The phenylcarbamate 43 was highly potent (IC50 31.6 nM) and slightly selective for AChE, and showed low acute toxicity. In enzyme kinetics assay, 43 exhibited uncompetitive inhibition and reacted by pseudo-irreversible mechanism. 43 also showed amyloid-β (Aβ) lowering effects (51.9% decrease of Aβ42) superior to phenserine  in SH-SY5Y-APP695 cells at 50 µM. The dual actions of 43 on cholinergic and amyloidogenic pathways indicated potential uses as symptomatic and disease-modifying agents.Multifunctional carbamate-type acetylcholinesterase (AChE) inhibitors with anti-amyloidogenic properties like phenserine are potential therapeutic agents for Alzheimer’s disease (AD). We reported here the design of new carbamates using pharmacophore model strategy to modulate both cholinesterase and amyloidogenesis. A five-feature pharmacophore model was generated based on 25 carbamate-type training set compounds. (−)-Meptazinol carbamates that superimposed well upon the model were designed and synthesized, which exhibited nanomolar AChE inhibitory potency and good anti-amyloidogenic properties in  Pathologically, AD is characterized by the progressive loss of basal forebrain cholinergic neurons41) is the first AChE inhibitor separated from natural products, but unacceptable toxicity limits its clinical use. Rivastigmine (14) is the only carbamate AChE inhibitor approved as anti-AD drug on the market.Current approved anti-AD drugs are all palliative treatments targeting cholinergic or glutamatergic neurotransmission thereby symptomatically improving memory and cognition in patients. Acetylcholinesterase (AChE) inhibitors  such as Over the last decade, much effort has been devoted to amyloidogenesis (APP generation/metabolism) and Aβ-induced neurotoxicity. Unfortunately, to date, Aβ-directed therapies, such as γ-secretase inhibitors and immunotherapies5)24), developed by Greig et al.Phenserine -in silico and validated through a test set of 16 structurally diverse compounds22–24,26,28,42 and 43, Based on three-dimensional (3D) quantitative structure-activity relationship (QSAR) analysis of 25 known carbamate-type AChE inhibitors42 and 43 for AChE and butyrylcholinesterase (BChE) were tested in vitro. Enzyme kinetic parameters, Michaelis constant (Km) and maximum velocity of reaction (Vmax), were measured on recombinant human acetylcholinesterase (rHuAChE). The association and dissociation rate constants, namely inhibit constant (ki), dissociation constant (k3), and affinity constant (KD), were determined using AChE immobilized disk. Anti-amyloidogenic experiments were conducted employing high content screening (HCS) in SH-SY5Y-APP695 cells, and enzyme-linked immunosorbent assay (ELISA) in the cell culture medium. Mechanisms for the actions of 42 and 43 on reducing APP and Aβ42 levels were further discussed.Inhibitory potencies of 50s tested by Ellman’s method50 values covered a range of three to four orders of magnitude and the activity uncertainty was set 3 as default. All two-dimensional (2D) structures of the compounds were built using ISIS Draw v2.2  and exported into DS to be converted into 3D format. A maximum of 255 conformers were generated for each compound over a 20 kcal/mol range using the BEST conformational analysis method. As an exception, (−)-meptazinol and its carbamate derivatives were calculated by both BEST and CAESAR conformation algorithms. Chemical features including hydrogen bond acceptor (HBA), ring aromatic (RA), positive ionizable (PI) and hydrophobic (HYD) features were selected and each feature was given parameters from a minimum of 1 to a maximum of 5. The minimum interfeature distance was set as a value of 2.50 and the maximum excluded volumes was set to 5. Different weights were assigned to the features and weight variation was the default value of 0.302. Otherwise default parameters were used.Pharmacophore model generation and validation were performed using 3D QSAR Pharmacophore Generation module and Ligand Pharmacophore Mapping module, respectively, in Discovery Studio v2.5  software package. Carbamate-type AChE inhibitors with comparable ICα]D) was determined on a JASCOP-1020 rotatory apparatus. IR data were recorded on an AVATAR 360 FT-IR spectrometer (KBr). NMR data were recorded on a Mercury Plus 400 instrument. Chemical shifts (δ) are expressed in parts per million (ppm), relative to tetramethylsilane (TMS) as an internal standard. Signals of active hydrogen disappeared after D2O exchange. Mass spectrum was measured on an Agilent 1100 Series LC/MSD 1946D spectrometer. Elemental analysis was tested on vario EL III element analyzer. Purity of the target compound was verified via HPLC. The elution with acetonitrile-0.01 mol/L KH2PO4 (pH =4.0) (33:67) was running through a Diamonsil® C18(2)  column at a flow rate of 1.0 mL/min and at the temperature of 30 °C using UV detection at 233 nm.All reagents except phenyl isocyanate were of commercial quality. Phenyl isocyanate was prepared from aniline and bis(trichloromethyl) carbonate. Rivastigmine hydrochloride standard was available from Sunve (Shanghai) Pharmaceutical Co., Ltd. Melting points were measured in open glass capillary tubes with Thiele-Dennis tube, and were uncorrected. Specific rotation . Adding dry HCl-ether to a solution of 42 in dry ether (pH adjusted to 4) afforded the hydrochloride salt 42·HCl . Crystallization of 42·HCl (0.50 g) from acetone gave needlelike crystals . mp 163–165 °C; [α]D20 = −17.95 ; 1H NMR : δ 10.17 , 8.62 , 7.43–7.35 , 7.26–6.99 , 3.98–3.58 , 3.48–3.38 , 3.18–3.04 , 2.90 , 2.83 , 2.41–1.41 , 0.52–0.47 ppm ; 13C NMR (DMSO-d6): 154.0 (C = O), 151.7 & 151.5 (CAr), 145.3 & 143.8 (CAr), 129.3 & 129.1 (CAr), 123.6 & 123.1 (CAr), 120.5 (CAr), 120.2 (CAr), 66.2 & 63.0 (NCH2), 59.5 & 57.9 (NCH2), 47.1 & 46.4 (NCH3), 44.1 & 43.8 (C), 36.3 (CON-CH3), 36.1 (CON-CH3), 35.9 & 35.3 (CH2), 33.5 & 32.9 (CH2), 26.3 & 24.7 (CH2), 20.6 & 20.5 (CH2), 8.2 & 8.0 (CH3); MS (ESI): m/z 305.2 [M + H]+; Anal. C18H28N2O2•HCl•1/4H2O .To a cooled and stirred mixture of 80% sodium hydride  in 10 mL dry tetrahydrofuran, a solution of (−)-meptazinol  in 10 mL dry tetrahydrofuran was added dropwise. The mixture was stirred in ice-water bath for 30 min, then N,N-dimethylcarbamoyl chloride  was added. After stirring at room temperature for 2 h, solvents were removed under reduced pressure. Then, water (20 mL) was added, and the mixture was extracted with EtOAc (15 mL × 2). Combined organic layer was washed by brine, dried over anhydrous Na2O were added to destroy any trace of remaining unreacted phenylisocyanate and pH was adjusted to 3 by adding 1N HCl. The mixture was washed with ether (10 mL × 3), basified with saturated Na2CO3 aqueous solution (adjusting pH to 9), and then extracted with ether (10 mL × 3). The latter ether layer was washed with brine, dried over anhydrous Na2SO4, and filtered to obtain a clear ether solution of the product. Evaporation of the solvent gave 43  as a white solid. Acidification of 43 in dry ether using HCl-ether (adjusting pH to 4) afforded 43 hydrochloride as a white powder : mp 122–127 °C; [α]D20 = −18.94 ; 1H NMR : δ 10.23 , 9.98 , 8.59 , 7.50–7.40 , 7.33–7.21 , 7.16–7.10 , 7.04 , 4.00–3.60 , 3.50–3.40 , 3.11 , 2.84 , 2.19–1.43 , 0.51 ppm ; 13C NMR (DMSO-d6): 151.7 (C = O), 150.9 & 150.7 (CAr), 145.5 & 144.0 (CAr), 138.6 (CAr), 129.6 & 129.4 (CAr), 128.9 (2 CAr), 123.9 & 123.4 (CAr), 123.0 (CAr), 120.6 (CAr), 120.3 (CAr), 120.2 (CAr), 118.5 (CAr), 66.2 & 62.9 (NCH2), 59.5 & 58.0 (NCH2), 47.1 & 46.4 (NCH3), 44.2 & 43.9 (C), 35.9 & 35.4 (CH2), 33.5 & 33.0 (CH2), 26.3 & 24.8 (CH2), 20.6 & 20.4 (CH2), 8.2 & 8.0 (CH3); MS (ESI): m/z 353.2 [M + H]+; Anal. C22H28N2O2•HCl•1/2H2O . HPLC: tR = 8.5 min, 98.4% purity.(−)-Meptazinol  was dissolved in anhydrous ether (15 mL), and a piece of Na metal (approximately 5 mg) was added. The mixture was stirred under nitrogen at room temperature for 10 min, then phenyl isocyanate  was added. The reaction mixture was stirred at room temperature for 3 h till the starting material had disappeared. 5 mL of H42, 43, or rivastigmine, six to seven concentrations) were mixed adequately. After incubation for 20 min at 37 °C, Ellman’s reagent  (DTNB), 0.5 mM in 0.1 M PB, pH 7.4) and acetylthiocholine iodide (ATCh) (300 µL of 0.5 mM water solution) were added successively, and percent inhibition was determined by absorbance changes at 412 nm detected by UV spectrophotometry compared with control. BChE inhibition assay was similarly carried out using butyrylthiocholine iodide (BTCh) (0.5 mM) as the substrate and BChE  as the enzyme source. The concentration of a compound that produced 50% inhibition of the enzyme activity, namely IC50 value, was calculated by nonlinear least squares regression of the response-concentration (log) curve. Results are reported as the mean ± SEM (standard error of the mean) of IC50 obtained from at least three independent measuresInhibitory activities of the compounds toward AChE and BChE were evaluated by Ellman’s methodKm and Vmax values for AChE inhibition were calculated by regression analysis of Lineweaver–Burk plots (1/velocity versus 1/[substrate]).10 µL inhibitors of different concentrations and 50 µL rHuAChE enzyme solution of 0.5 U/mL were mixed and incubated for 20 min, then 75 µL of DTNB solution and 100 µL of ATCh solution (concentration ranging from 0.057 mM to 0.2 mM) were added to the mixture. Enzyme activity was determined right after ATCh was added by modified Ellman’s spectrophotometrical method−4 M DTNB in PB (buffer A) for 10 min. Reference enzyme activity was assessed by injecting 20 µL of saturating substrate . The peak area was calculated and noted as A0. Then, the mobile phase was switched to the one containing selected concentrations of inhibitor (carbamoylation phase). Aliquots of 20 µL of saturating substrate were injected every 10–20 min and the time-dependent decreasing of peak area (Ai) was monitored down to a plateau. Once a constant plateau was reached, mobile phase was switched to buffer A again (decarbamoylation phase). Aliquots of substrate were kept injecting into the system to monitor the recovery of AChE activity over time. Percent inhibition of enzyme activity [(A0−Ai)/A0×100%] was plotted versus time. The experiment was carried out for two different concentrations of 42 and 43 (50 and 100 nM). The process of AChE carbamoylation and decarbamoylation is generally described as Scheme 1The EDA CIM disk was first connected to a syringe pump and equilibrated with 10 column volumes (CV) of triple-distilled water and 10 CVs of 0.5 M Na-PB (pH 8.0). The flow rate was set at 0.5 mL/min. Then, the disk was activated by a 1% glutaraldehyde solution in PB  . The activated disk was washed with 10 CVs of PB. 2 mL of rHuAChE in PB (10 U/mL) was then added into the column and left to react overnight (4 °C). The Schiff bases were reduced by 5 mL of 0.1 M sodium triacetoxyborohydride (STAB) in PB (2 h at room temperature) and 5 mL of 0.2 M monoethanolamine in PB (3 h at room temperature)ki and k3 was performed by applying Perola’s mathematical equationKD) was calculated following Calculation