Carbon nanostructures are promising ballistic protection materials, due to their low density and excellent mechanical properties. Recent experimental and computational investigations on the behavior of graphene under impact conditions revealed exceptional energy absorption properties as well. However, the reported numerical and experimental values differ by an order of magnitude. In this work, we combined numerical and analytical modeling to address this issue. In the numerical part, we employed reactive molecular dynamics to carry out ballistic tests on single, double, and triple-layered graphene sheets. We used velocity values within the range tested in experiments. Our numerical and the experimental results were used to determine parameters for a scaling law. We find that the specific penetration energy decreases as the number of layers (N) increases, from ∼15 MJ/kg for N = 1 to ∼0.9 MJ/kg for N = 350, for an impact velocity of 900 m/s. These values are in good agreement with simulations and experiments, within the entire range of N values for which data is presently available. Scale effects explain the apparent discrepancy between simulations and experiments. However, the rapid strain increase found in these applications can lead to unexpected behavior. For instance, experiments in this regime revealed unzipping of carbon nanotubes (CNTs) into nanoribbons2. While the high-strain-rate behavior of CNTs, either isolated4 or in composites8, has been studied for years, investigations on graphene mainly date from 201416. Of particular interest is the study by Lee et al.9, in which silica spheres were shot at multilayered graphene sheets. Exceptional energy absorption capabilities were found: the specific penetration energy of graphene was ten times greater than that of macroscopic steel. This was due in part to the impact energy being dissipated over an area much larger than that of the projectile cross-section.The combination of very high Young’s modulus (1 TPa), ultimate strength (130 GPa), and low density values  studies elucidated the atomistic structures formed during penetration of graphene monolayers and the role played by defects17. Our analytical model fits well all existing results for graphene.In the MD part of our study, we shot metallic projectiles at single, bilayer, and trilayer graphene sheets. We have considered different projectile velocities and impact angles, as well as sheets and projectiles of different dimensions . As further discussed below, we also obtained MD specific penetration energy values that are one order of magnitude larger than those from experiments, but the difference decreased progressively for the cases with two and three layers. From these results, we were able to extract parameters to apply in a scaling law proposed by Pugnod) of 140 Å. For other simulations, a spherical  m/s we obtained average cone velocities of 1.99 ± 0.15 (2.64 ± 0.10) km/s, values that are again close to those estimated by Lee et al., 1.95 (2.92) km/s9. It should be remarked that Haque et al.14 also found constant vc values in their MD simulations. However, under the higher velocity conditions they used, the obtained vc values were 18, suggesting a limit of validity for this expression.Results of this analysis are presented in Fig. 15 and experimental9 reports, we normalized the absorbed energy by the graphene mass within the projectile cross-sectional area, obtaining the Specific Penetration Energy (SPE). This comparison is presented in Table et al.14. Note that currently reported numerical values are an order of magnitude larger than experimental amounts, although the difference decreased for the considered bilayer and trilayer systems. It is important to remark that direct comparison between numerical  and experimental (up to now from 30 up to 300 layers) results is not presently possible, due to computational/technological limitations.In order to contrast our results against other theoreticald = 70 Å, θ = 0°, and v = 900 m/s) against square graphene sheets of varying length (20 and 40 nm). We observed reflection of impact-generated elastic waves at the system boundaries in both cases. For the larger structure, however, waves returned to the impact region only after fracture completion. See videos in the Supplementary Information. The smaller structure also absorbed less energy during these ballistic tests: increasing system size increased SPE from 12.9 MJ/kg to 14.1 MJ/kg. Regarding the structures employed to obtain the SPE values presented in Table We have also carried out tests to investigate whether our results could be affected by the limited size of the investigated structures. To this end, we examined the collision of a Nickel projectile 22. See the Supplementary Information for more details on this procedure. Thus, we can writeη is the ratio between the area of the projectile cross-section and the area of the damaged zone. This number is lower than one if an area larger than the cross-sectional area of the projectile is uniformly impacted. Notice that we are determining an expression for fixed impact velocity, as dN should also depend on v9. An issue from our MD simulations is that the projectile changes velocity during impact. This change is observed in experiments as well: in one instance, the measured projectile kinetic energy decreased from 9 nJ to 4.5 nJ9. In order to mitigate this issue, we considered large projectiles with d = 140 Å.One possible way to define the specific penetration energy of an ρ ≈ 2200 kg. m−3) and our d1 and d2 simulation values for v = 900 m/s, we can derive σ1 and σ2 from Eq. 3η = 1.For instance, by using graphene density  simulations were carried out using the Reactive Force Field (ReaxFF)As the projectile, we used nickel nanoparticles of varying size. For the results presented in Fig. NVT ensembleWe minimized and thermalized the nickel nanoparticle for 200 ps at 300 K in the NPT ensemble. To reduce the initial stress, we set a null pressure at the edges of the structureWe minimized and thermalized the graphene sheet for 200 ps at 300 K in the NVT ensembleWe thermalized the graphene sheet for an additional 200 ps at 300 K in the We fixed the edges of the graphene unit cell, to prevent uniform translation of the sheet during impactNVE ensemble, with velocity v and angle θ. Different v and θ values were considered.We shot the projectile against the graphene sheet in the We employed the following procedure in our simulations:29.For steps 1 to 3 we used a timestep of 0.5 fs while in step 5 we used a timestep of 0.02 fs. Temperature and pressure were controlled through chains of three Nosé-Hoover thermostats and barostatsWe would like to stress that the used simulation setup was devised to mimic  the experimental conditions used to investigate the mechanical behavior of graphene under high strain-rate conditions. In the experiments, graphene is also suspended and its edges are glued to a sample holder.θ ≠ 0 or ϕ ≠ 0, in some instances the vertical deformation reached the fixed end. In those cases, boundary effects may have played a role in determining the final outcome. For ballistic tests with varied azimuthal angle (ϕ) of impact, we fixed the velocity (v = 1100 m/s) and the polar angle (θ = 30°) of impact. We considered ϕ values of 0°, 15°, 30°, 45°, and 60°.For collisions considering an angle Ekin,projectile) and potential  energies. The energy absorbed by graphene is minus their sum13: ΔEgraphene = −ΔEprojectile = −.In order to calculate the energy absorbed by graphene, we first determine the change in the projectile’s kinetic (ΔdN), this energy has to be divided by the graphene mass within the projectile cross section. This mass is equal to m = πR2NLρA, where R is the projectile radius, NL is the number of layers, and ρA = 0.77 mg/m2 is the area density of graphene. For v = 5000 m/s, we obtained from the manuscript that NL = 1 and NL = 2. After dividing these results by the mass, we get d1 = 40.8 MJ and d2 = 25.2 MJ. More 14.In that paper, the energy transferred to the graphene sheet during a ballistic test is et al.9, Fig. 4a of Yoon et al.13, and Fig. 9a of Xia et al.15, we used the web app WebPlotDigitizer30.In order to extract data from Fig. 4c of Lee Video 1Video 2Video 3Video 4Supplementary Information


Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has provided unprecedented results in cancer treatment in the recent years. Development of chemical inhibitors for this pathway lags the antibody development because of insufficient structural information. The first nonpeptidic chemical inhibitors that target the PD-1/PD-L1 interaction have only been recently disclosed by Bristol-Myers Squibb. Here, we show that these small-molecule compounds bind directly to PD-L1 and that they potently block PD-1 binding. Structural studies reveal a dimeric protein complex with a single small molecule which stabilizes the dimer thus occluding the PD-1 interaction surface of PD-L1s. The small-molecule interaction “hot spots” on PD-L1 surfaces suggest approaches for the PD-1/PD-L1 antagonist drug discovery. Moreover, in 2015, after demonstrating unprecedented results in clinical trial in the metastatic squamous NSCLC, nivolumab has gained FDA acceptance in this indication, becoming the first monotherapy in more than 15 years to demonstrate proven superior overall survival compared to the standard of care [http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm436566.htm]. Numerous further clinical trials are currently in progress . TOngoing trials evaluating the PD-1 and PD-L1 targeting antibodies in multiple indications in cancer portend further rapid development. Moreover, initial results demonstrate association between PD-L1 status and the response to the treatment . A reliaThis impressive clinical development of the antibodies that interfere with the PD-1/PD-L1 axis is in contrast to the development of small-molecule modulators for this interaction. Several low-molecular weight immunomodulators targeting the PD-1/PD-L1 signaling pathway were reported. These are based on peptidomimetics –15 and mClearly, progress of small molecule modulators of PD-1/PD-L1 pathway is lagging behind that of antibodies, which is partially related to insufficient structural information to guide rational design and development. The structure of the complex of the murine PD-1 and human PD-L1 revealed the overall binding mode for the PD-1/PD-L1 interaction , but relin vitro. We further show that they inhibit the PD-1/PD-L1 interaction by inducing PD-L1 dimerization through PD-1 interacting surface. We provide the first crystal structures of a small-molecule inhibitors bound to its target PD protein, in this case the PD-L1 dimer. Together with our biochemical data, the provided insight into this protein-inhibitor interaction and detailed definition of the binding site “hot spots” should facilitate more dynamic progress in the development of the PD-1/PD-L1 immune checkpoint modulators.Herein, we report characterization of the interaction of Bristol-Myers Squibb (BMS) compounds with the target protein. We show that they act by directly binding to PD-L1 and not PD-1 and effectively dissociate a preformed PD-1/PD-L1 complex 50 towards the PD-L1/PD-1 complex in the range of 18 to 200 nM, the lowest IC50 values in the series of about 200 compounds. The compounds that we have studied are 8, 37, 202 and 242 in the patent, and these are designated herein as BMS-8, BMS-37, BMS-202 and BMS-242, respectively methanol scaffold . In the 1H-15N HMQC spectra of 15N-labeled proteins upon interaction with tested ligands. The 15N labeled PD-L1 was titrated with increasing amount of tested compound while the 1H-15N signals were monitored using 2D HMQC NMR experiment. Significant shifts in the correlation NMR signals of PD-L1 upon addition of each tested compound documented their direct binding to PD-L1 .To unambiguously validate if these compounds bind to PD-1 or PD-L1, we used NMR, a method that monitors the direct binding of a small molecule to its