researchers .We recorded only 36 instances of competition between at least two people to ask a question and 20 of these were between male and female audience members. In 12 of these 20 instances, a man was selected to ask the question. This ratio is similar to the overall bias in questions, however, the sample size is too small to draw any statistical conclusions. Nonetheless, this small sample suggests that there was not a substantial amount of competition to ask questions.By observing question and answer sessions at a large international conference we show that, even accounting for the gender ratio of the audience, male researchers ask more questions than their female counterparts. This confirms previous findings and, for the first time, demonstrates that this pattern is seen in both younger and more senior researchers, and is therefore unlikely to be a result of demographic inertia.Decades of research has shown a decline in classroom participation with age for female students , and ouret al. [We found that on average a male researcher asks an estimated 1.8 questions for each 1 question asked by a female researcher. Two similar studies at astronomy conferences estimated that men asked more questions than women  and men et al. . Howeveret al. ; 22 wereet al. . Ours isGiven that even amongst the younger age bracket, men asked significantly more questions than women, our findings suggest that behavioural patterns are not only a result of ‘demographic inertia’, in which historical inequalities persist due to a lag time ; 21; 22.et al. [We observed differences in participation throughout our sample and also found no evidence that the gender of the session chair or speaker affected the number of questions from male and female audience members. This result is in contrast to Davenport et al.  who founDifferences in participation in question sessions may be supported by other studies of conference participation that have found that women are more likely to apply to present their work in conference poster presentation sessions rather than oral presentations ; 46. SimHere, we do not assume that ‘male’ behaviour is correct and that women need to behave more like men; we recognise that there are other methods of participating at a conference and that the use of different communication strategies may need further research. We also reject the idea that our findings are linked to the women in our sample being less intelligent than the men, as the relationship between gender and intelligence is widely accepted to be complex . Whilst Our findings of gender differences are of particular interest, as the conference in question had taken steps to address inequality and discrimination in its most visible forms, using a clear code of conduct . It is cWe acknowledge that further work is needed to fully explore the issues surrounding gender and participation, and address some of the limitations of our study. In particular, further work to understand the underlying causes of the gender differences that we observed would be of benefit. For example, our observation methods were designed to ensure that attendees behaved normally, but future work could also carry out follow-up surveys with attendees to look at motivations for asking, or not asking, a question. In addition, we did not record the types of question asked, which may provide more information on the purpose of questioning  and how this might link to the gender of both the speaker and the questioner. The use of observation in our study also meant that we relied on gathering data on age and gender based on appearance, which may have led to some errors. In particular, we note that gender and appearance do not always align. Following on from this, we also note that we have treated gender as a binary dichotomy, and, we recognise that an increasing number of people do not identify with a single gender. These individuals, as well as other race demographics, are also likely to suffer discrimination and bias in academic science, however the evidence and sample sizes for these groups are extremely small. Here we examine women, who represent a larger proportion of the conservation biology community, as an example of how gender-aligned behaviour might be affected by and contribute to bias and discrimination.We found that behavioural differences in gender exist in younger researchers, and may be affected by self-esteem, or discrimination in school or professional environments. Therefore, our findings support assertions that the issues surrounding gender imbalances in STEM and particularly in academia are complex and in need of better understanding . HoweverS1 TableModel selection table for Generalized Estimating Equations (GEE) with data from 24 sessions that included data on chair gender and speaker gender and either male or female sessions chair(s) . After removing questions asked in a general discussion session, 188 questions were included in the model.