of 695 gene (SH-SY5Y-APP695)4/well  were plated in 200 µL culture medium  and incubated for 48 h at 37 °C in 5% CO2. Compounds 42 and 43 dissolved in culture medium were applied to the cells and incubated at 37 °C in 5% CO2 for 16 h. The cells were fixed and permeabilized. Mouse anti-APP monoclonal antibody N-terminus  and Goat anti-Mouse IgG conjugated to Alexa Fluor 555 were used as primary antibody and secondary antibody to detect APP while Hoechst Dye was used to stain nucleus simultaneously. KineticScan HCS System was employed to automatically find, focus, image, and analyze the cells on double-fluorescence channel guided by Target Activation Bioapplication. Fluorescence Unit of APP versus that of nucleus was used to evaluate the expression of APP, in order to balance the background.Human neuroblastoma SH-SY5Y cells transfected with wild-type APP40/Aβ42 in the medium was measured by Human/Rat βAmyloid (40/42) ELISA Kit Wako and calculated according to the standard line.The cell culture medium was collected and added with phenylmethylsulfonyl fluoride (PMSF) to the final concentration of 0.1%. The concentration of Aβad libitum and maintained on a 12/12 h light/dark cycle at 22 ± 1 °C. They were randomly assigned to one of the five concentrations between 0% and 100% lethal rate according to our preliminary studies (data not shown). Compounds 42 and 43 were dissolved in a 1:10 propylene glycol-normal saline mixture at 0.1 mol/L and then diluted to the final stepwise concentrations with normal saline. Each concentration of the compound was orally or intraperitoneally administered to a group of 10 animals. After two weeks of observation, the lethal rate for each group was measured. A 95% confidence interval for 42 or 43 after oral or intraperitoneal administration was calculated by Bliss method.Acute toxicity was evaluated in mice of both sexes . All animals were housed in plastic cages with food and water in silico using Discovery Studio v2.5  on 25 carbamate-type AChE inhibitors with diverse scaffolds. Carbamates with comparable IC50s tested by Ellman’s method22,1–11). Rivastigmine derivatives (D)12–23). Phenol compounds without carbamoyl group, such as (−)-eseroline (24)25)50 values of all the training set compounds covered a range of three to four orders of magnitude, spanning from 8 nM to 40 µM  , and best correlation coefficient. Fixed and null costs are two important theoretical cost values to evaluate pharmacophore hypotheses, and the difference between null and fixed cost (cost difference) represents the goodness of a pharmacophore model. A cost difference of 40–60 means a predictive correlation probability of 75–90%. Correlation coefficient is based on linear regression of experimental versus estimated activities. In this study, top-ranked hypothesis was selected as the best pharmacophore model due to the highest cost difference (Δcost: 46.16), lowest RMS (RMS: 0.78), and best correlation coefficient (r: 0.95) .21 was underestimated with an error of +10. The most active compound 15 (IC50 8 nM) had a fit value of 9.20, whereas the least active compound 25 (IC50 40 000 nM) showed lesser value of 5.55.Experimental and estimated activities, fit values, and corresponding error values of the training set molecules are listed in The best pharmacophore model consists of five chemical features  and two excluded volumes . A pair 25  is inevitably the most important feature since carbamoyl group is the basis of covalent carbamylation. Thus phenol compound 25 , which f25 (15  was the 1) to the pharmacophore model. Carbamoyl group and phenol ring overlapped to HBA and RA features, respectively. Nitrogen atom in 1-position instead of 8-position was aligned to the PI feature. (3aS)-methyl group in (−)-physostigmine (1) perfectly matched the HYD region (50 28 nM22). In contrast, the R-enantiomer (+)-physostigmine (2) is much less potent (IC50 9900 nM22). It demonstrated that configurational inversion of the two asymmetric centers (3a and 8a) caused crucial conformational changes which led to pharmacophore model mismatch. When (+)-physostigmine (2) yielded to map into the HYD region as shown in Unexpectedly, a small but important region of HYD (weight: 2.58) was identified in the model very near to the PI and RA features, which explained the activity difference between enantiomers. D region  and thus,26,28,26–41). Estimated AChE inhibitory activities based on fit values are listed in The proposed pharmacophore model was then validated by a test set of 16 compounds22–243) was supposed similar to that of (−)-bisnorcymserine since they had a common leaving group. Atom coordinates of the crystal AChE structure were fitted to those of the pharmacophore model through heavy atom superimposition of crystal (−)-bisnoreseroline structure upon pharmacophoric (−)-bisnorphysostigmine (3) conformation -bisnorcymserine  and AChEormation . Figure 50 41 µM42) and phenylcarbamate (43) -meptazinol is a moderate AChE inhibitor (ICate (43)  were des43)  center and HYD (cyan) region. The only nitrogen in azapane ring of 43 resembled the N1-nitrogen of phenserine. Mimicking the (3aS)-methyl group presented in phenserine, (3S)-ethyl group of 43 occupied the HYD feature.43) , and the43)  and 43 (43) 42, 43 and (−)-meptazinol. As a result, (−)-meptazinol was predicted an IC50 of 18 µM, similar to the experimental activity. By CAESAR algorithm, the estimated IC50s of 42 and 43 were 75 nM and 370 nM, respectively -meptazinol with N,N-dimethylcarbamoyl chloride in the presence of sodium hydride at room temperature. The coupling of (−)-meptazinol with phenyl isocyanate at room temperature in the presence of sodium gave 43 in 72% yield. 42 and 43 were prepared as hydrochloride salts for the following in vitro and in vivo assays. Structures of the hydrochloride salts were characterized by [α]D, IR, 1H NMR, 13C NMR, MS, and elemental analysis.Methodology employed for the synthesis of 42 and 43 were tested in vitro for AChE/BChE inhibitory potency and selectivity . The phenylcarbamate 43, showing an IC50 of 31.6 nM, was about 170 times more potent than rivastigmine and almost five times less potent than 42. Compared with the parent compound (−)-meptazinol, 42 and 43 showed a 5900-fold and 1300-fold increase, respectively, in the inhibition of mice brain AChE. With regard to activities reported by Yu et al.42 was four times more potent than physostigmine (IC50 27.9 nM), while 43 was 1.3 times less potent than phenserine (IC50 24.0 nM).The synthesized carbamate compounds ectivity . Mice br42 was slightly more selective (twofold) to BChE similar to rivastigmine and physostigmine, while 43 was an AChE-selective inhibitor, showing a twofold selectivity for AChE versus BChE (IC50 67.1 nM). Similar to phenserine, 43 would have less peripheral side effects and lower acute toxicity than those BChE-selective carbamates, such as physostigmine and 42.As for selectivity, 42 and 43 were revealed by enzyme kinetics assays. The plots of residual enzyme activity versus enzyme concentration at different concentrations of 42 and 43 gave a family of straight lines with a y-axis intercept, suggesting that both compounds were reversible AChE inhibitors. Their enzyme inhibitory properties were further modeled using double-reciprocal plots. Variance of the velocity of control group could be explained as the degradation of enzyme. Increasing the concentrations of 42 and 43 led to a decrease in Vmax and an unvaried Km (x-intercepts)  . 42 decr1) was first selected as a reference compound to verify the reliability of our disk. Percent inhibition of enzyme activity [(A0−Ai)/A0×100%] was plotted versus time. The curve was fitted to Perola’s mathematical equation. The calculated ki, k3, and KD of physostigmine were (4.78 ± 1.13) × 105 M−1 min−1, (1.94 ± 0.36) × 10−2 min−1 and (4.09 ± 0.22) × 10−8 M–1, respectively, consistent with the previously reported results43,42 and 43.The inhibition of AChE by carbamates involves carbamoylation of the enzyme and production of a covalent adduct. The carbamoylated enzyme is then hydrolyzed to regenerate the free enzyme. The process is time-dependent, therefore, determination of the kinetic parameters is of utmost importance to assess time of action. Bartolini et al.42 and 43 fitted well to Perola’s equation42 and 43 at 50 nM. The carbamoylation half-times of 42 and 43 were found to be 23.5 min and 20.3 min, respectively, longer than that of physostigmine (3.9 min) and rivastigmine (11.4 min) reported by Bartolini et al.42 and 43, respectively, similar to physostigmine (2 h) but much shorter than rivastigmine (34 h). The ki, k3 and KD values of 42 and 43 are shown in 43 could bind to and dissociated from AChE faster than 42, indicating that the enzyme was more affinitive to 42 than 43. And, 42 inhibited AChE more strongly than 43. There was no obvious difference of the ki and k3 values between 42 and 43. The enzyme affinity, carbamoylation and decarbamoylation rates and the duration of the inhibition of 42 and 43 were similar to that of physostigmine, suggesting that both the compounds reacted with the enzyme as a pseudo-irreversible inhibitor, in a way typical to carbamates, through quick formation of an addition complex and subsequent slow decarbamoylation.The data of the carbamoylation and decarbamoylation of AChE by 42 and 43 were evaluated with HCS in SH-SY5Y-APP695 cells42 and 43 for 16 h, intracellular APP levels were markedly reduced by 23.5% and 26.9%, respectively . The Aβ lowering effect of 43 was better than that of phenserine  and oral (p.o.) administration, and corresponding results are reported in 42 and 43 escalating, peripheral cholinergic side effects such as salivation, twitch, and incontinence were observed. 42 showed high acute toxicity (LD50 1.4 mg/kg) after i.p. administration. When administered orally, 42 (LD50 12 mg/kg) was almost three times less toxic than physostigmine (LD50 4.5 mg/kg)42 showed four times higher potency than physostigmine in in vitro test. The LD50 of 43  was 12–24 times higher than that of rivastigmine 43 (LD50 45 mg/kg) was slightly less toxic compared with phenserine (25 mg/kg)43 showed low acute toxicity and deserved further studies in cholinergic impairment animal models to evaluate its in vivo cognitive enhancement function.The LDIn summary, (−)-meptazinol carbamate derivatives were designed based on a 3D pharmacophore model built using 3D QSAR Pharmacophore Generation module in DS from 25 known carbamate-type AChE inhibitors. The best pharmacophore model consists of five chemical features  and two excluded volumes. The existence of a HYD region near the PI feature have been recognized as essential chemical characteristics in the model to differentiate enantiomers. Merging of carbamoyl groups onto the (−)-meptazinol scaffold generated new bifunctional ligands with dual actions on both cholinesterase and amyloidogenic pathways.42 and 43 were verified as nanomolar cholinesterase inhibitors in in vitro assay. 42 and 43 showed uncompetitive inhibition and reacted with the enzyme as a pseudo-irreversible inhibitor, such as typical carbamates, through quick carbamoylation and subsequent slow decarbamoylation. 42 (IC50 6.93 nM) was more potent in inhibiting AChE than 43, and was slightly selective to BChE (two-fold). In acute toxicity test, 42 had lower LD50 values  and showed more peripheral cholinergic side effects. However, the phenylcarbamate 43 was more promising and exhibited significant anti-cholinesterase and anti-amyloidogenic properties.The synthesized compounds 43 (IC50 31.6 nM) was 170 times more potent than rivastigmine in inhibiting AChE, and was 1.3 times less potent than phenserine. 43 exhibited a twofold selectivity for AChE, therefore milder peripheral side effects and lower acute toxicity were observed for 43 . 43 also showed Aβ lowering effects (51.9% decrease of Aβ42) superior to phenserine  at the concentration of 50 µM. Even at a lower concentration of 5 µM, 43 still reduced APP level by 20.0% and reduced Aβ42 by 30.5%. The dual actions of cholinesterase inhibition and anti-amyloidogenesis indicated a potential use of 43 as symptomatic and disease-modifying agent for the treatment of AD, which deserved further studies in cholinergic impairment animal models.