target protein –22. We pto PD-L1 . No sign15N labeled PD-1 was first titrated with the non-labeled PD-L1 until no further changes in the linewidth of the 1H-15N resonance peaks was observed as monitored by HMQC  of 35.4°C (m=44.8°C), whereas BMS-202 by 13°C (Tm=48.4°C). PD-L2 was characterized by a comparable melting temperature as PD-L1 (38.2°C), however, neither BMS-8 nor BMS-202 significantly affected the Tm value   . DSF monf 35.4°C . BMS-8 sctively) . These rHaving confirmed the affinity of BMS-202 towards PD-L1 and its ability to dissociate the PD-1/PD-L1 interaction, we crystallized the compound in the complex with the target protein. The obtained crystals diffracted to the 2.2 Å resolution Table . No addiATyr56 on the opposite side . The 2-methylbiphenyl core of the inhibitor anchors at the very bottom of the pocket acetamide moiety of the inhibitor provides additional electrostatic interactions, but only with the A monomer (and corresponding C monomer for the second inhibitor molecule contained in the asymmetric unit). A water-mediated interaction with the backbone carbonyl of APhe19 is observed for both inhibitor molecules contained in the asymmetric unit, but further interactions of the distal parts of the acetamide group differ between the monomers. This is unrelated to crystal packing since no symmetry related molecules locate near the described moiety, but rather seems an effect of its flexibility  with that of CThr20. Overall, the inhibitor-protein interaction is best described as bimodal, spatially divided into hydrophobic and electrostatic parts following the inhibitor bimodal design.Both inhibitor molecules are well defined by the electron density allowing unambiguous positioning of all moieties . The inht Figure . The dis3 Figure . MoreoveThe crystal structure of PD-L1/BMS-202 complex suggests that BMS-202 induces dimerization of PD-L1. To assess if the observed dimerization is induced by crystallization conditions or rather is specific for this class of compounds we crystallized BMS-8 in complex with PD-L1 Table . The disTo further assess if BMS compounds induced dimerization of PD-L1 is an effect of tight packing within the crystal or rather constitutes the actual mechanism of action, we checked whether BMS-202 and BMS-8 induce the PD-L1 dimerization in solution. To this end PD-L1 was characterized by size exclusion chromatography in the presence and absence of tested compounds. Apo-PD-L1 yielded a single peak corresponding to a protein of molecular weight of 17 kDa, suggesting a monomeric state of the apoprotein in solution. In the presence of each compound the peak shifted to shorter retention time corresponding to a protein of around 34 kDa, consistent with a calculated molecular weight of a dimer . This in1H resonance linewidth by NMR. In all the cases, the well resolved narrow resonance peaks in the aliphatic region of 1H NMR spectrum of apo-PD-L1 exhibited significant broadening upon addition of each compound indicating significant increase in the molecular weight of the complex methanol. The capability to block the PD-1/PD-L1 complex formation was demonstrated in the HTRF assay in the original disclosure, but no further confirmatory data or structural information was provided to date. Among their most potent examples, BMS-8 and -202 inhibited the formation of the PD-1/PD-L1 complex with the ICin vitro BMS-8 and BMS-202 are capable of dissociating the PD-1/PD-L1 interaction at stoichiometric concentration, consistent with the estimated KD of the PD-1/PD-L1 complex of 8 μM pyridine-3-carbaldehyde, 3-bromo-4-methoxy)benzaldehyde and the final compounds were prepared according to procedures described in BMS patent  with minCoordinates and structure factors were deposited in the Protein Data Bank with accession numbers 5J89 (BMS-202) and 5J8O (BMS-8).


Programmed death-ligand 1 (PD-L1) plays an essential protein for immune evasion, contributing to tumor development and progression. Recent studies have reported MET as an upregulator for PD-L1 overexpression through an oncogenic pathway. However, an association between PD-L1 expression with MET has not been reported in gastric cancer.The prognostic significance of PD-L1 and its association with Epstein-Barr virus (EBV), microsatellite instability (MSI), and mucin phenotype remain controversial.in situ hybridization for EBV-encoded RNA and immunohistochemistry in tissue microarrays for 394 gastric cancers. A multiplex polymerase chain reaction with five quasimonomorphic markers was performed for MSI.We performed PD-L1 expression was observed in 123 cases (31.2%), and clinicopathological features such as MET overexpression, high pT stage, and a lack of lymphatic invasion represent significant risk factors associated with PD-L1 overexpression in gastric cancers. No associations of EBV, MSI, or mucin phenotype with PD-L1 expression were statistically significant. PD-L1 expression was a strong indicator for worse overall survival (OS) but borderline significant in disease-free survival (DFS). A combined analysis of PD-L1 and MET expression indicated that the PD-L1+/MET+ subgroup showed the worst prognosis when compared to the PD-L1-/MET- subgroup, which had the best clinical outcome. Furthermore, PD-L1 overexpression exhibited poor prognosis in terms of both OS and DFS in EBV-negative, microsatellite stable, and intestinal mucin phenotype tumors. In conclusion, this is the first study to evaluate the overexpression of MET as a risk factor for PD-L1 positivity in gastric cancer tissue as well as the reliability and prognostic relevance of PD-L1/MET co-expression after surgery. Gastric cancer is the fourth most common human malignancy and the second leading cause of global cancer mortality, and is notorious for a dismal prognosis with a five-year survival rate of less than 40% despite multimodality treatments . In the Immune evasion by cancer cells plays an important role in the development and progression of tumors . An immuIn the present study, we investigated the expression of PD-L1 and its prognostic relevance in gastric cancers, and explored the clinicopathological factors affecting PD-L1 overexpression. Further stratified survival analyses of PD-L1 expression with regards to MET expression, EBV, MSI status, and mucin phenotype were conducted.The male-to-female ratio was 2.3:1. The mean age of the patients with gastric cancer was 60 years . The patient cohort consisted predominantly of those with middle to distal stomach tumors (93.9%). Tumors were either located at the upper third , middle third , lower third , or the whole stomach .Seventy-seven tumors (19.5%) were well-differentiated tubular adenocarcinoma, 94 (23.9%) were moderately differentiated tubular adenocarcinoma, 160 (40.6%) were poorly differentiated tubular adenocarcinoma, 56 (14.2%) were signet ring cell carcinoma, and 7 (1.8%) were mucinous adenocarcinoma. Intestinal, diffuse, and mixed type gastric adenocarcinomas according to the Lauren classification system comprised 51.5%, 32.0%, and 16.5%, respectively, of the samples. Of the 394 gastric cancers, 232 (58.9%) were diagnosed as stage I, 55 (14.0%) as stage II, 94 (23.8%) as stage III, and 13 (3.3%) as stage IV. Out of these patients, postoperative adjuvant chemotherapy was administered in 21 (9.1%) with stage I, 35 (63.6%) with stage II, 64 (68.1%) with stage III, and 9 (69.2%) with stage IV. Tumors consisted of pT1 , pT2 , pT3 , and pT4 . The pN status were pN0 , pN1 , pN2 , and pN3 . The mean follow-up period was 94.1 ± 34.4 months. A total of 128 (32.5%) patients experienced a recurrence, of which 117 patients (91.4%) died and 11 patients (8.6%) are alive. One hundred twenty-five patients (31.7%) patients died of the disease, of which 8 patients (6.4%) did not have any recurrence.P < 0.001 and P = 0.020, respectively). EBV was positive in 26 of 394 samples (6.6%), and of these EBV-positive cases, 11 (42.3%) were PD-L1-positive. MSI-H was observed in 37 (9.4%) of the 394 gastric cancers, whereas microsatellite-stable (MSS) status was observed in 357 cases (90.6%). Of the 37 MSI-H cases, 9 (24.3%) were PD-L1-positive. There were no correlations between PD-L1 positivity and EBV or MSI-H .The clinical and pathological characteristics of the patients were analyzed according to their PD-L1 and MET status Table -1L, 49 (P = 0.042), whereas there were no associations with other mucin markers (data not shown). Taking into account of the observed combinations of these phenotypic markers, 394 gastric cancers were classified into 157 intestinal phenotypes (39.8%), 57 gastric phenotypes (14.5%), 124 mixed phenotypes (31.5%), and 56 unclassified mucin phenotypes (14.2%), where PD-L1 positivity was most commonly observed in 56 intestinal phenotypes (45.5%), followed by 40 mixed phenotypes (32.5%), 15 unclassified phenotypes (12.2%), and 12 gastric phenotypes (9.8%). This difference of PD-L1 expression among the mucin phenotype was marginally statistically significant (P = 0.067).The expression levels of CDX-2, MUC2, MUC5AC, and MUC6 were demonstrated in 280 (71.1%), 70 (82.2%), 181 (45.9%), and 63 (16.0%) of 394 cases, respectively. Among these markers, PD-L1 positivity was most frequently co-expressed with CDX-2 , followed by MUC5AC (42.3%), MUC6 (20.0%), and MUC2 (18.7%). The tendency for PD-L1 to be co-expressed with CDX-2 was statistically significant (P = 0.043).PD-L1 positivity was not significantly correlated with any clinical characteristics except for lymphatic invasion. However, PD-L1 positivity was inversely correlated with lymphatic invasion  = 1.032–4.723; P = 0.032, odds ratio = 0.502, 95% CI = 0.268–0.941; and P < 0.001, odds ratio = 2.688, 95% CI = 1.641–4.403; respectively).The clinicopathological factors affecting PD-L1 overexpression were investigated by multivariate analyses using a logistic regression model Table . The mulP = 0.003 and P = 0.009, respectively) , age (P = 0.004), tumor size (P < 0.001), unclassified mucin phenotype (P = 0.006), advanced stage (P < 0.001), lymphatic invasion (P < 0.001), vascular invasion (P = 0.003), perineural invasion (P < 0.001), and MSI-H (P = 0.033) significantly affected OS. On the other hand, age (P = 0.046), tumor size (P < 0.001), unclassified mucin phenotype (P = 0.024), advanced stage (P < 0.001), lymphatic invasion (P < 0.001), vascular invasion (P = 0.003), perineural invasion (P < 0.001), and MSI-H (P = 0.022) were associated with DFS. Those clinical and pathological statistically proven variables were consistent with the previously reported prognostic factors in gastric cancers [P = 0.688 and P = 0.467, respectively).The overexpression of PD-L1 had a prognostic impact on the overall survival (OS) and disease-free survival (DFS) rate in patients with gastric cancers Table . Patient) Figure . Clinica cancers -21. HoweP = 0.006, hazard ratio (HR) = 1.709, 95% CI = 1.169–2.499), while there was borderline statistical significance with DFS . In addition, older age, larger tumor size, and advanced stage were also found to be independent prognostic factors for worse OS, whereas large tumor size and advanced stage were independent prognostic factors associated with DFS in gastric cancers. However, EBV status, MSI status, and MET or HGF expression were not independent prognostic factors for OS or DFS.By multivariate analysis, PD-L1 overexpression was confirmed to be an independent negative prognostic factor affecting OS (P = 0.022), whereas the differences were not statistically significant when compared with the PDL1-/MET+  and PDL1+/MET- subgroups  , while the differences were not statistically significant compared with those in the PDL1-/MET+  and PDL1+/MET- subgroups  ; PDL1-/MET+ ; PDL1+/MET- ; and PDL1+/MET+ . A subgroup survival analysis according to the PD-L1 and MET expression status showed that co-expression of PD-L1 and MET was a significant prognostic factor of OS and DFS. Among the four subgroups, patients in the PDL1+/MET+ subgroup had a drastically worse prognosis in terms of both OS and DFS than those in