(DOCX)Click here for additional data file.S1 FileFurther details of data collection and analysis methods.(DOCX)Click here for additional data file.S2 FileDataset used for the analysis. Columns include the session number, the gender of the chair, gender of the speaker, number of questions to that speaker from female audience members, number of questions to that speaker from: male audience members, female audience members under 50, male audience members under 50, the proportion of the audience that was male, the estimated and adjusted proportion of young audience members that were male.(TXT)Click here for additional data file.


Density functional theory (DFT) calculations indicated that the energy barrier (Eb) for dissociative adsorption of H2O was significantly lower in case of reconstructed graphene with a monovacancy compared to pristine graphene. Cross-sectional transmission electron microscopy of graphene transferred to the counterface revealed a partly amorphous structure incorporating damaged graphene layers with d-spacings larger than that of the original layers. DFT calculations on the reconstructed bilayer AB graphene systems revealed an increase of d-spacing due to the chemisorption of H, O, and OH at the vacancy sites and a reduction in the interlayer binding energy (EB) between the bilayer graphene interfaces compared to pristine graphene. Thus, sliding induced defects facilitated dissociative adsorption of water molecules and reduced COF of graphene for sliding tests under ambient and humid environments but not under an inert atmosphere.Sliding contact experiments and first-principles calculations were performed to elucidate the roles of structural defects and water dissociative adsorption process on the tribo-chemical mechanisms responsible for low friction of graphene. Sliding friction tests conducted in ambient air and under a dry N The low coefficient of friction (COF) of graphite arises from shearing of the basal planes but the COF is not an intrinsic property4 as it depends on the presence of dissociated water and/or oxygen molecules in the environment8. Similarly, low COF values of polycrystalline diamond (PCD) and diamond-like carbon (DLC) coatings observed under the ambient conditions were often attributed to the saturation of the dangling carbon bonds at the contact surfaces by dissociatively adsorbed gas molecules11. Ideally, graphene is a two dimensional array of carbon atoms, characterized by Raman peaks at 1580 cm−1 and 2700 cm−112. While the 2D peak of graphite at 2700 cm−1 consists of two separate components, namely 2D1 and 2D2, a single sharp 2D peak is the distinguishing feature of graphene13. Low-load (1 nN) sliding experiments conducted using friction force microscope showed that the multilayered graphene with more than four layers  exhibited a COF of 0.2 when tested under an ambient atmosphere with a relative humidity (RH) of 25% compared to a higher COF of 0.4 for the monolayer graphene14. Ethanol processed monolayer graphene tested using a ball-on-disk tribometer at a load of 1N under a dry H2 atmosphere showed a low COF of 0.22, whereas once the same sliding tests were conducted on three or four-layer graphene the COF decreased to 0.1515. CVD deposited multilayer graphene tested using a pin-on-disk tribometer at 1N showed a high COF of 0.52 in a dry N2 atmosphere, but exhibited a low steady state COF value of 0.11 in an air atmosphere with 45% RH16.The computational studies based on density functional theory (DFT) showed that hydroxyl groups on pristine graphene could reduce the adhesion between graphene layers contributing to low friction17, although the relation between adhesion and friction should be interpreted with caution, as it has been suggested that the friction force would depend on other factors such as adhesion energy hysteresis18.The reduction of friction by using carbon-based materials either in the bulk form, or as surface coatings, is an important technological alternative to the usage of liquid lubricants219. A high COF of 0.70 for nanocrystalline diamond sliding in high vacuum (10−9 Pa) was reported in another study, which also observed that introduction of water vapour reduced the COF to <0.05 while in a dry H2 atmosphere COF values <0.01were recorded20. The low COF of nanocrystalline diamond in