According to multiple studies, the objective response rate of PD‐1/PD‐L1 inhibitors in the second‐line treatment of unscreened non‐small cell lung cancer (NSCLC) is only approximately 20%. Predictive biomarkers of treatment efficacies are still under investigation. In selected NSCLC patients with PD‐L1 expression ≥ 50%, the response rate of pembrolizumab in first‐line treatment can reach 44.8%. Moreover, patients with a higher tumor mutation burden (TMB) tend to achieve a better response with nivolumab. Besides PD‐L1 expression and TMB, taking all these indicators into consideration would hypothetically maximize the clinical response in a specific subgroup of patients. Our study aims to accumulate large and complete samples and clinical data to verify the biomarkers and their cutoff values related to the efficacy of PD‐1/PD‐L1 inhibitors in Chinese NSCLC patients, and to construct a comprehensive predictive model by combining multi‐omics data with contemporary machine learning techniques. NSCLC patients administered treatment of anti‐PD‐1/PD‐L1 antibodies or a combination with other drugs have been enrolled. The estimated enrollment is 250 participants. A sophisticated predictive model of immunotherapy response in the Chinese population has not yet been developed. It is clinically and practically imperative to comprehensively evaluate the possible indicators of Chinese NSCLC patients through multiple test platforms, such as next generation sequencing, PCR, or immunohistochemistry. This study is registered in the Chinese Clinical Trial Registry (ChiCTR1900021395). EGFR mutation‐positive subgroup, patients administered gefitinib had longer PFS than others administered chemotherapy , but in the EGFR mutation‐negative subgroup, patients administered gefitinib had shorter PFS than others administered chemotherapy .Lung cancer ranks first worldwide in terms of morbidity and mortality among all malignant tumors. In China in 2015, approximately 4.292 million new cases and 2.814 million deaths from cancer were estimated, including approximately 700 000 new lung cancer cases, of which non‐small cell lung cancer (NSCLC) accounts for approximately 85%.Recently, research in cancer immunotherapy has been thriving. Inhibition of the immunological checkpoint pathway represented by PD‐1/PD‐L1 in cancer immunotherapy is considered one of the most promising treatment methods. There are currently three PD‐1/PD‐L1 inhibitors approved by the FDA for NSCLC, namely nivolumab,These results, among others, suggest that not all NSCLC patients benefit from PD‐1/PD‐L1 inhibitors. A better clinical response might be achieved with parallel tests and analyses concerning other biomarkers, including PD‐L1 expression,JAK1/2 mutations are associated with resistance to anti‐PD‐1/PD‐L1 antibodiesMDM2/MDM4 and EGFR alterations may correlate with hyperprogression.EGFR or ALK mutations, and pembrolizumab for patients with PD‐L1 expression ≥ 1% as second‐line treatment. In January 2018, multiple studies confirmed that the composition of specific intestinal microbiota in Western populations significantly prolonged PFS in patients treated with PD‐1/PD‐L1 inhibitors.The expression levels of some proliferation‐associated genes are associated with response to immune checkpoint inhibitors in NSCLC.The main purposes of this prospective observational study are to accumulate large and complete samples and clinical data to verify the biomarkers and their cutoff values related to the efficacy of PD‐1/PD‐L1 inhibitors in Chinese NSCLC patients, and to construct a comprehensive predictive model from the data. The secondary purposes are to explore the relationship between numbers and/or frequencies of certain genetic variations  and immunotherapy response, and to verify the influence of known DNA repair‐related genes on immunotherapy resistance and/or hyperprogressive disease. This study will also focus on the application of liquid biopsy, especially the mutation spectrum and TMB in circulating tumor DNA (ctDNA) for discriminating pseudoprogression and predicting resistance or progression of immunotherapy. An evaluation of intestinal microbiota metagenomic sequencing, based on the efficacy of PD‐1/PD‐L1 inhibitors and changes in intestinal microecology in Chinese tumor patients, will also be conducted in conditional research centers.EGFR or ALK genetic alteration (common or uncommon); administered anti‐PD‐1/PD‐L1 antibodies or a combination with other drugs; with available tumor tissue samples within two years before treatment or undergoing biopsies; with no less than 15 formalin‐fixed paraffin‐embedded samples; with available peripheral blood samples obtained within one week before treatment, and six weeks, three months and six months after treatment, and at the time point of withdrawal (10 mL each sample); at least one measurable lesion according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; Eastern Cooperative Oncology Group performance status (ECOG PS) score of 0 or 1; life expectancy of at least 12 weeks; and with adequate organ function and hematopoiesis. Subjects with a history of hematologic or primary solid tumor malignancy within five years prior to the first study treatment are excluded. The ethics committees at Zhongshan Hospital of Fudan University and each institution approved this study and written informed consent will be obtained from each patient.The estimated enrollment is 250 participants. Enrollment is limited to patients: aged 18–75 years old; with histologically or cytologically confirmed metastatic or local advanced NSCLC; without any EGFR or ALK genomic tumor aberrations treated with PD‐1 or PD‐L1 inhibitors  somatic mutation analysis with tumor DNA using whole exon sequencing; (ii) somatic mutation analysis with ctDNA using NGS for 620 cancer‐associated genes; (iii) gene expression profiling analysis using RNA‐Seq; (iv) microsatellite instability analysis using PCR; and (v) PD‐L1 and CD8 expression analysis using immunohistochemical staining (Fig All of the data obtained from six layers will be statistically analyzed to develop a robust clinical predictive model of immunotherapy using the following framework. We will identify the biomarkers for predicting patient response to PD‐1 or PD‐L1 inhibitors from each layer. We will then combine multi‐omics data with contemporary machine learning techniques to develop predictive models. We intend to focus on drug resistance mechanisms; in particular, we will explore acquired mutations during treatment via ctDNA analysis. This will hopefully allow us to understand the mechanisms of resistance and highlight the potential mutations that can be targeted by novel therapeutics. Furthermore, we will investigate whether the effectiveness of immunotherapy can be predicted based on changes in ctDNA levels. We will compare the timing and magnitude of changes in ctDNA levels and radiographic tumor size measurements longitudinally during treatment. We will examine whether patients with downtrending ctDNA levels are more likely to gain a prolonged treatment benefit. We will also examine whether such patients have improved PFS. Furthermore, we seek to determine if baseline and early therapy measurements of ctDNA can differentiate pseudoprogression from true progression in patients treated with immunotherapy.The study design has been validated in numerous clinical trials. Multiple platforms will be introduced, such as NSG, Sanger sequencing, metagenomic sequencing, PCR, RNA‐seq, and IHC. TMB will be detected by NGS, and immune‐related gene expression profiles by the RNA‐seq platform. PD‐L1 and CD8 expression will be detected by IHC. The detection of thousands of samples has validated these platforms. By means of these platforms, DNA and RNA in different types of samples will be detected and we can then obtain adequate information. We will analyze valuable information in the view of genomics, RNomics, and proteomics and screen biomarkers to predict therapeutic effects of immunotherapy. Moreover, testing samples are available. CtDNA from peripheral blood and microbiome DNA from excrement will be obtained and detected. Finally, the medical records of each patient will be reviewed and a paper case report form completed by well‐trained study physicians, which will be updated manually. Professional data entry staff will manually review the form and transcribe clinical and sequencing data into an electronic data capture system specifically designed for this study.The primary goal of this study is to establish a multicenter cohort of NSCLC patients in China. This result will complement current understanding of the use of PD‐1/PD‐L1 inhibitors to predict biomarkers in advanced Chinese NSCLC patients and explore the relevant influencing factors. Through long‐term follow‐up of patients, we can further improve real‐world multicenter data of the efficacy of advanced NSCLC immunotherapy in China.This multicenter prospective study will deepen the mining of multiple biomarkers, establish a comprehensive forecasting model, and predict the sensitivity of treatment in multiple dimensions. We believe that biomarkers indicating the efficacy of PD‐1 can be integrated into a mathematical model, which will greatly improve the predictive value of immunotherapy.However, this study does have potential limitations, such as the inconsistencies and incompleteness of clinical practice data sources; diagnostic bias among different institutions; the fact that blinding is not performed in a real‐world study; and intervention by external clinical trial participants. Research methods with higher statistical power will help to reduce these biases.No authors report any conflict of interest.