the PDL1-/MET- subgroup. Patients in the PDL1+/MET+ subgroup showed shorter OS  than those in the PDL1-/MET- subgroup  Figure . Similar) Figure .P = 0.001) and DFS .The PD-L1/MET status and the abovementioned variables  that correlated significantly with OS or DFS on the univariate analyses were further analyzed by multivariate analyses. In the multivariate analyses, PDL1+/MET+ overexpression was a worse independent prognostic factor for OS . However, there were no significant differences in the OS or DFS for EBV-positive gastric cancers .We further analyzed the prognostic value of PD-L1 expression for OS and DFS according to EBV, MSI status, or mucin phenotype Figure . In EBV-P = 0.007 and P = 0.018, respectively; 101.1 ± 5.2 months vs. 114.7 ± 3.4 months for OS; 99.8± 5.4 months vs. 111.7 ± 3.6 months for DFS). There was also significant difference in the DFS in MSI-H gastric cancers (P = 0.041), while there was borderline statistical significance with OS (P = 0.057).For MSS gastric cancers, the OS and DFS were significantly worse in patients with PD-L1-positive tumors compared to those with PD-L1-negative tumors . In those with the mixed mucin type, PD-L1 positivity was associated only with decreased DFS (P = 0.045) but not with OS (P = 0.100). PD-L1 expression was not statistically associated with OS or DFS in other mucin phenotypes such as gastric type (P = 0.268 and P = 0.428) and unclassified type (P = 0.500 and P = 0.866).Furthermore, for the mucin phenotypes, PD-L1 positivity was correlated with shorter OS or DFS in the patients with gastric cancer showing the intestinal mucin phenotype . The paradoxical correlation of PD-L1 expression with a lack of lymphatic invasion but poor prognosis may suggest that PD-L1-positive tumors may be related to other mechanisms coupled with other oncogenic pathways leading to metastatic potential.PD-L1 positivity was observed in 31.2% gastric cancers, of which frequency was within the range (5.1%-65.0%) of previous studies in worldwide , 22, 23 PD-L1 expression has been reported to be driven by the MET/HGF oncogenic signaling pathway . MET andin vitro have shown that MET can modulate the survival of cancer cells through the regulation of PD-L1 [Furthermore, PD-L1 overexpression in tumor cells inhibits T cell-mediated anti-tumor immunity via PD-1 on tumor infiltrating lymphocytes, which protects tumor cells from cytotoxic lysis and ultimately influences patients’ clinical outcomes . The mecof PD-L1 , 14. Oneof PD-L1 , 36. Anoof PD-L1 . MET-indof PD-L1 . Taken tCDH1, RHOA, HER2, EGFR, VEGFR, MET, and FGFR2, all of which may directly or indirectly affect the MAPK and PI3K/Akt survival pathways [A recent phase Ib clinical trial (KEYNOTE-012) has shown that Pembrolizumab, an anti-PD-L1 monoclonal antibody, can result in an antitumor response rate as high as 22% in patients with PD-L1-positive advanced gastric cancer, thereby demonstrating the robust, durable responses of antibody therapy in patients with gastric cancer . That stpathways , 41. HowThe limitations of our study include the retrospective procurement of archival samples. Nevertheless, our results raise some interesting points. The PD-L1 expression had clear poor prognostic significances in patients with gastric cancer, indicating the potential of PD-L1 as a prognostic tissue marker. The subgroup analysis of PD-L1/MET co-expression could suggest the detailed prognostic prediction after surgery. This may help to guide the selection of treatments for high-risk patients in need of more intensive therapy and the possibility of anti-PD-L1 targeted therapy in this subset of gastric cancers.th edition of the American Joint Committee on Cancer (AJCC) cancer staging manual. This study was approved by the Institutional Review Board of Hallym University Sacred-Heart Hospital (2017-I037).Gastric cancer tissue samples were retrospectively collected from 394 patients (stages IB to IVa) who underwent gastrectomy with extensive node dissection (D2) consecutively from 2003 to 2011 at Hallym University Sacred-Heart Hospital, Korea. Only those patients who were diagnosed with primary gastric cancers and that were not treated with chemotherapy and targeted drug therapy at the time of the surgery, and whose formalin-fixed, paraffin-embedded (FFPE) tumor tissue blocks were available for analysis were included in this study. Medical records of each patient were reviewed for demographic information, radiological data, treatment details, tumor recurrence, or survival status. All of the slides stained with hematoxylin and eosin were reviewed by two pathologists (MJK and JYC) for diagnostic confirmation, the re-evaluation for histopathological characteristics , and the selection of a representative section for subsequent immunohistochemical and molecular studies. The diagnostic criteria and tumor differentiation metrics used were those indicated by the World Health Organization classification of tumors of the digestive system. Tumor stage was defined according to the TNM classification of malignant tumors as described in the 7Tissue microarrays were constructed for each FFPE block as described previously . After rPD-L1 was evaluated based on the intensity and proportion of membranous staining and/or cytoplasmic staining in tumor cells. PD-L1 was scored as 0 (no staining or any staining less than 10% of cells), 1+ (weak staining in more than 10% of the tumor cells), 2+ (moderate staining in more than 10% of the tumor cells), or 3+ (strong staining in more than 10% of the tumor cells) , 28 Fig. Cases wStaining score of MET and HGF expression using modified criteria used in clinical trials involving the MET inhibitor was defined as the following: 0, absence of staining or staining intensity in <50% tumor cells; 1+, weak-to-moderate staining intensity in >50% tumor cells; 2+, moderate to strong intensity staining in >50% of tumor cells; 3+, strong staining intensity in >50% tumor cells based on membranous and/or cytoplasmic staining Figure  for HGF CDX-2 expression was regarded as positive if nuclear staining was observed in ≥5% of the tumor specimens; otherwise, it was classified as negative according to previous reports , 45. ForIn situ hybridization for EBV-encoded RNA was also performed on the BenchMark XT autostainer using fluorescein-conjugated oligonucleotide probes . In brief, 3-μm-thick sections from the tissue microarray blocks were deparaffinized and rehydrated, and then the sections were digested with a proteolytic enzyme (proteinase K at 37°C for 25 min). Thereafter, the slides were incubated with the probe at 55°C for 25 min and washed with a stringent solution. The slides were labeled with an anti-alkaline phosphatase-conjugated antibody to fluorescein. A chromogen (5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium) was then added and counterstained with Mayer’s hematoxylin. Only cases with a strong signal within almost all tumor cell nuclei were considered positive , as previously described , 26. In The MSI evaluation was performed using multiplex PCR comprising five quasimonomorphic mononucleotide repeat markers  . Each seP <0.05) in univariate analysis were included in subsequent multivariate logistic regression analysis to identify independent variables associated with PD-L1 and MET expression. Survival analyses were performed using the Kaplan-Meier method and were compared using a log-rank test. OS was defined as the interval from the first day of surgery until death or the end of the follow-up period. DFS was defined as the interval from the first day of surgery until tumor progression, death, or end of follow-up period. OS and DFS were analyzed until February 2015. Univariate and multivariate analyses using the Cox proportional hazard regression model were applied to determine the HR and 95% CI for specific variables with respect to OS and DFS. SPSS version 18  was used for all statistical analyses. A P value <0.05 was considered statistically significant.Categorical variables were compared using Pearson’s chi-squared test or Fisher’s exact test, and continuous variables, which are presented as means ± SD, using the t-test. Factors found to be significant (


Ericaceae are common in arctic and alpine regions. Many of these plants are associated with ericoid mycorrhizal (ERM) fungi, which allow them to take nutrients and water from the soil under harsh environmental conditions and, thus, affect host plant survival. Despite the importance of ERM fungi to alpine plant communities, limited information is available on the effects of microhabitat and host identity on ERM fungal communities. We investigated the communities of putative ERM fungi isolated from five dwarf shrub species  that co-occur in an alpine region of Japan, with reference to distinct microhabitats provided by large stone pine (Pinus pumila) shrubs . We obtained 703 fungal isolates from 222 individual plants. These isolates were classified into 55 operational taxonomic units (OTUs) based on the sequencing of internal transcribed spacer regions in ribosomal DNA. These putative ERM fungal communities were dominated by Helotiales fungi for all host species. Cistella and Trimmatostroma species, which have rarely been detected in ERM roots in previous studies, were abundant. ERM fungal communities were significantly different among microhabitats (R2=0.28), while the host effect explained less variance in the fungal communities after excluding the microhabitat effect (R2=0.17). Our results suggest that the host effect on ERM fungal communities is minor and the distributions of hosts and fungal communities may be assessed based on microhabitat conditions.Dwarf shrubs of the family  Ericaceae and Diapensiaceae (e.g. Schizocodon and Diapensia)  is a symbiotic association between fungi and the roots of plants in the families apensia) , 53, 57.apensia) , 39, 59.apensia) . ERM hosapensia) , 34, 52. regions , 47, 59.Helotiales is the most dominant group (Rhizoscyphus ericae (=Hymenoscyphus ericae) and Oidiodendron species have frequently been isolated from ERM roots and have been confirmed as ERM-forming mycobionts in inoculation experiments  have recently been identified and sequenced  shrubs and ericaceous dwarf shrubs. Strong winds lead to desiccation in these habitats, which makes plant establishment difficult, leaving large areas of bare ground. However, once Japanese stone pine or Ericaceae plants are established, their shrubs alleviate the harsh environmental conditions locally and facilitate further seedling establishment, as reported by Perkins  co-existing in an alpine ecosystem. The hypotheses examined in this study are as follows: putative ERM fungal communities are affected more by microhabitats than by hosts, and host effects on putative ERM fungal communities become evident after excluding microhabitat effects.In the present study, we investigated putative ERM fungal communities with reference to microhabitat and host identity using five dwarf shrub species (Three study plots (0.5 ha each) were established on the exposed mountain ridges of Mt. Norikura, Kikyougahara , Daikokudake , and Fujimidake .Ericaceae species, i.e. A. nana, D. lapponica, E. nigrum, L. procumbens, and V. vitis-idaea, were found in all study plots and were used in this study. ERM formation in these plant species has already been reported  in January and the highest (12.0°C) in August, and mean annual precipitation is 2,738 mm, according to observations by the Japan Meteorological Agency . The site is typically covered by snow packs until late June . Five reported , 53, 57.2) were collected from four microhabitat categories: pine shrub (the center of P. pumila shrubs under dense shade from pine leaves), the east edge , the west edge (the western and windward edge of P. pumila shrubs), and open (the bare ground away from P. pumila shrubs). In the pine shrub habitat, we only collected V. vitis-idaea because no other Ericaceae plants were found. All five plant species were collected in the east edge, west edge, and open