water vapour and hydrogen atmospheres were attributed to OH and H termination of surface C atoms. The non-hydrogenated grades of diamond-like carbon (NH-DLC) coatings showed a reduction in COF with an increase in the humidity10. It was proposed that the dangling sp3 carbon bonds in PCD were terminated by a physisorbed water layer while for the NH-DLC the sp2 hybridized carbon atoms were converted to sp3 as a result of moisture adsorption21.Considering the friction behaviour of other carbon based materials, polycrystalline diamond (PCD) tested in an ambient air with 37% RH against a ceramic counterface was shown to exhibit a COF of 0.10, but a COF of 0.55 was recorded in a dry N2 compared to 4.08 J/m2 for C without H termination22. For a diamond (111) surface, DFT computations predicted a low H2O dissociation energy barrier (Eb) of 0.122 eV23. Contrary to diamond, pristine graphene does not possess dangling bonds. According to DFT calculations a large Eb is required for dissociative adsorption of H2O on pristine graphene24.Density functional theory (DFT) based calculations showed that the chemisorption of C atoms by H atoms on a diamond (111) surface would reduce the work of separation between an Al interface and H terminated C atoms to 0.02 J/m16. Edge fracture of graphene plates within the wear tracks were also reported25. Static atomic force indentation experiments revealed a reduced elastic modulus for graphene upon the introduction of vacancies26. A decrease in the fracture strength of graphene was reported to occur as a result of introduction of Ar+ irradiation defects27. It was shown that vacancy formation and adsorption of the hydroxyl groups would reduce the strength and elastic modulus of graphene28.Formation of sliding-induced defects should be considered as an important part of friction mechanisms of graphene. Raman spectroscopy of the worn graphene surfaces indicated generation of D, and split G peaks due to the formation of defects during sliding contact2O dissociation processes may be facilitated by the formation of defects in graphene29. Comparision of Eb values calculated for water dissociation on graphene using different computation techniques indicated that water dissociation process favoured the monovacancy graphene but not the pristine graphene31. Among various types of defects that may exist in graphene Stone-Wales defects were shown to have lower formation energy than a monovacancy32, and since C atoms at these defects do not possess dangling bonds, the binding of OH- or H- groups to the C atoms would be less likely. The reactivity of defective graphene with H, F and phenyl groups was reported to decrease in the following order; monovacancy > hydrogenated zigzag edge > double vacancy > Stone-Wales > hydrogenated armchair edge > defect-free graphene33. Monovacancy graphene (MG) was frequently used to simulate the dissociation mechanisms of molecules like H2O and O236, and different Eb values were reported depending on the simulation methods  used. An Eb of 0.04 eV for H2O dissociation was found by means of DFT calculations without non-local dispersion terms using a cell size of 3 × 324, but a higher value was calculated using a larger cell size of 4 × 430.H37 were used to model the interfacial tribo-chemical mechanisms. The effects of dissociative adsorption of water molecules between the graphene layers proved to be an important factor in rationalizing the friction reduction mechanisms of graphene.The current manuscript elucidates for the first time the friction reduction mechanisms in graphene by considering the roles of dissociated water molecules on the sliding induced defect sites in graphene. The research unraveled the significant roles of sliding-induced graphene defects and transfer layers generated on the counterface and water dissociation in reducing the friction. Using atomistic calculations, and sliding friction experiments it was shown that dissociated water molecules can reduce the graphene interlayer biding energy and may increase the interlayer distance between the adjacent layers as observed by analytical high resolution microscopy techniques. First-principles calculations with vdW functionalR) where the COF values reached a peak followed by a steady state period. The graphene provided a low running in COF, µR and the transition to the steady state regime was immediate. The average steady state COF was low at µs = 0.10 for this sample. The curves shown in Fig. R and µs values obtained from all friction tests are presented in Fig. R for graphene was 0.20, with a tR of 1–2 revolutions, and the average µs was 0.15. DLC coatings exhibited