Furthermore, the propagation bandgap between the upper and the lower modes can be tuned by changing the chirality parameter. The effect of the chemical potential (μc)and the relaxation time (τ) on the normalized propagation constant, propagation length, and the effective refractive index is studied. The present work may have potential applications in optical and chiral sensing in the terahertz frequency range.Theoretical investigations are carried out to study hybrid SPP wave propagation along the Chiral-Graphene-Metal (CGM) interface. The Kubo formulism is used for the physical modeling of single-layer graphene and the impedance boundary conditions approach is applied at the CGM interface to compute the dispersion relationship for hybrid SPP waves. It is demonstrated that the chirality ( Its Dirac cone-like band structure with zero band gap distinguishes it from other ordinary materials because the electrons in graphene behave as massless fermions3. Recent developments in graphene-based optical devices have revolutionized optical societies by its ultrafast response4, broadband absorption5, and broadband communication6, terahertz lenses8, chemical and biological sensors9, ultra-broad photo detectors10, and terahertz phase shifters and modulators12.Atom-thick purely 2D carbon’s allotrope with a honeycomb structure, known as Graphene, is of interest in many research communities due to its extraordinary electronic, optical, chemical, and mechanical properties14. Wave propagation on the surface of graphene is evident from many theoretical and experimental studies that are commonly known as graphene plasmonics. The strongly confined modes and low loss propagation are remarkable features of graphene SPP waves compared to ordinary metal dielectric SPP waves15. Many researchers have studied different graphene-based structures for the exploitation of different types of surface wave and wave guide modes under different frequency ranges .Graphene optics exploits a new area of research, i.e. terahertz sciences, which deals with the propagation and modulation of terahertz waves for optical sensing, biochemical sensing, THz communication, and THz spectroscopy applications−2 to 10−7, depending upon their nature and structure22. Such molecules are modeled as chiral materials and have been the interest of many scientific communities as chiral sensing, chemical or analyte sensing tool and Enantiomer detection23.The chiral sculptured thin film-based metal dielectric interface geometry is examined for the optical sensing of an analyte by generating multiple SPP waves and Tamm Waves under different conditions27. Recently, the isotropic chiral-metal interface has been studied for the propagation of hybrid SPP wave generation for the detection of chiral sensing and enantiomeric detection it is reported that chirality is sensitive to the propagation length28. To further enhance the sensitivity, control and modulation of surface waves, the graphene’s dynamical tuning of conductivity, introduces new degrees of freedom to manipulate and control the optical properties of the graphene layer15. Due to these extraordinary properties, a graphene-based planar structure is motivated in the present work, i.e. the Chiral-Graphene-Metal (CGM) structure.Generally the organic, inorganic and biochemical molecules exhibit the phenomenon of dichroism which is scaled by the strength of chirality parameter, which, typically ranges from 10ξ) and chemical potential (μc) of single-layer graphene on the dispersion curve. The behavior of the normalized propagation constant, effective refractive index, and propagation length as a function of the frequency range are studied under different values of chirality (ξ), chemical potential (μc) and relaxation time (τ). The time dependence is taken as ejωt in all formulations.This paper investigates wave propagation along the planar CGM interface. Section 2 contains details of the mathematical formulation of the hybrid SPP wave propagation along the interface of the MGC structure, while Section 3 presents the results of numerical simulations to study the effect of chirality (x > 0) with parameters of the permittivity of chiral material (εc), permeability (μo), and chirality (ξ). While regions II and III represent the single-layer graphene (SLG) deposited on the metal half space x ≤ 0 with parameters σg as the graphene conductivity, μo as the permeability of free space and εm as the permittivity of metal to characterize the type of metal. In this geometry shown in Fig. The planar distribution of different materials and the mathematical formulation for the hybrid surface wave propagation along the CGM interface is given in this section. On the basis of the electromagnetic properties, the whole geometry can be divided in terms of three regions with respect to the material as region I represents the chiral material half space  and Left circular polarization (LCP) wave propagation, which means that the Transverse Magnetic (TM) and Transverse Electric (TE) modes cannot be studied independently, which is clear from the constitutive relationships i.e., The remaining field components in the chiral medium can be obtained from coupled wave equation asχ = ξ/ηc is the normalized chirality admittance, k± stands for the RCP and LCP propagation constants and expressed as x < 0; i.e. the electromagnetic fields in metal are given below asIn above equations, 28εm and μ0 are the electric permittivity of metal and permeability of the free-space, 1, A2, B1 and B2 are unknown constants. To compute the dispersion relationship of the CGM interface, the impedance boundary condition approach is used to physically model the graphene-based interface as in Zhou et al.17. The boundary conditions with continuity at x = 0 are given asg is the optical conductivity of graphene which is the function of incident frequency, chemical potential, scattering rates and temperature, as described by the Kubo formula15. Its mathematical modeling consists of two parts, inter-band & intra-band, which are given as:ħ is the reduced Planck constant, e is the electronic charge, τ is the momentum relaxation time, μ is the chemical potential, ωis the optical frequency, kB is the Boltzmann constant, and T is the temperature. The chemical potential can be obtained as vF is the Fermi velocity and n is the carrier density. It is clear that chemical potential of graphene can be tuned by electronically gated voltage or by doping of carrier density from 0 eV to 2 eV and much higher values has been discussed in30. The relaxation time is associated with the quality of graphene and phenomenological scattering rate as reported by Idzuchi et al.31.While the other associated field components can be derived by using Max well’s equation as reported by Mi & VanThe following dispersion relationship for the CGM interface is obtained after implementing the above boundary conditions.σg = 0,then the above dispersion equation transforms into the dispersion relation presented in Mi and Van28. The next section presents simulation results for further studying the physical behavior of hybrid surface wave propagating modes.If the graphene monolayer is removed i.e., 33. Therefore, in present simulations these values will be used. In all graphs, gold is assumed as the supporting metal with its plasma frequency ωp = 1.30 × 1016 rad/s and damping factor γ = 2.80 × 1013 rad/s as described by the Drude Model28.Numerical simulations are executed in the Wolfarm Mathematica® software package, to explore the characteristics of hybrid SPP wave propagation along the CGM interface. Generally the organic and biochemical materials has refractive index from 1 to 1.6 as given inω/ωsp) and the normalized propagation constant (β/ksp) in Fig. ω) is normalized by the surface plasmon resonance frequency β) is normalized by ξ = 2 × 10−3 Ω−1, ξ = 4 × 10−3 Ω−1, ξ = 6 × 10−3Ω−1, and ξ = 8 × 10−3 Ω−1) it can be observed that the propagation bandgap between modes increases in proportion to the increase of chirality and decreases with the decrease in strength of the chirality parameter. The propagation band gap between the upper and lower can be tuned by tuning the chirality of the chiral medium. Further, it is clear that with the decrease of chirality i.e., low order chiral values  the forbidden region between the modes starts squeezing as presented in the Fig. ξ = 0.0Ω−1 & σg = 0) the propagation band gap between the upper and lower modes becomes zero and the dispersion curve only presents a single mode of propagation (i.e. the standard dielectric Surface Plasmon Polariton (SPP)28 as depicted in Fig. Ex|. The Fig. Ey| component as compared to lower mode.To study the possible modes of propagation along the CGM interface, the dispersion relationship is plotted between the normalized frequency  of the intraband) on the hybrid modes of surface wave propagation for the normalized wave propagation constant (β/ksp) is plotted under the terahertz frequency range (0.1–8 THz) and presented in Figs μc = 0.2 eV, μc = 0.4 eV, μc = 0.6 eV, μc = 0.8 eV, μc = 1.0 eV & μc = 1.2 eV). The influence of the chemical potential on upper modes is more dominant at low frequencies compared to high terahertz frequencies, while the lower modes have a significant difference in all terahertz frequencies.The graphene mainly contributes in the terahertz region for the propagation of surface wave generation, thus the influence of graphene parameters  on the normalized propagation constant (β/ksp) under the terahertz frequency range (0.1–8 THz). The normalized propagation constant (β/ksp) for upper modes has a significant effect at 0.1–2 THz but the relaxation time has no effect on the wave propagation constantfor frequencies higher than 2 THz. While the lower modes have a distinct wave propagation constant in the 0.1–3 THz range above this frequency, the relaxation time has no effect on the wave propagation constant.Figure Neff = Re{β}/ko) as a function of the normalized frequency (ω/ωsp) is presented in Fig. ξ = 1 × 10−4 Ω−1, ξ = 3 × 10−4 Ω−1, ξ = 5 × 10−4 Ω−1 & ξ = 7 × 10−4 Ω−1) and chemical potential (μc = 0.6 eV). The change in the effective refractive index can be used to modulate the propagating phase speed of different modes at different values of chiral and graphene parameters along the CGM interface, which is clear in Fig. ω/ωsp), the effective refractive index (Neff) increases and the phase speed of the propagating hybrid modes decreases.The effective refractive index (ω = 0.1 − 10 THz) for different values of chirality  and chemical potential  is presented in Fig. ξ) and the chemical potential (μc) can significantly modulate the propagation length of both hybrid modes. With the increase of chirality and chemical potential,the propagation length of the upper mode increases in contrast to the lower propagating modes. The propagation length profiles of the upper and lower modes follow the exponential decay. The incorporation of graphene can be used to increase the propagation length of hybrid modes compared to ordinary chiral-metal interface modes.The propagation length c) as function of normalized frequency (ω/ωsp) under different values of refractive index i.e., . The cut off condition is obtained by assuming the n+ > n− and setting the values of β = k+, kx1 = 0 and 28. The cut off chiral values varied from 10−8 to 10−3 Ω−1, and it is obvious from fig. that it exhibits the sensitive behavior towards the chemical and optical changes in the chiral material.With the increase of normalized frequency the cut off chiral values increases and for lower frequency range shows adistinct behavior for different values of refractive index.In conclusion of these results, proposed scheme can be used for analyzing the different analyte on the basis of their chiroptic and chemical properties.The Fig. equation  as provi(i)Hybrid surface wave modes (the upper and lower) propagate along the CGM interface.(ii)The frequency difference between the upper and lower modes (viz. the propagation bandgap) can be tuned by changing the chirality or chemical potential of graphene.(iii)The normalized propagation constant is very sensitive to graphene’s chemical potential and time relaxation for the lower terahertz frequency range, but these parameters have no significant effect at the higher terahertz range.(iv)34.The effective mode index and propagation length as a function of the terahertz frequency range is studied and concluded that under appropriate parameters one can modulate the effective refractive index, the phase speed of surface waves, and the propagation length. The present method of surface wave modulation is quite simple as compared to corrugated structures(v)The proposed geometry can be used for optical sensing and wave propagation in the terahertz regime.The CGM interface is used to excite the hybrid SPP waves. The dispersion relationship is computed by implementing the impedance boundary conditions approach andthe following conclusions can be drawn:


S. aureus (MSSA) isolates.We aim to assess the similarities of proportional, phenotypic, and molecular characteristics between the school environment and schoolchildren on methicillin-sensitive A cross-sectional study was conducted between March 2016 and August 2016 in eight elementary schools in Guangzhou, China. Nasal swabs from students and environmental swabs from school environments were collected. Univariate and multivariate logistic regression analyses under a multistage stratified cluster cross-sectional survey design were performed to access the prevalence relationship and influencing factors, respectively. Phenotypic and molecular characterizations of MSSA isolates were conducted using the Kirby-Bauer disk diffusion method and polymerase chain reaction assays, respectively.P = 0.010) and 1.04  for the school or class environment and students, respectively. Similar phenotypic and molecular characteristics were identified between schoolchildren and the environment. A cause and effect relationship could not be established because the study design was cross-sectional.In total, 1705 schoolchildren and 1240 environmental samples from 40 classes in eight elementary schools obtained between March and August 2016 were include in this study. The rates of MSSA prevalence among schoolchildren and the environment were 11.44% (195/1705) and 4.60% (57/1240), respectively. The odds ratios and 95% confidence intervals (CIs) on the prevalence of MSSA isolates were 1.11 (95% CI, 1.05–1.29; Because of the cross-sectional design, we can reveal the association between school environment and schoolchildren on MSSA, but it is not a cause and effect relationship. Staphylococcus aureus (S. aureus) has become one of the leading causes of bacteremia with a 20–30% mortality rate  [DNA was obtained from all MSSA isolates using the sodium dodecyl sulfate method  and weren (Pvl)] , 22 usinhttp://www.mlst.net). Singletons or members of a clonal complex were determined using a related sequence types algorithm (http://eburst.mlst.net). Dendrogram analysis was performed based on STs to determine the clonal relatedness and potential epidemiologic origin.Multilocus sequence typing (MLST) was performed using previously published primers and conditions . Allelic2). Age- and gender-specific BMI cutoff points recommended by the Working Group on Obesity in China were used to define obesity [Anthropometric measurements were obtained using standardized techniques and calibrated equipment. Schoolchildren were weighted to the nearest 0.1 kg in light indoor clothing and bare feet or with stockings. Height was measured using a stadiometer. Schoolchildren stood in erect posture without shoes, and the results were recorded to the nearest 0.1 cm. Measures were obtained twice, and the average was considered in the analysis. Body mass index (BMI) was calculated as the ratio of weight in kilograms to the square of height in meters  from STATA package. A two-sided P-value for statistical significance was defined as P-value of < 0.05. All analyses were performed using STATA 14.2 .The significance of the difference between proportions was assessed using Pearson’s Chi-square test for categorical variables. There were two clusters in the study; a class cluster and a school cluster. We drew linear prediction plots to intuitively elucidate the dose-response relationship of MSSA isolates between nasal colonization among schoolchildren and environment in class or school clusters. The association between influencing factors and MSSA nasal colonization was examined using multivariable logistic regression models. We performed multivariable logistic regression analysis of all variables with a 2 = 53.01, P<0.001; class: χ2 = 95.23, P<0.001) nasal colonization was noted among schoolchildren in different schools and classes. More details of the characteristics of participants are presented in In total, 1705 schoolchildren and 1240 environmental samples from 40 classes in eight elementary schools were included in the study. The prevalence of MSSA nasal colonization among schoolchildren was 11.44% (195/1705). A significant difference in MSSA  environmental contamination were noted in different schools, classes, and locations. However, no significant differences in MSSA  environmental contamination were noted in different places. A more specific distribution of MSSA isolates in the environment is presented in The prevalence of MSSA environmental contamination was 4.60% (57/1240). Significant differences in MSSA  were associated with MSSA nasal colonization among schoolchildren.We found that obese [odds ratio (OR), 2.01; 95% confidence interval (CI), 1.72–5.59; To account for the potential confounding factors among influencing factors, we further analyzed the relationship between the potential predictors using a multivariate logistic regression model. This model demonstrated that when controlling for the effects of the other influencing factors , the relationships found in the univariable analyses changed. Obese and family members who were healthcare workers were no longer associated with MSSA nasal colonization. More specific details are presented in P = 0.010) and 1.04 , respectively.A positive relationship between MSSA isolates from the school or class environment and schoolchildren was observed. ORs and 95% CIs for the school or class environment and schoolchildren on the prevalence of MSSA isolates were 1.11 , CC6 , CC45 , CC188 , and CC121 . The five most predominant CC types of MSSA isolates in the environment were CC30 , CC188 , CC6 , CC5 , and CC398 . The proportions of different CC types of MSSA isolates between schoolchildren and the environment exhibited no significant differences. The five most predominant STs of MSSA isolates from schoolchildren were ST45 , ST6 , ST188 , ST30 , and ST7 . The five most predominant STs of MSSA isolates from the environment were ST30 , ST188 , ST6 , ST398 , ST5 , and ST217 . There was no significant ST of MSSA isolates between schoolchildren and the environment. Note that ST5, ST6, ST25, ST30, ST45, ST59, ST188, ST217, ST398, ST489, ST535, ST573, ST944, ST946, ST950, ST1295, and ST2114 were identified in MSSA isolates from both schoolchildren and the environment. More specific details are presented in The prevalence of MSSA nasal colonization among schoolchildren was 11.44% (195/1705). The rate was lower compared with that of secondary school students in Iraq  and university students in Italy  . HoweverS. aureus colonization [S. aureus [S. aureus [Our study contributes to the existing literature because we assessed a population with a high burden of obesity and MSSA colonization in which the association has not been examined in previous publications. Obesity was not a risk factor for MSSA nasal colonization among schoolchildren in the model of multivariate logistic regression analysis, which differed from other observed studies , 32. Ournization . Obesity. aureus . Accumul. aureus , 35. Oth. aureus , 37. FamGiven that the proportions of antibiotic resistance in MSSA isolates from schoolchildren and the environment were not different, the antibiotic resistance patterns of MSSA isolates from schoolchildren and the environment were similar, which might indicate the homology of MSSA isolates between schoolchildren and the environment.The proportions of toxin genes in MSSA isolates were increased compared with other observed studies , 38, revThe dominant CC types and STs in this study were similar to other studies , 39. TheTherefore, according to the positive relationship of MSSA isolates, prevalence between schoolchildren and the environment and the homology of MSSA isolates between schoolchildren and environment based on phenotypic, genetic, and molecular characteristics, we suggest that both the decontamination of MSSA in the environment and the increasing hygiene of schoolchildren could be beneficial to reduce the prevalence of MSSA nasal colonization among schoolchildren.One of the strengths of this study is that the sample size is relatively large and representative. Given considerable variability in BMI, it enabled us to make robust estimates of the relationships. The relatively large number of schoolchildren with a high response rate yields high statistical power to infer the characteristics of the study population. Moreover, this study is the first to elucidate the quantitative association of isolates between schoolchildren and the environment. We also performed phenotypic and molecular characterization of MSSA isolates to further assess the relationship.This study also has several limitations. First, we cannot establish a cause and effect relationship because the study design is cross-sectional. Second, one of the strengths of the study is the use of multistage stratified cluster regression analysis, which overcomes the limitations of the standard regression analysis previously performed. Finally, given limited financial support, we could not offer decolonization measurements to schoolchildren, and whether such intervention would improve health of schoolchildren could not be scientifically proven.Despite these limitations, our findings suggest that both schoolchildren and the school environment were colonized with MSSA. Therefore, environmental decontamination, including enhancement of disinfection and cleaning, may be considered as strategies to decrease and prevent MSSA nasal colonization in schoolchildren.S1 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 Data(XLS)Click here for additional data file.