habitats. Individual plants were excavated with the surrounding soil, placed separately in plastic bags, and sealed and stored at 4°C during transport to the laboratory. A total of 26, 72, 76, and 48 individuals were collected in the pine shrub, east edge, west edge, and open habitats, respectively. Of the 222 plants, 60, 12, 48, 22, and 80 were A. nana, D. lapponica, E. nigrum, L. procumbens, and V. vitis-idaea, respectively. The number of plant samples collected for each species/microhabitat combination is listed in In early August 2014, mature plant samples  based on ≥97% similarity  using ATet al.  to test for significant differences in fungal communities among habitat categories and among hosts. Singletons and doubletons were excluded from community data matrices. These statistical analyses were performed using R with 9,999 permutations, applying the Bray–Curtis distance as a community dissimilarity index.The occurrence of an OTU was quantified as the number of individual plants isolated from that OTU. The biased occurrence of OTUs was tested using a weighted chi-squared test, in which the number of individuals collected was used as the expected occurrence. Chao2 values, which is a species richness estimator based on the occurrence of rare taxa, were calculated for each host and microhabitat category with sample-based rarefaction analyses using Estimate S var. 8.20  with 1,0The identified sequences were deposited at DDBJ under the accession numbers KY522913–KY522967.Capnodiales and one to Chaetothyriales. All remaining fungi belonged to the order Helotiales, apart from two OTUs, which were assigned only at the class level. Phialocephala fortinii sp. 1 had the most frequent occurrence and was found in 54 plants, followed by Cistella sp., found in 47 . NMDS ordination indicated that putative ERM fungal communities were clearly separated based on microhabitat , which was found to be significant in an Adonis test   in pineshrub, east edge, west edge, and open habitats were 20 (37.3), 29 (39.9), 37 (74.4), and 16 (25.1), respectively. No significant differences were found in putative ERM fungal communities between the east edge and west edge habitats  .A. nana, D. lapponica, E. nigrum, L. procumbens, and V. vitis-idaea were 27 (42.3), 4 (4.0), 21 (41.5), 9 (10.9), and 42 (67.9), respectively. Each of the hosts was uniquely associated with 6, 1, 5, 2, and 15 OTUs, respectively  were found in all hosts. Differences in occurrence between hosts were found in seven OTUs   on ectively . Two OTUa sp. 1) .p<0.001). However, the host effect was confounded by that of microhabitat because some hosts preferred a certain microhabitat. Nevertheless, even for an Adonis test with the effects of microhabitats excluded, the host effect was significant (p=0.001), but explained less variance in the community data (R2=0.17). When only edge habitat data were used, the fungal communities of E. nigrum were distinct from those of A. nana and V. vitis-idaea (p<0.001). The fungal communities of A. nana and V. vitis-idaea were tightly clustered in the NMDS plot (p=0.800). When fungal communities were compared among hosts in just the open habitat samples, the communities in D. lapponica were distinct from those in other hosts , while no significant difference was found among A. nana, E. nigrum, and L. procumbens.Overall, the host effect was significant in an Adonis test that did not exclude the effect of microhabitat , supporting our first hypothesis that habitat has an effect on putative ERM fungal communities. Ishida and Nordin , but was confounded by the effect of microhabitats because some host species showed a habitat preference. For example, V. vitis-idaea was the only species that was found under pine shrubs, while D. lapponica was mostly located in open habitats. Previous findings on potential host effects on ERM communities have been inconsistent, being significantly different in Bougoure et al.  and Cistella species in ERM roots. Capnodialean species have been reported to be the root-associated fungi of arctic plants, including ERM hosts, but at low frequencies (Cistella species are typically identified as soil fungi (Another interesting result is the dominance of quencies , 59. Capquencies , 21 and quencies , 21, 28.il fungi  and leafil fungi . AlthougAlpine dwarf shrubs of ericaceous plants under similar environmental conditions shared the majority of putative ERM fungi. This result indicates that most hosts are associated with generalist ERM fungi, which may, in turn, facilitate seedling establishment in a broad range of hosts and allow these hosts to co-exist. This facilitation is often reported in ectomycorrhizal systems, but not in ERM associations , 38, 41.


LOXL1/LOXL2 gene expression and protein levels were increased in IPF versus non-IPF. Increased collagen fibril thickness in IPF versus non-IPF lung tissues correlated with increased LOXL1/LOXL2, and decreased LOX, protein expression. β-Aminoproprionitrile  but not Compound A (LOXL2-specific inhibitor) interfered with transforming growth factor-β-induced collagen remodelling in both models. The β-APN treatment group was tested further, and β-APN was found to interfere with stiffening in the decellularized matrix model. LOXL1 activity might drive collagen remodelling in IPF lungs. The interrelationship between collagen structural remodelling and LOs is disrupted in IPF lungs. Inhibition of LO activity alleviates fibrosis by limiting fibrillar collagen cross-linking, thereby potentially impeding the formation of a pathological microenvironment in IPF.Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with few effective therapeutic options. Structural remodelling of the extracellular matrix [i.e. collagen cross-linking mediated by the lysyl oxidase (LO) family of enzymes ] might contribute to disease pathogenesis and represent a therapeutic target. This study aimed to further our understanding of the mechanisms by which LO inhibitors might improve lung fibrosis. Lung tissues from IPF and non-IPF subjects were examined for collagen structure (second harmonic generation imaging) and LO gene  and protein (immunohistochemistry and western blotting) levels. Functional effects (collagen structure and tissue stiffness using atomic force microscopy) of LO inhibitors on collagen remodelling were examined in two models, collagen hydrogels and decellularized human lung matrices.  Summary: Transforming growth factor-β-induced collagen remodelling is driven by enhanced lysyl oxidase expression in idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is a specific form of progressive fibrosing lung disease of unknown cause that leads to chronic respiratory failure and death , with a Like many fibrotic disorders, IPF is characterized by enhanced deposition and remodelling of the extracellular matrix (ECM). Although data are emerging about the altered composition of the ECM in IPF , less isin vitro, ex vivo and in vivo (Fibrillar collagen structures (predominantly collagens I and III) can be visualized and quantified using second harmonic generation (SHG) microscopy . SHG ima in vivo  and can  in vivo . This te in vivo  and even in vivo . We have in vivo .Maturity of collagen fibres and thus the mechanical properties of the fibres are dependent on the level of intermolecular cross-linking. Although the intermolecular fibrillar organization and certain cross-linking processes can happen spontaneously, enzymes such as lysyl oxidases (LOs) are necessary for further cross-linking. The LO family of enzymes consists of five known paralogues: lysyl oxidase (LOX) and LOX like 1-4 (LOXL1-4). During normal development, the expression of LOs is tightly regulated, with aberrant expression and activity being associated with pathological manifestation in various diseases, including cancer  and, as ClinicalTrials.gov NCT01769196 .To investigate the collagen structural remodelling in IPF, SHG microscopy was performed on lung tissue sections from non-IPF and IPF subjects. The SHG forward (F) signal is predominantly from mature/organized fibrillar collagen, whereas the backward (B) signal is from immature/disorganized fibrillar collagen. Thus, increased F/B area corresponds to increased amounts of mature/immature collagen, whereas increased F/B intensity ratios correspond to an increased degree of collagen maturity/thickness and vice versa . QuantifLOXL1 gene expression was increased in IPF lung tissue compared with healthy controls in two independent studies [false discovery rate (FDR) adjusted P-value <0.25], whereas the LOXL2 gene expression was increased in IPF lung tissue in a single data set. No differences were found in LOX gene expression levels.Fibrillar collagen maturity is catalysed by the LO family of enzymes. To investigate the levels of LO enzymes in lung tissues, lung tissue gene expression profiles of IPF and healthy controls were analysed from two publically available data sets . LOXL1 gTable S1). In vitro, primary human lung parenchymal fibroblasts from IPF patients produced significantly greater levels of LOXL2 but not LOX or LOXL1 in the cell lysate compared with non-IPF controls .To confirm the findings from the gene expression study, tissue sections from the same samples used for the examination of collagen structural organization were examined for LOX, LOXL1 and LOXL2 protein levels. Quantification of the captured images of the stained lung tissue sections showed that LOX percentage surface area and density were decreased in IPF compared with non-IPF, whereas the percentage surface area and density of LOXL1 was increased in IPF compared with non-IPF tissues A. In agrFig. S3) and LOXL2  densities and LOX/LOXL1 percentage surface areas were significantly decreased in IPF lung tissue samples compared with non-IPF subjects . The ratio of LOX/LOXL1 percentage surface area inversely correlated with collagen maturity/organization .The ratio of LOX/LOXL1 B and LOXion F/B; C. There Table S2 shows the strength of the contribution of the variables  to the two relationship groups. The strengths of contributions of the variables were used to determine the weighted average values for each relationship group . The pan-inhibitor of LOs, β-APN, returned TGF-β-induced collagen remodelling to baseline. In addition, the LOXL2-specific inhibitor blocked TGF-β-induced remodelling of fibrillar collagen; remodelling levels in the presence of either inhibitor without TGF-β were not different compared with control  was measured by atomic force microscopy . TGF-β induced a significant increase in tissue stiffness that was inhibited by the pan-inhibitor of LOs, β-APN . IndividLOXL1 and LOXL2 gene and protein levels are increased in the lungs of IPF compared with non-IPF subjects, with a positive correlation between increased protein expression and increased fibrillar collagen organization.Although increased collagen deposition is well appreciated in fibrotic lung tissues, this study identifies, for the first time, alterations in the fibrillar organization of the collagen fibres in IPF lung tissue. In addition, this study reports that ClinicalTrials.gov NCT01769196) was halted early because of a lack of efficacy  compared with non-diseased subjects. It can be postulated that the increased collagen fibre maturity/thickness contributes to the increased stiffness characteristic of IPF lungs . This isLOXL1 gene and protein expression in IPF lungs, compared with non-IPF tissues, was observed, with a positive correlation of protein levels with collagen organization. By contrast, LOX gene expression was unchanged, whereas the LOX protein level was decreased in IPF lung, highlighting differential LO family expression in IPF lung tissue, which is similar to that reported during tumour progression and metastasis  culture express a 50 kDa proteolytically processed form of LOXL2 , rather Fig. S7), but the present experiment was not designed to address this hypothesis.It is worth noting that the stiffness measured in the reseeded decellularized matrices (mean 40-60 kPa) was