high µR values, i.e. 0.55 for hydrogenated diamond-like carbon (H-DLC) and 0.57 for NH-DLC, and tR for the DLCs were short, tR <10 revolutions. The µs values recorded for the both DLC coatings were 0.10. The PCD showed the longest tR of 40 revolutions  peak at 2923 cm−1. Neither of them was present in spectra of pristine graphene. A reduction in the intensity of the 2D peak (2700 cm−1) and formation of a split of G peak (1580–1600 cm−1) were also observed38. These results suggest that sliding contact altered the structure of pristine graphene by increasing the degree of disorder in the graphene network. A G peak appeared at 1563 cm−1 in the transfer layers of PCD , and a H-DLC counterface was selected to prevent metallic material transfer that would otherwise occur to the graphene surface under dry and inert atmospheres25. At the beginning of both experiments, a µR peak was observed during the running-in period where initial surface damage occurred and once the transfer layers were formed on the counterface the COF was reduced. For the sliding experiments that were continued to be carried out in a dry N2 the COF continuously increased and reached 0.14 after 1,000 revolutions, no steady state behaviour could be observed. However, when the chamber was purged with air with 52% RH at the end of the running-in period, a low and steady state COF ensued with µs = 0.05.During sliding of the graphene, easy formation of transfer layers does not automatically lend itself to a low COF, and the moisture in environment plays an important role in this process. As the above experiments were carried out at a constant humidity of 22% RH, a new set of experiments were conducted to elucidate the role of the transition between the inert-to-humid atmosphere on the frictional properties of graphene. The effect of humidity on controlling the friction of the graphene can be readily understood by considering the two experiments shown in Fig. 2O molecules were investigated using spin-polarized density functional theory (DFT) calculations. The details are given in the ‘Methods’ section. Starting with diamond structure as a reference, Fig. Eb for water dissociation at the bridge site of diamond (111) surface was low at 0.41 eV. The total energies and changes in the O-H1 and O-H2 bond lengths of an H2O molecule as it approaches the surfaces of pristine graphene (PG) and monovacancy graphene (MG) are plotted in Fig. 2O molecule approached the PG surface, the energy of the system progressively decreased and dropped to a minimum of −0.13 eV at a H2O-PG distance of 3.1 Å with no change in the O-H bond length as shown in Fig. 41. The energy then increased to 1.08 eV due to repulsion between H2O and graphene when the water molecule brought to 1.1 Å away from the PG surface with no change to the O-H bond length, but during this process graphene became subjected to out-of-plane deformation. At a distance of 1.0 Å, the O-H1 bond length increased abruptly to 2.77 Å while almost no change in the O-H2 bond length occurred, indicating that the H2O molecule was dissociated into -OH and -H and chemisorbed on the PG surface. The energy of the system increased to 3.53 eV once the water was dissociated. Thus, this process is endothermic and due to the inert nature of PG the energy barrier Eb for dissociative adsorption of water molecule is high. Here, Eb = 3.53 eV is defined as the energy difference between the maximum energy before water dissociation and the energy of the reference state. Using DFT calculations that employed B3LYP:DFTB-D42 an Eb of 4.4 eV was previously reported for water dissociating on graphite (per molecule) and the use of GGA/Perdew-Wang 91 provided an Eb of 3.64 eV24.The thermodynamics and structural aspects of interactions between graphene surfaces and HEb of 1.27 eV was observed for water dissociation. At this distance range, the water molecule reoriented itself above the vacancy site, but the O-H bond length did not change. However, at the separation distance of 0.6 Å, the bond length of O-H1 increased to 2.20 Å, indicating again that water molecule was dissociatively adsorbed at the vacancy site of graphene with formation of C-OH and C-H. Meanwhile the bond length of O-H2 remained unchanged. The Eb for water dissociation on MG was lower compared to the Eb for water dissociation on PG Fig. 2 to sp3 transformation. It is also observed in Fig. For a water molecule approaching the MG surface Fig. , the eneEb of 0.43 eV. Since the water dissociated structure in Fig. 30. Therefore, the structure needs to be relaxed for studying dissociation of OH molecule to O and H. This structure