We investigated the role of PD-L1 in the metabolic reprogramming of non-small cell lung cancer (NSCLC).low and PD-L1high NSCLC cells after transfection or knockdown of PD-L1, respectively. Jurkat T-cell activation was assessed after co-culture with NSCLC cells. The association between PD-L1 and immune response-related molecules or glycolysis were analyzed in patients with NSCLC and The Cancer Genome Atlas (TCGA).Changes in glycolysis-related molecules and glycolytic activity were evaluated in PD-L1PD-L1 in PD-L1low cells enhanced hexokinase-2 (HK2) expression, lactate production, and extracellular acidification rates, but minimally altered GLUT1 and PKM2 expression and oxygen consumption rates. By contrast, knocking-down PD-L1 in PD-L1high cells decreased HK2 expression and glycolysis by suppressing PI3K/Akt and Erk pathways. Interferon-γ (IFNγ) secretion and activation marker expression was decreased in stimulated Jurkat T-cells when co-cultured with HK2-overexpressing vector-transfected tumor cells rather than empty vector-transfected tumor cells. Immunohistochemistry revealed that PD-L1 expression was positively correlated with HK2 expression in NSCLC (p < 0.001). In TCGA, HK2 exhibited a positive linear association with CD274 (PD-L1) expression (p < 0.001) but an inverse correlation with the expression of CD4, CD8A, and T-cell effector function-related genes in the CD274high rather than CD274low group. Consistently, there were fewer CD8+ T-cells in PD-L1positive/HK2high tumors compared to PD-L1positive/HK2low tumors in squamous cell carcinoma.Transfecting PD-L1 enhances glycolysis in NSCLC by upregulating HK2, which might dampen anti-tumor immunity. PD-L1 may contribute to NSCLC oncogenesis by inducing metabolic reprogramming and immune checkpoint. In addition, the ORR was higher in the PD-L1positive/HK2low group than the PD-L1positive/HK2high group, although statistically insignificant , sarcoma , and stomach adenocarcinoma , but not in renal cell carcinoma and urothelial carcinoma (data not shown). These findings suggest that a functional link between PD-L1 and HK2 might occur in various cancers in a histologic type-dependent manner.Several studies have found that HIF-1α enhances glycolysis by upregulating glycolytic enzymes , 23, andIn this study, we confirmed that the changes in surface PD-L1 as well as total PD-L1 expression after PD-L1 overexpression and knockdown , in a co-culture system. These findings were further validated using TCGA data and IHC for lung cancer tissues. + T-cell, tumors can be divided into four groups: PD-L1high/CD8+ T-cellhigh (TMIT I), PD-L1low/CD8+ T-celllow (TMIT II), PD-L1high/CD8+ T-celllow (TMIT III), and PD-L1low/CD8+ T-cellhigh (TMIT IV). Patients with TMIT I tumors are considered the best candidates for PD-1/PD-L1-targeted therapy [high/CD8+ T-celllow TMIT III NSCLCs could be characterized by high HK2 expression and glycolytic signature.When classifying TME immune types (TMIT) based on PD-L1 expression and CD8 therapy , 26. By  therapy . The curpositive/HK2low NSCLC compared to those with PD-L1positive/HK2high NSCLCs, but no difference in the survival time between these groups. These results might be attributable to small number of patients, variable performance status and previous treatment history, laboratory-developed PD-L1 test, limitations of retrospective evaluation, and a possible occurrence of resistance mechanisms other than HK2 overexpression. Therefore, the clinical value of HK2 as a potential metabolic biomarker for anti-PD-1/PD-L1 therapy needs further validation.Regarding functional aspects of the immune responses, our results suggest that PD-L1-mediated HK2/glycolysis upregulation of tumor cells might dampen T-cell function in the TME of NSCLCs. In previous studies, tumor-derived lactate, a metabolite of glycolysis, inhibits T-cell function , and uprMoreover, HK2 itself could be a therapeutic target in cancer therapy. HK2 participates in aerobic glycolysis and can affect many other metabolic processes through its product, glucose-6-phosphate. Besides metabolic process, HK2 has been demonstrated to prohibit apoptosis. HK2 binds to a voltage-dependent anion channel (VDAC) and the HK2/VDAC interaction stabilizes mitochondrial membrane and prevents binding pro-apoptotic factors to VDAC –31. TherIn summary, this study demonstrates that PD-L1 increases glycolysis in NSCLC cells by upregulating HK2, which is associated with reduced expression of T-cell effector genes. These findings suggest that PD-L1 expression might enhance tumor cell survival by increasing glycolysis intrinsically and providing inhibitory signals to neighboring T-cells extrinsically, through interaction with PD-1 and metabolic competition. Thus, HK2 expression and glycolysis could be potential biomarkers or therapeutic targets in NSCLC patients in the era of cancer immunotherapy.Additional file 1: Supplementary material and methods. More detailed information about Patients and samples, FDG-PET/CT image analysis, Glycolysis assays, Oxygen consumption rate (OCR) assay, RNAseq and Gene set enrichment analyses, TCGA analyses, Immunohistochemistry (IHC) and Statistical analysis was addressed. (DOCX 45 kb)Additional file 2:Table S1. Clinicopathological parameters of patients with NSCLCs. Table S2 Clinicopathological features of NSCLC patients with PD-1 blockade. (DOCX 21 kb)Additional file 3:Figure S1. The expressions of HK2 and glycolytic activity are elevated in PD-L1high lung cancer cell lines. Figure S2. PD-L1 overexpression or knockdown does not affect oxidative phosphorylation. Figure S3. PD-L1 overexpression or knockdown does not affect the mRNA level of glycolysis-related genes, other than HK2. Figure S4. PD-L1 expression is positively correlated with glycolysis signature in NSCLC cells. Figure S5. Glycolysis-related parameters analyzed by PET scanning and immunohistochemistry in NSCLC patients. Figure S6. Basal expression of glycolysis-related molecules according to PD-L1 expression in pSqCC and pADC patients. Figure S7. Expression levels of T-effector immune response-related genes according to CD274 (PD-L1) and SLC2A1 (GLUT1) or PKM expression status in NSCLC from TCGA data. Figure S8. HK2 mRNA is higher in TIMT III (CD274high/HK2low) than in TIMT I (CD274high/HK2high) NSCLC from TCGA data. Figure S9. High HK2 expression is related to a lower response rate to PD-1 blockade in patients with NSCLC. Figure S10. A model figure of this study. (PDF 1278 kb)


Inkjet-printed wearable electronic textiles (e-textiles) are considered to be very promising due to excellent processing and environmental benefits offered by digital fabrication technique. Inkjet-printing of conductive metallic inks such as silver (Ag) nanoparticles (NPs) are well-established and that of graphene-based inks is of great interest due to multi-functional properties of graphene. However, poor ink stability at higher graphene concentration and the cost associated with the higher Ag loading in metal inks have limited their wider use. Moreover, graphene-based e-textiles reported so far are mainly based on graphene derivatives such as graphene oxide (GO) or reduced graphene oxide (rGO), which suffers from poor electrical conductivity. Here we report inkjet printing of highly conductive and cost-effective graphene-Ag composite ink for wearable e-textiles applications. The composite inks were formulated, characterised and inkjet-printed onto PEL paper first and then sintered at 150 °C for 1 hr. The sheet resistance of the printed patterns is found to be in the range of ~0.08–4.74 Ω/sq depending on the number of print layers and the graphene-Ag ratio in the formulation. The optimised composite ink was then successfully printed onto surface pre-treated (by inkjet printing) cotton fabrics in order to produce all-inkjet-printed highly conductive and cost-effective electronic textiles. Among them, wearable electronic textiles (e-textiles) that contain electronics and interconnections woven/knitted or printed/coated onto or into textiles11, are of great interest, as they provide better comfortability, durability and lighter weight as well as maintaining the desirable electrical property. Inkjet printing is one of the most promising techniques for the fabrication of flexible and wearable electronics, due to a number of advantages over conventional manufacturing techniques such as digital and additive patterning, reduction in material waste, deposition of a controlled quantity of materials and compatibility with various substrates13. Currently silver nanoparticles (NPs) based inkjet inks are the most reported and utilised conductive inks due to their excellent electrical conductivity and strong antioxidant characteristics16. However, a higher concentration of NPs and higher sintering temperatures are required in order to obtain a continuous metallic phase, with numerous percolation paths between the metal particles within the printed pattern18. Moreover, the higher price of silver increases the processing cost15. In addition, some of the printed substrates are also sensitive to higher temperature, which limited the choice of the substrates to be printed14.Printed flexible electronics have demonstrated great potential for further growth with the applications in health diagnostics2 carbon allotropes, has been the focus of extensive investigations in recent years due to its unique physical and chemical properties20. Inkjet printing of graphene-based inks is an encouraging research approach in the field of printed electronics as both the benefits of inkjet printing12 and the extra-ordinary electronic21, optical and mechanical properties23 of graphene can be exploited24. Several studies27 have reported inkjet printing of reduced graphene oxide (rGO) as a popular choice to fabricate flexible devices, as it is readily dispersible in water, and higher volume can be produced at lower cost28. Our recent studies have shown promise of using graphene derivatives such as GO or rGO for