higher than the values measured in native human lung parenchymal tissue . After formaldehyde fixation, isolated lung tissues were embedded in paraffin.Tissue isolation was performed on fresh explanted lung tissues. Human lung parenchymal tissues were isolated and cut into pieces no bigger than 1 cmPrimary human lung fibroblasts were isolated as previously described  and were2 incubator (5% CO2 in air) at 37°C. Growth media were aspirated from cells every 4 days and replaced with fresh growth media until confluence.Primary human lung fibroblasts were grown in growth media [DMEM low glucose (Gibco) with 0.025 M HEPES (Gibco) and 0.375% sodium hydrogen carbonate ] incubated in a humidified CO3VO4; Sigma), 1% (v/v) Triton X-100 (Sigma), 10% (v/v) glycerol (Sigma), 0.1% (w/v) SDS (Amresco), 0.5% (w/v) sodium deoxycholate (Sigma), 1 mM PMSF , 1% (v/v) Protease Inhibitor Cocktail Set III ] on ice, scraped into a tube, and centrifuged at 150×g for 5 min. The supernatants were collected and stored at −80°C for further experiments. Total sample protein was measured using a standard BCA assay (Sigma).Whole-cell lysates were prepared using protein extraction buffer  containing resuspended cells was then added at higher cell concentration for dilution in the collagen gel solution . Collagen gel mixed with cells  was pipetted into a 48-well plate and allowed to set in a humidified CO2 incubator at 37°C for 2 h. Once the gel had set, the gels were then released from the wells by gently running a sterile syringe needle around between the gel and the wall of the wells. Treatment media  of the same volume as the gel were pipetted gently on top of the gels in the wells. The collagen gels were collected at day 7, fixed in 4% paraformaldehyde solution and paraffin embedded.Collagen gel solution was prepared for a final collagen gel concentration of 2 mg mlDecellularized tissues from a non-diseased subject and an IPF subject were prepared as previously described . Essenti5 fibroblasts ml−1 in growth media at a ratio of 1 matrix:2 ml of cell solution  up to a maximum of 25 ml of solution in a 50 ml Falcon tube. The Falcon tubes containing the matrices and cell suspension were mounted on a suspension mixer  and constantly rotated at low speed (approximately one rotation every 20 s) at 37°C for 24 h. The reseeded ECMs were placed in separate wells of a 48-well plate with fresh media [0.01% DMSO +0.1% BSA ±10 ng ml−1 TGF-β  ±300 nM Compound A or 0.1 mM β-APN (Sigma)] and incubated at 37°C and 5% CO2. The reseeded decellularized matrices were collected at day 7; half were fixed in 4% paraformaldehyde solution for 24 h and paraffin embedded, whereas the other half were fresh frozen in optimal cutting temperature (OCT) compound and stored at −80°C.Decellularized lung slices from both non-diseased and IPF subjects were washed three times in sterile DMEM solution and were subsequently added to a suspension of 5.0×10AFM measurements were performed on decellularized human lung matrices with or without reseeded non-IPF cells. Matrices were embedded in optimal OCT compound, and 10-µm-thick tissue slices were cryosectioned at −21°C and mounted on poly-L-lysine-coated glass slides. PBS solution was added on the tissue slice to avoid drying.−1 by the thermal fluctuation method .Parenchymal tissue areas were identified under an optical microscope  and mechanically analysed with a BioScope Catalyst AFM . Microindentations were performed using a 2.5-µm-radius sphere-tipped probe  with a spring constant determined at ∼100 pN nmn method . For eacE, was determined by fitting of the force curve by the Hertz sphere model  or the Mann–Whitney U-test  as appropriate. Kruskal–Wallis and two-way ANOVA statistical analyses were performed where appropriate. Factor analysis was performed using SPSS software  using ‘principal axis factoring’ analysing ‘correlation matrix’ and extracting ‘2’ factors from the data set. Promax rotation was used for the factor analysis, using a regression method and excluding cases listwise where there were missing data points.Data were entered and sorted using Microsoft Excel . Sample sizes for comparing IPF versus non-IPF were determined based on a previous study . Statist


A 4 × 5 design was utilized, with four composites , and five water-ageing time periods . After each water-ageing period, the mechanical properties of the resins were measured in a three-point flexure, and antibacterial properties were tested via a dental plaque biofilm model using human saliva as an inoculum. After 12 months of water-ageing, NACP-QADM-NAg had a flexural strength and elastic modulus matching those of the commercial control (P>0.1). Incorporation of QADM or NAg into the NACP composite greatly reduced biofilm viability, metabolic activity and acid production. A composite containing both QADM and NAg possessed a stronger antibacterial capability than one with QADM or NAg alone (P<0.05). The anti-biofilm activity was maintained after 12 months of water-ageing and showed no significant decrease with increasing time (P>0.1). In conclusion, the NACP-QADM-NAg composite decreased biofilm viability and lactic acid production, while matching the load-bearing capability of a commercial composite. There was no decrease in its antibacterial properties after 1 year of water-ageing. The durable antibacterial and mechanical properties indicate that NACP-QADM-NAg composites may be useful in dental restorations to combat caries.Dental composites are commonly used restorative materials; however, secondary caries due to biofilm acids remains a major problem. The objectives of this study were (1) to develop a composite containing quaternary ammonium dimethacrylate (QADM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP), and (2) to conduct the first investigation of the mechanical properties, biofilm response and acid production  This approach enables a high antibacterial activity at a low Ag filler level, without compromising the composite colour and mechanical properties.33Dental caries is a prevalent worldwide problem,38 CaP biomaterials are important because of their excellent biocompatibility, bioactivity, and similarity to minerals in hard tissues.42 Composites containing nanoparticles of amorphous calcium phosphate (NACP) release calcium (Ca) and phosphate (P) ions similar to traditional CaP composites, but possess much better mechanical properties.43 NACP composites are “smart” and greatly increase the release of Ca and P ions at an acidic pH, at which these ions would be most needed to inhibit caries.42 In addition, NACP composites have achieved enamel lesion remineralization rates fourfold higher than that of a fluoride-releasing commercial dental composite.38Another approach to combat caries has been the development of calcium phosphate (CaP)-containing resins.43 However, long-term experiments are still needed to investigate the antibacterial durability of the dental resins. A previous study has shown that a composite containing MDPB has a strong antibacterial effect after 3 months of immersion in water.19 In another study, the antibacterial activity of a composite containing PEI nanoparticles has been found to be maintained after 1 month of water-ageing.25 In addition, the antimicrobial activity of a QAM-containing acrylic resin has been shown to be maintained after 3 months of water-ageing.44 However, whether the antibacterial effects decrease after 6 months is unclear, and longer term studies  have not been reported for antibacterial dental resins.To further enhance the anti-caries efficacy, the antibacterial approach and the CaP approach have been combined in the same resin, so that the antibacterial agent can reduce biofilms while the Ca and P ions remineralize lesions and inhibit caries.Accordingly, the objectives of this study were to develop a bioactive composite containing QADM, NAg, and NACP for the inhibition of caries, and to conduct the first investigation of the mechanical properties, oral biofilm response and lactic acid production as a function of water-ageing time from 1 day to 12 months. We hypothesized that: (1) an NACP composite containing QADM and NAg would have much less biofilm viability and acid production than a commercial composite control; (2) the antibacterial activity of the NACP-QADM-NAg composite would show no significant decrease with increasing water-ageing time from 1 day to 1 year; and (3) the NACP-QADM-NAg composite would possess similar mechanical properties to those of a commercial control composite after a long-term water-ageing treatment.3[PO4]2) were synthesized via a spray-drying technique, as described in a previous study.42 Briefly, calcium carbonate  and anhydrous dicalcium phosphate  were dissolved in acetic acid to obtain Ca and P ion concentrations of 8 mmol·L−1 and 5.333 mmol·L−1; respectively, yielding a Ca/P molar ratio of 1.5. This solution was sprayed into the heated chamber of a spray-drying apparatus, and an electrostatic precipitator was used to collect the dried particles. This process produced NACP with a mean particle size of ~116 nm.42 NACP consisted of individual particles as well as some clusters.43 The clusters contained numerous small particles that were probably stuck together in the spray-drying chamber before the particles were completely dried. Whereas the individual particles had sizes on the order of 10 nm, the clusters had sizes of ~100–300 nm.43 Measurement of 100 random particles in a previous study has shown an average size of 37 nm for the individual particles, and an average size of 225 nm for the clusters.43NACP  and triethylene glycol dimethacrylate (TEGDMA) at 1:1 mass ratio was rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4-32 The bis(2-methacryloyloxyethyl) dimethylammonium bromide was a dimethacrylate, which has been identified as QADM in previous studies.32 Briefly, 10 mmol of 2-ethyl methacrylate  and 10 mmol of 2-bromoethyl methacrylate  were combined with 3 g of ethanol in a closed vial. After stirring at 60 °C for 24 h for the reaction to complete, the solvent was removed via evaporation under vacuum. This process yielded QADM as a clear and viscous liquid. QADM was mixed with the BisGMA-TEGDMA resin at a QADM mass fraction of 20%. A preliminary study has shown that this mass fraction yields strong antibacterial properties without compromising the resin's mechanical properties. This resin is herein referred to as the BisGMA-TEGDMA-QADM resin.The synthesis of antibacterial bis(2-methacryloyloxyethyl) dimethylammonium bromide was as previously described.45 Briefly, 0.1 g of silver 2-ethylhexanoate (0.1 g) was dissolved into 0.9 g of 2-(tert-butylamino) ethyl methacrylate  by gentle stirring. Then, 1% mass fraction of this solution was added to the two resins: the BisGMA-TEGDMA resin and the BisGMA-TEGDMA-QADM resin. This process yielded a 0.1% mass fraction of the Ag salt in each resin. TBAEMA was used because it improved the solubility by forming Ag-N coordination bonds with Ag ions, thereby facilitating Ag salt dissolution in the resin solution. In addition, TBAEMA contains reactive methacrylate groups and can be chemically incorporated into the polymer network upon photo-polymerization.45 The 0.1% mass fraction of silver 2-ethylhexanoate in the resin was used in a preliminary study that showed a strong antibacterial activity, without a noticeable colour change to the resin or negative effects on its mechanical properties.NAg were formed in a dental resin as described previously.n-propylamine.42 Each resin contained a filler mass fraction of 70%, with 20% of NACP and 50% of glass, which were mixed to yield a cohesive composite paste. Because the resin matrix mass fraction was 30%, the QADM mass fraction in the final composite was 6%.To produce NACP composites, three resins were used: (1) the BisGMA-TEGDMA-QADM resin, (2) the BisGMA-TEGDMA-NAg resin, and (3) the BisGMA-TEGDMA-QADM-NAg resin. Each resin was filled with the NACP and with glass particles for reinforcement , which were silanised with 4% 3-methacryloxypropyltrimethoxysilane and 2% Composite containing remineralizing NACP and antibacterial QADM (referred to as NACP+QADM);Composite containing NACP and antibacterial NAg (referred to as NACP+NAg);Composite containing NACP, QADM, and NAg (NACP+QADM+NAg).Therefore, after filling with NACP and glass particles, these three resins resulted in three composites:In addition, a commercial composite served as a comparative control . This resin consisted of fillers of 40 nm to 0.2 micron particle sizes at 60% filler level by volume in a multifunctional acrylic resin of diurethane dimethacrylate and butanediol dimethacrylate. According to the manufacturer, Renamel is indicated for use in Class III, IV, and V restorations.For mechanical testing, each composite paste was placed into rectangular moulds of 2mm × 2mm × 25 mm. For biofilm testing, disk moulds were used with a diameter of 9 mm and a thickness of 2 mm. All specimens were photo-polymerized  for 1 min on each open side of the mould.The specimens were immersed in distilled water at 37 °C for 1 day, 3 months, 6 months, 9 months, or 12 months. This constituted a 4 × 5 full factorial design, with four composites and five water-ageing time periods.