relaxation at the start of Fig. The structure of MG with O-H2 and H1 chemisorbed on its surface was in a metastable state relative to the structure of completely dissociated water. The changes in the energy and the O-H2 bond length with the distance between O-H2 and MG are plotted in Fig. So far, DFT calculations investigated the dissociation of water molecule on PG and MG surfaces and showed that presence of monovacancy would reduce the dissociation energy, resulting in the saturation of the bonds. In multilayer graphene, dissociated OH, H, and O functional groups on each layer interact with the functional groups at the adjacent layer. To analyze the role of dissociated water molecules in modifying the interlayer binding energy between the graphene layers, 6 different types of AB graphene configurations incorporating functional groups with different orientations were considered in following section. It will be shown that the spacing between the graphene layers could increase and layers that were stuck together could separate due to the reduction of interlayer adhesion forces.43. As described in the previous section, adsorption of the dissociated water molecule occurred at the vacancy site of MG, hence all AB-stacked bilayer graphene configurations were constructed in such a way that each contained a monovacancy  between A and B layers was set to 5.3 Å to allow functional groups at both graphene layers to interact with each other. The interlayer binding energy (EB) between the graphene layers (bilayer interfaces) was calculated as N is the number of C, H, O atoms, 2 in gaseous state ; H-MG-O/O-MG-H had the lowest EB of 0.21 J/m2. EB of the other four graphene interfaces  were in the range of 0.27~0.28 J/m2. The interlayer spacing for H-MG-OH/OH-MG-H configuration with two OH initially facing the interface increased by 0.26 Å , H-MG-O/H-MG-O (0.89 J/m2) and H-MG-O/O-MG-H (0.94 J/m2). As the interface energies for all configurations are essentially similar (0.9–1.2 J/m2), it is conceivable that all bilayer graphene configurations considered may form concurrently and coexist.Another important piece of information that arises from the data shown in Table R but would not lend itself to a steady state friction regime. Raman spectra of the graphene wear track revealed the occurrence of a D peak at 1330 cm−1, which infers that defects were generated during sliding. It could be germane to comment here that graphene has a distinctly different Raman fingerprint that differentiates it from graphite as indicated in the introduction section. Pristine graphene is hydrophobic but a graphite structure consists of dangling carbon atoms. Dangling carbon atoms on the graphite surfaces have a tendency to promote dissociative adsorption of water molecules6. Creation of sliding induced defects is an essential step for friction reduction in graphene as demonstrated in this paper. First principles calculations suggested that adsorption of H, O, and OH groups that were dissociated from water molecules in the environment during sliding would reduce the interlayer binding energy between the adjacent graphene layers. The formation of C-O, C-H, and C-OH bonds could lead to distortion of the graphene planes, which may further facilitate dissociative adsorption of water molecules on the graphene surfaces. The sliding induced defects were important for the dissociation process as they reduced the energy required for this process compared to pristine graphene prior to the sliding. If the defect sites were not passivated the COF would increase as illustrated by the experiment carried on the graphene sliding under a nitrogen atmosphere  = 0.334 nm44. In contrast, the transfer layer that was generated under a N2 atmosphere was amorphized during sliding. No remnants of graphene stacks layers could be detected , and the Eb of graphene could be further lowered due to the generation of defects during sliding. This could be attributed to the adsorption of H2O and OH at the vacancy sites on thegraphene planes. In contrast, water can be easily dissociated on cleaved diamond (111) surface with a low Eb of 0.41 eV, indicating freshly cleaved diamond surface is more reactive than a monovacancy graphene surface. However, as the amorphous DLC and the PCD would need to be graphitized before being transferred to the counterface and the magnitudes of µR of DLC and PCD were higher and the duration of the running in-period were longer than that observed for graphene. Once the transfer layer was formed and the graphene planes were passivated by the dissociated water molecules at sliding induced defect sites on graphene