wearable e-textiles applications31. However, the large number of unreduced oxygen-containing functional groups and inter-sheet junctions between the graphene flakes limit the conductivity achieved with rGO32. In order to overcome such limitations associated with rGO inks, pristine graphene inks were developed and used for printing. However, a number of limitations still exist with such inks and their fabrication process developed so far: such as lower graphene concentration and residual solvent in the printed electronics34, time consuming process and toxic solvents24, repeated and complicated process12, a higher annealing temperature and a number of inkjet layers needed to achieve the desired conductivity35.Graphene, a single atom thick two-dimensional closely packed honeycomb lattice of sp39 reported composite formulations containing metal nanoparticles such as Ag and Cu, and carbon-based materials such as graphene and carbon nanotubes (CNT), however limited to lower concentration and not demonstrated for printing onto challenging substrates like textiles for wearable applications. Here we report inkjet printing of highly conductive graphene-silver nanoparticles-based composite ink for wearable e-textiles applications. We optimised the formulation using a cube film applicator first and then formulated a range of composite inkjet inks. Finally the optimised graphene-silver composite ink was printed on surface pre-treated (by inkjet) textiles for highly conductive wearable e-textiles applications.In order to overcome such challenges, here we propose the formulation of highly conductive silver nanoparticles decorated graphene-based composite inkjet ink that can achieve desired electrical conductivity with few layers of printing. Previous studiesA range of composite formulations were prepared using a silver nanoparticle-based ink  and a highly concentrated graphene dispersion . A simple triple reservoir cube film applicator was used to produce conductive films (~90 µm thick) of composite formulations on a glass substrate. The formulations were then optimised by measuring the sheet resistance of such conductive films. The conductive film with BS8 ink on glass substrate provides higher sheet resistance (~32 kΩ/sq). As expected, with the addition of SA-Ag to the composite ink formulation, the sheet resistance of the conductive film decreases with the increase of wt.-% of highly conductive SA-Ag (as received) nanoparticles (~0.7 Ω/sq), Fig. 14. PVP is a linear homopolymer of N-vinylpyrrolidone, and soluble in water and other polar solvents. It forms a coating on graphene flake surfaces to prevent aggregation in aqueous dispersion40. The presence of graphene flakes in the SA-Ag/BS8 composite increases the electrical conductivity, as the conductive film drawn with such ink imparted lowest sheet resistance (~7.73 Ω/sq) out of three formulations presented in Table 42, therefore in terms of volume the SA-Ag/BS8 composite formulation is mostly dominated by BS8 (~70.57 vol.-%) with less Ag (either vol.-% or wt.-%) content than the other formulations. Graphene flakes provide an effective pathway in both the length and width directions of the conductive film to avoid negative influence of the void15. Moreover, graphene has very high surface area with theoretical value ~2600 m2 g−1, which causes spread out of the transparent graphene in all directions of the film to create a bridge between the aggregates of Ag NPs. Thus good contact between graphene and silver is achieved to provide better conductivity.In order to demonstrate the effect of graphene flakes on the conductivity of composite formulations, SA-Ag inks were also diluted with TEGMME and 1 wt.-% PVP in TEGMME. Again the conductive patterns were drawn using a cube film applicator and sintered at 150 °C for 1 hr. The results are compared in Table 2 conductive pattern with formulated ink, even after adding a stabiliser (PVP). This may be due to the van der Waals forces between highly concentrated graphene sheets with a higher aspect ratio, which causes them to stick together to form large bundles or ropes, and clogs the nozzles of the inkjet print head43. The sheet resistance of the printed patterns is found to be ~49.26, ~40.99 and ~28.13 Ω/sq for 1, 2 and 3 layers, respectively, which are lower than those achieved with inkjet-printed pristine graphene in several previous studies reported in this review44. Moreover, BS8 provides lower sheet resistance on surface pre-treated PEL paper than glass, may be due to the better overlapping and connections between individual flakes. Please note, the thickness of the conductive film on paper cannot be measured accurately due to the roughness of the substrate.A Dimatix Materials Printer (DMP) was used for inkjet printing of conductive inks onto a PEL paper first. Therefore, the surface tension of BS8 graphene dispersions was adjusted to ~34.5 mN/m by adding 1 wt.-% non-ionic surfactant (Triton X-100) to make it inkjettable in DMP. However, the higher voltage was required to jet ink droplets through the nozzles and chaotic jets were observed after printing three layers of a 1 cmIn contrast, the viscosity and surface tension of SA-Ag inks are reported as ~10–18 cps and ~30–40 mN/m, respectively, as per the technical information supplied by Sigma-Aldrich, which are within the DMP printing range. The ink was jetted effectively through all the inkjet nozzles and 16 jets were working well at 25 V. The sheet resistance of the conductive pattern printed with SA-Ag inks are ~0.07, ~0.04, ~0.04, ~0.03 and ~0.03 Ω/Sq for 1 L, 2 L, 3 L, 4 L and 5 L, respectively Fig. . It is a45. In order to improve the stability of the formulated ink, a polymer stabiliser PVP (dissolved as 1 wt.-% in TEGMME) was then added to Ink A in different quantities in order to prepare a range of inks, Table via DMP inkjet printer and uninterrupted printing of a few layers of composite inks. Firstly, TEGMME (~31.4 mN/m) has lower surface tensions, which helps to reduce the surface tension of composite ink formulations and keep it within the range of DMP inkjet printer. Secondly, PVP improves the ink stability by forming a coating on graphene flake surfaces to prevent aggregation in aqueous dispersion40. In addition, PVP is the most commonly used stabiliser for metal NPs-based inks in various solvents, which interact with metal NPs through the oxygen atom of the carbonyl group46. However, the presence of a stabiliser in a higher quantity could have a negative effect on the conductivity, as it acts as an insulating organic layer on NPs surfaces that prevents close electrical contacts between NPs14.We then formulated a range of silver decorated graphene-based composites inks Table  on the b17. The coating of Ag NPs onto graphene sheets increases the contact interfaces and reduces the contact resistance between graphene sheets; thus improves the conductivity of the film37. Moreover, graphene also provides effective electrical networks between the silver flakes36, by reducing the negative influence of the voids present in the printed silver film17. The sheet resistance of a single layer (1 L) inkjet-printed conductive pattern with Ink B is found to be ~4.74 Ω/Sq, which is reduced approximately by 3 times to ~1.70 Ω/Sq when the bulk wt.-% and vol.-% of SA-Ag increases to 44.63% and 89.36%, respectively, in the formulation, Ink C  in order to form a continuous electrically conductive film. The temperature required for sintering varies from ink to ink due to the different boiling points of the organic solvents, dissociation temperatures of the salts, the diameter of the NPs and graphene flakes. Figure 38. The thermal decomposition of composite ink (Ink C) is found to be almost similar to that of SA-Ag ink; however, the second stage decomposition for that ink is observed from 265 °C to 380 °C with a slight decrease in weight (~2.45 wt.-%).As illustrated in Fig. SEM was used in order to analyse the surface topography of the printed pattern on PEL paper. Figure 12. However, inkjet printing of low viscosity inkjet inks onto a rough and porous textile surface is of great challenge, due to the orientation of fibres or yarns47 and the change of fibre morphology constantly due to the exchange of water molecules with surroundings9. In order to address these challenges, in our previous study10, we reported synthesis of a novel surface pre-treat (NP1) and inkjet printing of 12 layers NP1 onto rough and porous textiles with subsequent printing of rGO on surface pre-treated textiles in order to produce continuous conductive pathway onto textiles. However, rGO surfers from poor electrical conductivity due to the partial restoration of sp2 carbon after reduction of GO.Inkjet printing of electrically conductive pathway on textiles is of interest, as inkjet offers a number of advantages over conventional manufacturing techniques, such as high resolution printed circuit, deposition of controlled amount of conductive materials at precise locations, reduction of materials waste and usage of natural resources such as waters, gas and electricityLike previous study, here we printed 12 layers of NP1 on a rough and porous textile surface. However, we printed highly conductive graphene-silver composite (Ink C) as subsequent conductive layers (6 L). Table SEM images of inkjet-printed e-textiles with composite Ink C Fig.  provide 30. In order to demonstrate the suitability of the printed textiles for wearable strain sensor application, we mounted the printed fabric on a wrist joint and recorded the electrical resistance variation continuously during cyclic upward and downward bending of the wrist. Figure We further investigated the flexibility of graphene-silver composite ink printed wearable e-textiles. Figure We report inkjet printing of graphene-based composite inks decorated with silver nanoparticles for wearable e-textiles applications for the first time. The conductivity of graphene-based e-textiles can be increased by layer by layer inkjet