−1.42 Flexural strength (S) was calculated as:The water-aged rectangular specimens were fractured using a computer-controlled Universal Testing Machine  in three-point flexure with a span of 10 mm and a crosshead speed of 1 mm·minS=3PmaxL/(2bh2)Pmax represents the load, L represents the span, b represents the specimen width and h represents the thickness. The elastic modulus (E) was calculated as:where E=(P/d)(L3/[4bh3])P divided by displacement d is the slope of the load-displacement curve in the linear elastic region. Six specimens were tested for each composite at each water-ageing time period. The specimens were tested within a few minutes after being taken out of the water, and the specimens were wet while being fractured.where load in vivo plaques.46 Saliva was collected from 10 healthy adult donors possessing natural dentition without active caries or periopathology and who had not used antibiotics within the previous 3 months.48 The donors did not brush their teeth for 24 h and stopped any food/drink intake for at least 2 h before donating saliva. Stimulated saliva was collected during parafilm chewing and kept on ice. An equal volume of saliva from each of the 10 donors was combined to form the saliva sample. The saliva was diluted in sterile glycerol to a saliva concentration of 70%. Aliquots (1 mL) of diluted saliva were stored at −80 °C for subsequent use.48The dental plaque microcosm biofilm model was approved by the University of Maryland Baltimore Institutional Review Board. This model has the advantage of maintaining much of the complexity and heterogeneity of 49 The McBain medium contained mucin  at a concentration of 2.5 g·L−1; bacteriological peptone, 2.0 g·L−1; tryptone, 2.0 g·L−1; yeast extract, 1.0 g·L−1; NaCl, 0.35 g·L−1; KCl, 0.2 g·L−1; CaCl2, 0.2 g·L−1; cysteine hydrochloride, 0.1 g·L−1; haemin, 0.001 g·L−1; and vitamin K1, 0.000 2 g·L−1, at pH 7.49 Each disk was placed into a well of a 24-well plate. Each well was filled with 1.5 mL of the inoculum, and incubated under 5% CO2 at 37 ºC. After 8 h, each disk was transferred to a new 24-well plate with 1.5 mL of fresh medium and incubated under 5% CO2 at 37 °C for 16 h. Then, each disk was transferred to a new 24-well plate with 1.5 mL of fresh medium and incubated for 24 h. This totalled 2 days of incubation, which has been shown to be sufficient to form relatively mature biofilms on dental resins.33Composite disks after water-ageing were sterilized with an ethylene oxide sterilizer  and then degassed, following the manufacturer's instructions. The saliva-glycerol stock was added, using 1:50 final dilution, to a McBain artificial saliva growth medium as the inoculum.48 Imaging was performed via a confocal laser scanning microscope . Four randomly chosen fields of view were photographed for each disk, with six disks per material at each time period, yielding 24 images for each composite at each water-ageing time period.The composite disks with 2-day biofilms were rinsed with phosphate-buffered saline (PBS) and live/dead stained using the BacLight live/dead bacterial viability kit .28 MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to purple formazan. Disks with 2-day biofilms (n=6) were transferred to a new 24-well plate, and 1 mL of MTT dye (0.5 mg·mL−1 MTT in PBS) was added to each well and incubated at 37 °C under 5% CO2 for 1 h. During the incubation, metabolically active bacteria metabolized the MTT to formazan inside the living cells. Disks were then transferred to new 24-well plates, and 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. The plates were incubated for 20 min with gentle mixing at room temperature. Two hundred microlitres of the DMSO solution from each well was collected, and the absorbance at 540 nm was measured via a microplate reader . A higher absorbance indicates a higher formazan concentration, which is related to more metabolic activity in the biofilm on the composite surface.33A 3--2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the metabolic activity of the biofilms.n=6) were rinsed with cysteine peptone water (CPW) to remove loose bacteria, and then transferred to 24-well plates containing 1.5 mL of buffered-peptone water (BPW) plus 0.2% sucrose. The specimens were incubated for 3 h to allow the biofilms to produce acid. Then, the BPW solutions were collected for lactate analysis using an enzymatic method.33 The 340-nm absorbance of BPW was measured with a microplate reader . Standard curves were prepared using a standard lactic acid  as described in previous studies.33Disks with 2-day biofilms , by following the methods used in previous studies.33 Three types of agar plates were prepared. First, tryptic soy blood agar culture plates were used to determine the total number of microorganisms.33 Second, mitis salivarius agar (MSA) culture plates, containing 15% sucrose, were used to determine the total number of streptococci.50 This count was feasible because MSA contains the selective agents crystal violet, potassium tellurite, and trypan blue, which inhibit most Gram-negative bacilli and most Gram-positive bacteria except streptococci, thus enabling streptococci to grow.50 Third, cariogenic mutans streptococci are known to be resistant to bacitracin, and this property is often used to isolate mutans streptococci from the highly heterogeneous oral microflora. Hence, MSA agar culture plates plus 0.2 units of bacitracin per mL were used to determine the number of mutant streptococci.51 The bacterial suspensions were serially diluted and spread onto agar plates for colony-forming unit (CFU) analysis by following the methods used in previous studies.Disks with 2-day biofilms were transferred into tubes with 2 mL CPW, and the biofilms were harvested by sonication and vortexing P-value of 0.05.Statistical analyses were performed using Statistical Package for the Social Sciences . Two-way analyses-of-variance (ANOVA) were performed to detect the significant effects of the variables. Tukey's multiple comparison was used to compare the data at a n=6). For all four composites, there was a moderate decrease in strength and modulus from 1 day to 3 months of water-ageing (P<0.05). There was little subsequent decrease from 3 to 12 months (P>0.1). At each water-ageing time period, the flexural strength and elastic modulus of NACP-QADM, NACP-NAg, and NACP-QADM-NAg composites were similar to those of the commercial control composite (P>0.1).The flexural strength and elastic modulus results are plotted in Representative live/dead images of 2-day biofilms on the composites are shown in P<0.05). Furthermore, incorporating QADM and NAg together into the same composite resulted in the lowest metabolic activity and lactic acid production (P<0.05). For all composites, the biofilm metabolic activity was similar from 1 day to 12 months of water-ageing (P>0.1), and the lactic acid production from 1 day to 12 months was also not significantly changed over this period (P<0.05). These results demonstrate that incorporating QADM and NAg into the NACP composite substantially decreased the biofilm metabolic activity and lactic acid production, and the antibacterial potency was maintained and durable during water-ageing for 1 year.n=6). Adding QADM or NAg significantly reduced the CFUs compared with the commercial composite control, and combining both QADM and NAg into the same composite yielded the least CFU counts. For each composite, the CFU counts of biofilms did not vary significantly vs water-ageing time from 1 day to 12 months. For example, the NACP-QADM-NAg composite had a similar number of CFUs at 1 day and after 3, 6, 9, or 12 months of water-ageing (P>0.1).The CFU counts for 2-day biofilms on composite disks are plotted in 38In the present study, antibacterial and remineralizing dental composites were developed containing QADM, NAg, and NACP, which were demonstrated to maintain the anti-biofilm activity after 12 months of water-ageing. Incorporating QADM and/or NAg into the NACP composite did not decrease its mechanical properties, which matched those of a commercial composite control after water-ageing for 1 year. Quaternary ammonium salts are important compounds with a broad antimicrobial spectrum. The advantage of QAM-containing dental resins is that the antibacterial monomer is copolymerized with the resin matrix by forming a covalent bond with the polymer network, and therefore, the QAM is immobilized in the resin and is not released or lost over time. Therefore, this copolymerization and immobilization method may impart a durable antibacterial capability to the dental resin. The present study provides the first demonstration that after 1 year of water-ageing, the antibacterial potency of a composite containing QADM, NAg, and NACP is still as strong as it was at 1 day. Such sustained reduction in biofilm/plaque and acid production may help minimize tooth structure demineralization, and the addition of NACP may promote remineralization.28 The synthesis of QADM involves a modified Menschutkin reaction in which a tertiary amine group is reacted with an organo-halide, and the reaction products are generated at virtually quantitative amounts and require minimal purification.28 In addition, as a dimethacrylate, QADM can be beneficial as compared as quaternary ammonium monomethacrylates. QADM has reactive groups on both ends of the molecule, which can be incorporated into the resin matrix with less of a negative effect on the mechanical and physical properties of the composite.28 In contrast, a quaternary ammonium monomethacrylate has only one reactive group and may weaken the resin matrix when incorporated. Furthermore, QADM is compatible with common dental dimethacrylate monomers and is expected to have minimal monomer leachability, compared with other quaternary ammonium salts. The detailed antimicrobial mechanism of quaternary ammonium remains to be fully understood; however, quaternary ammonium materials appear to cause bacterial lysis by binding to the cell membrane and causing cytoplasmic leakage.25 This leakage is stimulated when the negatively charged bacterial cell contacts the positively charged (N+) sites of the quaternary ammonium resin. This disturbs the electrical balance of the cell membrane, and the bacterium then ruptures under its own osmotic pressure. The results of the present study involving water-ageing for 1 year showed that such an antibacterial mechanism indeed has a long term and durable inhibitory effect on oral biofilm growth.The present study used QADM because its synthesis method is fairly straightforward as compared with the synthesis of other QAMs.52 Its antimicrobial mechanism appears to involve Ag ions interacting with and inactivating the vital enzymes of bacteria, causing the DNA in the bacteria to lose its replication ability, leading