surface, a steady state friction regime was established.As stated earlier, dissociation of water at a monovacancy site of graphene showed a lower S would occur–a process that requires dissociative adoption of water molecules. The interpretations of the proposed friction reduction mechanisms are well supported by the experimental observations as well as by the results of the first principles calculations. However, the details of generation of sliding induced defects should be further studied.In summary, sliding induced defects generated on the contact surfaces of graphene in ambient air act favourably and assist with the formation of transfer layers. As a result a steady state friction regime with a low µSliding contact experiments and DFT calculations that used van der Waals interactions elucidated the friction reduction mechanisms of graphene by determining the roles of structural defects and dissociated water molecules. The sliding friction tests showed low steady state COF values in ambient and humid atmospheres but not in an inert atmosphere. Cross-sectional TEM studies revealed that a tribolayer was formed on the counterface and consisted of an amorphous carbon matrix with intermittent stringers of graphene stacks; the d-spacing between the graphene layers was larger compared to that of the pristine graphene. On the other hand, the transfer layer formed from PCD exhibited an amorphous carbon matrix incorporating metallic debris as inclusions.DFT calculations showed that compared to pristine graphene, water was more likely to be dissociatively adsorbed on graphene incorporating a monovacancy defect due to a lower energy barrier of water dissociation into OH, H and O. the dissociated moelcules served to passivate the dangling carbon bonds. Dissociated water molecules intercalated between the graphene layers increased the graphene layer spacing as observed experimentally, and reduced the interlayer binding energy by ~30% compared to pristine graphene. In comparison, dissociated water molecules was adsorbed only at the surface C atoms PCD (and DLC). The H, O, and OH intercalation between the graphene layers allowed the graphene layers to be transferred readily to the counterface resulting in a lower COF running-in period with shorter duration compared to those of other carbon based materials, namely DLC and PCD.−1. The ratio of G peak at 1580 cm−1 to 2D peak was equal or greater than one in many locations indicating a multi layered structure45. There was no evidence for the formation of a D peak at 1330 cm−1 suggesting that the defect density of the pristine multilayer graphene plates was low. Sliding friction tests were also conducted on polycrystalline (35° rhombic) diamond, PCD and diamond-like carbon (DLC) coatings. The DLC coatings were deposited using an unbalanced magnetron sputtering system that used one chromium (to deposit an interlayer) and two graphite targets. Butane precursor gas was used to produce H-DLC type coating with 40 at.% hydrogen measured by elastic recoil detector (ERD). The hydrogen content of the NH-DLC type coating was <2 at.%.Sliding friction tests on graphene were performed against both Ti-6Al-4V and H-DLC coated tool steel. Graphene samples were deposited on Ni foils using a chemical vapour deposition (CVD) process. Methane gas was utilized as the source of carbon. Micro-Raman spectra obtained using a Horiba Raman micro-spectrometer with 50 mW Nd–YAG laser (532 nm excitation line) indicated a single sharp 2D peak at ∼2700 cmS, values were calculated from the arithmetic mean of the COF signals of each curve following the running in stage. Each set of tests on a given type of sample was repeated three times. The average values of µR and µS were calculated. Tests conducted using H-DLC counterfaces in a dry N2 atmosphere are described in Results and Discussion section.A pin-on-disk tribometer was used to measure the COF of multilayered graphene samples and other carbon surfaces against Ti-6Al-4V alloy pins with hemispherical tips of 4.05 mm in diameter. A constant speed of 0.05 m/s and a normal load of 1.00N were applied for 500 revolutions. Sliding tests were conducted under the ambient atmospheric conditions with 22% RH. A particular focus of the work was to understand of the formation of sliding induced structural defects as well as to determine the origin of the tribolayers generated on the contact surfaces. For this purpose, special emphasis was placed on studying the running-in behaviour of graphene in the initial stage of friction where the tribolayers were first generated. The highest value of COF reached during the running-in stage