to cell death.52 Although the present study indicates that the NAg in the composite possesses antibacterial activity even after 12 months of water-ageing, further studies are needed to investigate how long the antibacterial effect of NAg resin lasts and whether the antibacterial potency decreases after 2 or 3 years. Furthermore, the results of the present study showed that the use of two antibacterial agents (QADM+NAg) achieved a significantly stronger antibacterial potency than using either single agent, and the activity of the dual agents was maintained after water-ageing for 1 year. This was achieved because both QADM and silver, through TBAEMA's reactive methacrylate groups, which copolymerize with the resin matrix.45 These results suggest that copolymerization with the resin matrix and the use of dual agents appear to be promising approaches to maximize the antibacterial function of the resin without compromising its mechanical properties or long-term durability.Ag is another effective and widely used antibacterial agent with a strong toxicity to a wide range of microorganisms.53 In contrast, commercial restorative controls fail to raise the pH.53 Other studies have shown that the NACP composite remineralizes enamel lesions38 and inhibits secondary caries at the composite-enamel margins in the oral environment, in a human in situ model.54 The NACP composite in these previous studies had no antibacterial agent in the composite. In the present study, incorporating both QADM and NAg into the NACP composite achieved double benefits: remineralization capability due to NACP and anti-biofilm activity due to QADM and NAg. Therefore, we expect that its caries-inhibition efficacy will be further enhanced when compared with NACP alone. Further studies are needed to compare the inhibition of secondary caries at the tooth-restoration margins by NACP vs NACP+QADM+NAg in a human in situ model.In addition to the QADM and NAg incorporation reducing biofilm acids and combating caries, NACP incorporation may also contribute to the inhibition of caries. A previous study has shown that a NACP composite can release Ca and P ions and neutralize an acid, rapidly increasing the pH of an acidic solution from a pH of 4 to one above 6.55 Gingival recession exposes more tooth roots, which have a higher solubility and are less resistant to biofilm acids than tooth enamel.56 In addition, reduced saliva flow tends to increase biofilm and plaque buildup.57 Indeed, root caries increase from 7% among young people to 56% in seniors.58 Class V restorations with subgingival restorative margins have pockets for bacterial growth that are difficult to clean and can lead to periodontitis.59 Therefore, there is a need to develop novel bioactive restoratives for Class V lesions that are antibacterial to inhibit cariogenic biofilms as well as periodontal pathogens in cases with subgingival margins. The antibacterial function of the NACP+QADM+NAg composite, as well as the ability of NACP to neutralize acids and to raise the pH above 6  in the same composite resulted in a significantly stronger antibacterial capability than using QADM or NAg alone. The antibacterial results were not significantly different after water-ageing for 12 months, compared with 1 day, thus indicating a long-term durability. The durable antibacterial and mechanical properties, plus the calcium and phosphate ion release and acid neutralization reported previously for remineralization, indicate that the novel NACP-QADM-NAg composites may be useful in dental restorations to combat caries.


Direct growth of spherical graphene with large surface area is important for various applications in sensor technology. However, the preparation of transfer-free graphene on different substrates is still a challenge. This study presents a novel approach for the transfer-free graphene growth directly on cemented carbide. The used simple thermal annealing induces an in-situ transformation of magnetron-sputtered amorphous silicon carbide films into the graphene matrix. The study reveals the role of Co, a binding phase in cemented carbides, in Si sublimation process, and its interplay with the annealing temperature in development of the graphene matrix. A detailed physico-chemical characterisation was performed by structural , morphological (SEM) and chemical (EDS) analyses. The optimal bilayer graphene matrix with hollow graphene spheres on top readily grows at 1000 °C. Higher annealing temperature critically decreases the amount of Si, which yields an increased number of the graphene layers and formation of multi-layer graphene (MLG). The proposed action mechanism involves silicidation of Co during thermal treatment, which influences the existing chemical form of Co, and thus, the graphene formation and variations in a number of the formed graphene layers. The transfer of graphene generates impurities and defects that vitiate the mechanical and electrical properties3. Another problem is low bonding strength between graphene and different substrates.Synthesis of graphene on different substrates is a challenging and attractive approach that can broaden application spectra of currently existing engineering materials. Versatile experimental approaches were used, but major constraints are related to synthesis of adequate graphene sheets and subsequent application problems caused by the transfer of synthesised grapheneet al.4 reported graphene formation on Ni surface by the mechanism of carbon dissolving from SiC substrates by rapid heating. Machač et al.5 demonstrated a feasible route for graphene growth by annealing of Ni/SiC structures, highlighting the role of Ni layer thickness and a shortage of Ni amount in the structure. Surface Ni-silicidation reaction is also an effective mean for preparation of few-layer graphene (FLG) by controlled thermal annealing of SiC crystals, which was successfully demonstrated in Ni/6H-SiC (0001) system, with the best graphene structure obtained at 800 °C6. The similar approaches are also valuable for the transfer-free synthesis of graphene on SiO27, sapphire and Cu2O (111) substrates8. Besides Ni and Cu, another transition metal, Co, was investigated as an attractive catalyst for transfer-free graphene growth. By deposition of Co layer on SiC, the FLG was grown via selective reaction between Co film and SiC substrate at high temperatures followed by the rapid cooling9. The question that arises here is related to control of carbon diffusion process since it affects number and quality of graphene layers; particularly, formation of the top CoO layer is actually critical for the synthesis of a high-quality graphene layer10. Having in mind these results, a successful synthesis of transfer-free graphene layer by the metal-silicidation approach requires several steps, including deposition of selected metal layer/metal oxide layer, control of processing parameters and carbon diffusion process as well.Metal-catalysed graphene growth from SiC followed by a simple but controlled annealing step is a useful approach towards the synthesis of large area graphene layer. Among different metals, Ni, Cu and Co effectively catalysed graphene generation. Juang 12. Development of a suitable material on the surface of cemented carbide tools could enable acquisition of drilling information, detection and automated correction of certain failure modes, such as auger choking, auger jamming, and bit wear15. Among different materials, graphene-based composites due to their remarkable mechanical, electrical, piezo-resistive and other physical properties become the best option regarding the inherent conventional sensor defects, such as low sensitivity, short service life, and rapid attenuation19. Especially three-dimensional (3D) graphene with hollow spheres on the surface shows enhanced sensing performances due to large surface area that provides more binding sites, which has great application potential20. However, there are no acceptable methods to synthesise in-situ a graphene layer on the cemented carbide surface, especially in regards to following aspects: (1) using readily available a-SiC instead of costly SiC single-crystal as carbon source, (2) the impact of annealing temperature on the number of produced graphene layers and (3) the understanding of Si atoms role in graphene growth and change in the number of graphene layers. If the mechanism of Si acting is not identified, the graphene layercannot be directly grown from a-SiC.Cemented carbides are extensively used materials in space drilling applications for providing a first-hand insight into the geological structure and mineral distribution of planets but also as cutting tool materials22. The clear advantage of this study is an elegant approach towards utilisation of currently existing engineering material and scientific data to develop a sustainable new aspect of cemented carbides without any need of depositing metal oxide layer to control carbon diffusion. The graphene layer with graphene balls on top is formed in-situ by simple and controlled annealing. An extensive physicochemical characterization, including structural, morphological and chemical aspects, was performed to reveal the influence of temperature on the number of formed graphene layers and mechanism of Si atom acting during the graphene matrix generation. This paper opens new horizons towards further enhanced industrial applications of the cemented carbides.In this study, we propose a novel approach towards the in-situ synthesis of graphene layer on cemented carbides by utilisation of Co as a binding phase in cemented carbides to catalyse the in-situ growth of the graphene film. Herein, Co binding phase catalyses graphitization of amorphous SiC (a-SiC), which is deposited on the cemented carbide at low temperature by the magnetron sputtering technique. In addition, this does not change the carbide performance due to a trace amount of Co involvedGraphene sheets with graphene balls on top were grown directly on the cemented carbide surface by a new approach that uses Co as a binding phase in the cemented carbides to catalyse the formation of graphene from a-SiC. Moreover, the exact mechanism of graphene formation by annealing a-SiC was revealed. The influence of annealing temperature on morphology, film composition, structural changes and number of graphene layers was systematically studied. Furthermore, the role of Si atoms in graphene formation was discussed regarding the phase transformations and the number of graphene layers.23.In the present study, Co from cemented carbides is used to in-situ catalyse graphene formation from magnetron-sputtered a-SiC films by an annealing process. The Raman spectra of the deposited a-SiC films annealed at five different temperatures are shown in Fig. 2D/IG), full width at half maximum (FWHM) of the peak and the number of fitted Lorentzian peaks24. The general equation for determination of the crystallite size, La using any laser line in the visible range can be written as25:λ is 532 nm. The Raman spectrum at annealing temperature of 1000 °C showed that I2D/IG value was higher than 1, FWHM was 45 cm−1 and the 2D peak was at 2700 cm−1. For a single layer, the intensity of the 2D peak was four times higher than that of the G peak, whilst the intensity of the 2D peak was 1 to 2 times higher than that of the G peak for two layers26; FWHM of the 2D peak for a single layer of graphene is in the range of 30–45 cm−1, for two layers is in the range of 45–60 cm−1 and for three layers is in the range of 65–70 cm−1, whilst the FWHM of the 2D peak of graphite was lower than 30 cm−1 27. Inset 2-a indicates that 2D peak may be deconvoluted into four peaks with maxima at 2659, 2687, 2704 and 2725 cm−1, respectively. In general, a 2D peak of a single layer of graphene can be fitted by a single Lorentzian profile, a 2D peak of two layers of graphene can be fitted by four Lorentzian profiles, and a 2D peak of three layers of graphene can be fitted into six Lorentzian profiles, whilst a 2D peak of four or more graphene layers can be fitted only with two Lorentzian profiles23. Therefore, it can be concluded that by annealing at 1000 °C, the quality graphene with 2 layers was successfully generated on the cemented carbide.Data of the D, G and 2D peaks of the graphene obtained at 1000 and 1150 °C are listed in Table −1 (Inset 2-b), respectively. It is suggested that the number of graphene layers was increased. In addition, the crystallite size of graphene (La) decreased and the ID/IG ratio increased, indicating that the number of film defects increased with the annealing temperature28. The reason for the increase of a number of defects with temperature is the temperature increase leads to the increase in the number of graphene layers. This induces the formation of boundaries among the graphene layers and the increase of the ratio of ID/IG.The intensity ratio of the 2D and G peaks at annealing temperature of 1150 °C is lower than that at 1000 °C, whilst the FWHM of the former is greater than of the latter; thus, it is clear that graphene is generated, but the number of layers produced at 1150 °C is obviously higher. The 2D peak may be fitted by two Lorentzian profiles with maxima at 2689 and 2710 cm2D/IG ratio and FWHM of Raman mapping of the graphene film synthesised at two annealing temperatures, 1000 and 1150 °C, with the size of 100 × 100 μm2. As shown in Fig. 2D/IG ratio is in the range of 1.1–1.5, and FWHM, Fig. −1. At 1150 °C, Fig. 2D/IG ratio value drops and ranges from 0.7 to 1.2, whilst FWHM value, Fig. −1 but also less than 30 cm−1. From these findings can be seen that the graphene film has high quality and consists of 2 layers at 1000 °C, whilst at 1150 °C, the film consists of several layers of graphene and graphite phases.Figure Thus, in the suitable annealing temperature range (1000–1150 °C), the graphene film was formed on the cemented carbide by Co-catalysed a-SiC transformation. When the annealing temperature was increased from 1000 to 1150 °C, the number of graphene layers, as well as the number defects of the graphene films, were increased, and the quality of the graphene layer was degraded.The number of graphene layers is an important parameter to distinguish between graphene and graphite phases and to gauge the quality of graphene. Therefore, it was attempted to investigate how the annealing temperature influences the number of graphene layers. The following approaches were applied: (1) probing the impact of the annealing temperature on the graphene layer structure by XRD; (2) SEM monitoring of morphological changes of the graphene layer upon the annealing process; and (3) analysis of the annealing temperature impact on the graphene film composition by EDS.2Si and Co, respectively, appeared in the XRD spectra, denoting that within this temperature range Co diffused from cemented carbide into the SiC film and reacted with SiC, thus preventing the crystallisation of a-SiC. However, the diffraction peak of β-SiC appeared on the surface of the Si slice at 1000 and 1150 °C,  is a thermodynamically spontaneous reaction. If the reaction temperature is higher than 600 °C, Co2Si is generated (2Co + SiC → Co2Si + C), and if the temperature is higher than 800 °C, the materials further react to form CoSi (Co2Si + SiC → 2CoSi + C)29. The formation of graphene is based on the reaction mechanism where C is isolated on the surface of the Co/CoxSiy film, owing to the decrease in solubility of C with temperature decrease30. As for the annealing temperature of 1000 °C, the Raman analysis shows that graphene was synthesised from the a-SiC film, which is the process catalysed by Co. Because the number of graphene layers was low, the graphite phase was not detected by XRD at approximately 26.5 ° (2θ). The Co reacts with the a-SiC film to form Co2Si, and the Co/Si atomic ratio after reaction may contribute to this reaction product31. When the Co/Si atom ratio increases, a compound phase in the Co-Si bi-component system approaches Co, and the CoSi compound transforms to Co2Si28. Thus, at the annealing temperature of 1000 °C, Co sufficiently reacts with the a-SiC film to catalyse graphene generation, generating carbon-saturated Co2Si.Different products were generated at different annealing temperatures by the catalytic reaction of Co and a-SiC. The structure of precipitated graphene layer was also changed with temperature. Systematic research on the impact of the annealing temperature on the structure of precipitated graphene layer could assist in revealing the mechanism by which the temperature causes the change in the number of graphene layers. Upon heating, Co and SiC interact, generating the Co-Si compound saturated with C atoms2Si compound. The same as afore-mentioned, Co reacts with the a-SiC film at 800 °C to preferentially generate CoSi. At the annealing temperature of 1150 °C, the CoSi compound in the film disappeared, and the Co phase appeared. Meanwhile, the number of the generated graphene layers increased and the graphite phase could be also detected.Unlike that at 1000 °C, Co reacts with a-SiC at 1150 °C, and there is only the Co phase without Co2Si compound saturated with C atoms, and the quality graphene layer with low ID/IG (0.339) and high I2D/IG (1.304) ratio is obtained. When the annealing temperature is further increased, the Co2Si compound transforms into Co, and the amount of precipitated C is increased, as well as the number of graphene layers.Therefore, in the suitable annealing temperature range (1000–1150 °C), Co diffuses in a-SiC film, catalysing graphene formation. At 1000 °C, Co reacts with a-SiC to form the CoThe impact of the annealing temperature on graphene morphology is shown in Fig. 32.The presented results show that at annealing temperatures of 1000 and 1150 °C, Co diffused into a-SiC film and catalysed the formation of graphene thereon. The metal reacted with a-SiC film and metal silicide spontaneously agglomerated to reduce the surface energy, which is in agreement with previously reported results that the graphene film is generated preferentially at areas where the metal disappeared after contraction2Si particles agglomerated on cemented carbide; hollow graphene balls were generated and graphene sheets with graphene balls were obtained. When the annealing temperature was increased to 1150 °C, Co2Si particles disappeared, the film of hollow graphene balls became thicker and the number of graphene layers increased. Such graphene sheets, with hollow graphene balls that meet the requirement of the high specific surface area for an ideal graphene film, can be beneficial for improvement of the efficiency of strain sensors by the reduction of size and power consumption23.Thus, it can be concluded that at the annealing temperature of 1000 °C, the graphene balls were precipitated from Co2Si compound disappeared, so the Co phase appeared in the film, and the number of generated graphene layers increased. In the Co-Si system, different Co/Si ratios resulted in different Co-Si phases29. The mechanism by which the precipitation layer structure of segregated C was altered with temperature may be revealed only by a systematic research of the impact of the annealing temperature on the atomic content during the synthesis of graphene. Figure 2Si and the reaction with SiC is not possible, so it does not appear in the a-SiC film. When the annealing temperature was increased, Co atoms appear in the film, the content of Si atoms decreases and the content of C atoms becomes higher, which implies that Co diffused into the a-SiC film.Influence of annealing temperature on the chemical composition of graphene film was monitored by EDS. In the temperature range of 1000–1150 °C, when the annealing temperature was increased, the Co–4 Pa) conditions for Si sublimation from SiC film to enable restructuring of C atoms and graphene formation, graphene can be synthesised at low temperature using a SiC film with Co catalyst33. This study shows that at 1000 °C, Co diffused into the a-SiC film and at Co concentration of 11.43%, the content of Si dropped from 30.5% to 4.22%, whilst the amount C atoms increased to 84.35%. It suggests that Co catalysed the sublimation of Si and the restructuring of C atoms, decreasing the content of Si and rearranging C atoms to generate graphene. Compared to that, at 1150 °C the content of Co atoms remained essentially unchanged, 10.83%, the amount of Si was even lower, 0.93% and the amount of C atoms increased to 88.24%. Having in mind the afore-mentioned results of Raman and XRD measurements, it can be stated that at the annealing temperature of 1150 °C, the Co-catalysed sublimation of Si was accelerated, inducing Co2Si cracking and the increase in a number of segregated carbon atoms in Co layer, as well as the number of graphene layers.Unlike conventional graphene epitaxial growth by SiC that requires high temperatures (>1200 °C) and high vacuum  graphene is directly generated from the a-SiC film on the surface of cemented carbide catalysed by Co binding phase via the annealing process in the temperature range of 1000–1150 °C. When the annealing temperature is increased (1150 °C), the number of graphene layers increases and the graphene quality becomes poorer. (2) Influence of the annealing temperature on the number of graphene layers is related to changes in the film composition and structure. In the suitable temperature range (1000–1150 °C), Co catalyses Si sublimation, and C particles segregate to generate graphene. The related changes in atomic and phase compositions at the higher annealing temperature induced the increase in the number of graphene layers. (3) The content of Si influences the Co pattern and causes the generation of graphene and change in the number of graphene layers. The mechanism of Si atoms acting actually includes three subsequent parts: (a) when the content of Si particles decreases, C particles segregate in the Co layer to generate graphene; (b) Si particles react with Co to generate Si-Co compound, so the free Co layer disappears, and the number of precipitated graphene layers is low; and, (c) when Si particles disappear, no reaction with free Co metal occurs, and the number of graphene layers increases.This research study provides a new deep insight into the transfer-free direct growth of graphene on the surface of cemented carbides, explaining the mechanism of chemical processes behind, especially underlying the structural, chemical and morphological aspects of the graphene layer formation.−3 Pa), Ar ion bombardment duration (20 min), bias (−800 V), deposition pressure (0.5 Pa), C2H2 flow rate (35 SCCM), substrate bias (−100 V), target current (22 A) and deposition temperature (150 °C). The film thickness was approximately 2.0 μm.The a-SiC films were deposited by a mid-frequency magnetron sputtering technique. There were two kinds of substrate materials: monocrystal Si wafer was used for control analysis of the structure, whilst cemented carbide YG 8 (WC-Co 8%) was used for the analysis of the graphene structure. The sample preparation method included following steps: (1) the substrate was cleaned in both acetone and absolute ethanol by ultrasonication in each medium for 10 min, and (2) the substrate was dried by nitrogen and placed in a vacuum chamber for deposition. The reaction of Si target and acetylene, which provided the source of silicon and carbon atoms, respectively, was used for the a-SiC film deposition. The main experimental parameters include basic pressure , 2θ range 20–80° and a step size of 0.02°). Valence bond and the number of layers of graphene films were determined by Raman spectroscopy  using 532 nm laser excitation at room temperature, and layer uniformity is revealed by Raman mapping; surface morphology was identified by scanning electron microscopy , while compositions of the film surfaces were analysed by energy-dispersive X-ray spectrometry (EDS). The atomic composition was obtained by taking the average of six measurements on the surface of the individual sample.