and the duration of running-in stage were noted. The steady state COF, µA FEI Quanta 200 FEG scanning electron microscope (SEM) equipped with an energy-dispersive X-ray EDAX SiLi Detector spectrometer was used to study the contact surfaces. Cross-sectional transmission electron microscopy (TEM) samples taken from the transfer layers were excised using a focused ion beam (FIB) lift out technique in a Carl Zeiss NVision 40 dual beam work station. The samples were ion-milled from both sides to a thickness of about 100 nm. The final milling of the samples was conducted at a low beam current of 40 pA. TEM observations were performed using FEI Titan 80–300 HR-TEM, with a lateral resolution <1 Å, operated at 300 kV.46 as implemented in the Vienna Ab Initio Simulation Package (VASP)48. The generalized gradient approximation (GGA)49 of the exchange correlation described by Perdew, Burke, and Ernzerhof (PBE)50 was used with a plane-wave cut off energy of 500 eV to reach the energy convergence of 1–2 meV/atom. The exchange-correlation energy 51, where 37 exchange functional was used. The electronic degrees of freedom were converged to 10−5 eV/cell, and the Hellman-Feynman forces were relaxed to less than 0.05 eV/Å via the conjugate gradient method. A 24 × 24 × 10 Monkhorst-Pack grid of k-points was used for unit cell of graphite, whereas a grid of 4 × 4 × 1 k-points was used for all water/(6 × 6)graphene slabs. For diamond, 20 × 20 × 20 k-points mesh was used while a mesh of 8 × 8 × 1 was sufficient to converge the energies of water/(3 × 3)diamond (111) slabs. Spin polarized calculations were performed to account for the magnetism incurred by vacancy formation in graphene53, and were employed for all applicable simulation systems.These calculations were performed using the projector-augmented wave (PAW) method with the exchange correlations that incorporated van der Waals interactions in the framework of vdW-DF2 functionala = 2.48 Å, and interlayer separation is c = 3.518 Å. The calculated interlayer binding energy per carbon atom of 52.6 meV/atom was consistent with 52.0 meV/atom calculated using the vdW-DF2 method54, and agreed well with the experimental value of 52 meV/atom55, although the vdW-DF2 slightly overestimated the interlayer separation by about 6% compared with the experimental value of 3.34 Å56. The lattice constant obtained for diamond, a = 3.61 Å, was the same as that reported in the literature calculated by vdw-DF2, which was 2% higher than the experiment value of 3.54 Å57.Prior to using vdW-DF2 for calculating the thermodynamic and structural properties of multilayered graphene, lattice constants and binding energy between graphite layers were calculated. We obtained the lattice constants of bulk (1 × 1) graphite 2O molecule and the bond angle of 104.8° (by relaxing the water molecule in a cubic box with 10 Å cell edge) agreed with the experimental bond length value of 0.958 Å and bond angle of 104.8°58, and these values were used in the simulation of the reaction mechanisms between water and the graphene. The reaction of water with graphene with monovacancy was simulated by gradually bringing a water molecule from a large separation distance (7 Å) towards the vacancy site on (6 × 6) graphene plane. A vacancy was created by removing one C atom at the center of (6 × 6) graphene cell (with 71 C atoms) as shown in Fig. 30. The same water orientation with O and H1 located at the center of a carbon hexagonal ring of the (6 × 6) pristine graphene with O-H2 making the same angle of 14.8° in the Z direction was maintained. Therefore, the initial configurations of water molecule in the pristine graphene and graphene with monovacancy were the same. Consequently, the energy barrier calculations for physisorption and water dissociative adsorption of water molecule on pristine  graphene cell (with 72 C atoms) was selected to accommodate possible deformation of graphene in the normal direction (Z direction) in Fig. Eb for water dissociation. A 15 Å vacuum layer was added between the carbon layers. The plane view of O-H1 of a H2O molecule at bridge site of diamond (111) surface and the side view of a water molecule approaching to diamond surface are shown in Fig. For studying water reaction with diamond, a cell of four double-layers of diamond (111) surface with (3 × 3) cell size and dimension of 7.65 Å × 7.65 Å × 21.78 Å was used to simulate bulk diamond by fixing the bottom two double-layers of carbon atoms in all directions. Here, the reconstructed diamond (111)−2 × 1 surface (Pandey structure) was not used because this structure provided a high Supplementary Information