deposition of SA-Ag/BS8 composite ink. The graphene-silver composites approach could potentially reduce the production cost of inkjet-printed wearable e-textiles by reducing the silver consumption. This will open up so many potential applications of graphene-based wearable e-textiles where higher electrical conductivity is desired.10.Highly concentrated graphene dispersion  was supplied by BGT Materials Limited, UK. The graphene dispersion (BS8) was stabilised using a non-ionic polymer-type surfactant. Silver nanoparticle inks , Triethylene glycol monomethyl ether (TEGMME), Polyvinylpyrrolidone (PVP) of 10 K molecular weight and Triton X-100 were purchased from Sigma-Aldrich. Nano 60 PEL paper was purchased from Printed Electronics Limited, UK. 100% Cotton woven fabrics (BD022) were supplied by Royal TenCate, Netherlands. Hydroxyl functionalised cross-linked styrene/divinylbenzene nanoparticles (NP1) were synthesized by following our previously reported methodIn order to find the optimum percolation threshold for diluted SA-Ag ink, a series of composite inks was formulated by blending BS8, TEGMME and 1% PVP (in TEGMME) with SA-Ag inks. The formulated composite inks were deposited onto PEL paper using a triple reservoir cube film applicator  and cured at 150 °C for 1 hr to form 90 µm thick conductive films.2 area and thermally-cured at 150 °C for 1 hr in an oven to sinter the conductive inks.Composite inkjet inks were formulated with an optimised concentration of SA-Ag inks and various concentrations of BS8 dispersion and 1 wt.-% PVP in TEGMME. The formulated inks were inkjet-printed using a Dimatix DMP-2800 inkjet printer  which can create and define patterns over an area of 200 × 300 mm and handle substrates up to 25 mm thick, being adjustable in the Z direction. This printer is equipped with a disposable piezo inkjet cartridge and the nozzle plate consists of a single row of 16 nozzles of ~21.5 µm diameter spaced 254 µm with typical drop diameter ~27 µm and 10 pl drop size. Print head height was adjusted to 0.75 mm and the formulated inks were jetted at 37 °C temperature, using frequent cleaning cycles during the printing. A few layers (1–5 L) of composite inks were printed to produce a conductive pattern of 1 cm10. Subsequently, six layers (6 L) of graphene inks (formulated from BS8 dispersion) or composite inks C were inkjet-printed onto hydrophobic areas of cotton fabrics.In order to demonstrate the potential electronic textiles applications of graphene-based composite inks, a hydrophobic coating was inkjet-printed onto 100% cotton plain twill fabrics (B022) by depositing 12 layers of nanoparticles (NP1) as reported in our previous studyThe Raman spectra of BS8 was obtained from a low power (<1 mW) He-Ne laser  in Renishaw 2000 spectrometer. The viscosity of formulated inks was measured using a Brookfield DV-II+PRO programmable digital viscometer at 25 °C temperature and surface tension was measured by using a torsion balance (model OS) for surface and interfacial tension measurement. Thermogravimetric Analysis (TGA) was conducted to investigate the thermal stability of formulated inks using a TGA Q500 . A Philips XL 30 Field Emission Gun Scanning Electron Microscope (SEM) was used to analyse the surface topography. Printed samples were gold-palladium (Au-Pd) coated for 90 seconds and assessed under FEG SEM with the following operating parameters: 6.0 KV, spot size 2.0, 10 mm WD and magnification: X500 to X40000. A Jandel four-point probe system  was employed to measure the sheet resistance of the conductive patterns. The sheet resistance was calculated from the average of six measurements and multiplied resistance by a correction factor of ~4.5324.A Zwick/Roell tensile tester  was used to measure the change of the resistance during bending and unbending of graphene/silver composites ink printed textiles. A National Instrument 9219 data acquisition card  was used to record the change of the resistance during bending, folding and upward-downward movement of the wrist joint.


Twisted bi-layer graphene (tBLG) has attracted much attention because of its unique band structure and properties. The properties of tBLG vary with small differences in the interlayer twist angle, but it is difficult to accurately adjust the interlayer twist angle of tBLG with the conventional fabrication method. In this study, we introduce a facile tBLG fabrication method that directly picks up a single-crystalline graphene layer from a growth substrate and places it on another graphene layer with a pre-designed twist angle. Using this approach, we stacked single-crystalline graphene layers with controlled twist angles and thus fabricated tBLG and twisted multi-layer graphene (tMLG). The structural, optical and electrical properties depending on the twist angle and number of layers, were investigated using transmission electron microscopy (TEM), micro–Raman spectroscopy, and gate-dependent sheet resistance measurements. The obtained results show that the pick and place approach enables the direct dry transfer of the top graphene layer on the as-grown graphene to fabricate uniform tBLG and tMLG with minimal interlayer contamination and pre-defined twist angles.  Bi-layer graphene, in which two sheets of monolayer graphene are stacked, has unique physical and electrical properties that are different from monolayer graphene ,2. TypicIn this study, we introduce a facile pick and place method for fabricating precise and uniform twist bi-layer and a multi-layer graphene structure with a clean interface. We were able to selectively separate the desired shapes of the graphene on as-grown large-area single-crystalline graphene by the pattern pick up process. After that, the tBLG was fabricated by rotating the angle and placing it on the as-grown graphene again. The van der Waals interaction of the tBLG fabricated on the catalyst substrate was stronger than the interaction between the catalyst substrate and the graphene, thus the tBLG could easily be separated from the catalyst substrate and transferred to the target substrate. The fabricated tBLG has an accurate and uniform twist angle, which was confirmed by Raman spectroscopy and transmission electron microscope analysis.2 square pattern on the graphene grown on Ge wafers. A 40 nm thick gold (Au) film was deposited onto the pattern using a thermal evaporator. Then, Au-patterned graphene substrate was annealed in a hydrogen (H2) and argon (Ar) atmosphere at 200 °C and 100 torr for 2 h.Graphene was synthesized on Germanium (Ge) (110) substrate by the CVD method . E-beam 2 plasma treatment  and polypropylene carbonate (PPC) was spin-coated  on the surface of the PDMS stamp. A 10:1 ratio mixture of SYGARD 184 base and curing agent was dropped onto a slide glass and cured at 70 °C to produce a polydimethylsiloxane (PDMS) stamp. The surface of the PDMS stamp was hydrophilized by O2 plasma cleaning . For the pick and place process, we used home-made transfer equipment consisting of an optical microscope, a sample stage  manual stage with 360° rotation and hot chuck), and a stamp stage . The PPC/PDMS stamp immobilized on the stamp stages was contacted with the gold pattern on the as-growTn graphene. Then, the temperature was slowly raised to 50 °C, and the stamp was lifted to exfoliate the gold pattern and the underlying graphene from the Ge substrate. The gold/graphene on the stamp was attached to the target substrate at 130 °C. As the adhesion between the PPC and gold/graphene weakened at the high temperature, gold/graphene could be placed on the target substrate. It was then soaked in acetone for 20 min to remove any polymer on the surface of the gold . The rem2/Si 300 nm substrate at 2000 rpm for 1 min. The spin-coated substrate was baked at 100 °C for 5 min and floated on the etching solution (HF:H2O2:H2O = 1:1:1). As the SiO2 etched, the PMMA/tBLG was separated from the substrate and floated on the DI-water for cleaning. The PMMA/ tBLG was placed onto a TEM grid, and then the PMMA was removed by using acetone vapor and solution.Polymethyl methacrylate (PMMA) was spin-coated on the tBLG/SiO2/Si 300 nm substrate by standard photolithography and metal electrode deposition . The sheet resistance of the graphene FET was measured using a Keithley SCS-4200 system at room temperature. The charge carrier density was calculated by using a simple Drude model and the carrier mobilities were extracted at a high carrier concentration of n = 3 × 1012 cm−2.The back gated graphene field-effect transistor (FET) were fabricated on SiOThe high-resolution transmission electron microscopy (HR–TEM) images and the selected area electron diffraction (SAED) patterns of graphene were taken using a JEOL ARM 200F  at 80 kV. A Raman microscope  with excitation energy of 532 nm at the MEMS·Sensor Platform Center of Sungkyunkwan University , was used for the Raman measurements. X-ray photoelectron spectroscopy (XPS) analysis was carried out by ESCA2000 spectrometry  using Al-Kα radiation (1468.6 eV).2 atmosphere resulted in hydrogen intercalation between the graphene and the Ge substrate [2 substrate after the gold pattern was removed. Alternatively, the tBLG was produced by transferring the picked-up gold/graphene to the as-grown graphene on Ge again with a controlled twist angle. The resulting gold/tBLG on the Ge substrate was thermally annealed in a mixed H2 and Ar atmosphere to enhance the van der Waals interaction between the two graphene layers and weaken the adhesion between the graphene and Ge. As van der Waals interaction between graphene layers is stronger than the adhesion between graphene and Ge, the gold/tBLG was easily picked up from the Ge substrate in the same way as gold/graphene. The gold on tBLG prevents the contamination of the graphene surface that may occur during the pick and place process [2 substrate, after the gold pattern was removed, shows a darker color than the monolayer graphene  seen in the HR–TEM image of the 12° tBLG is 1.187 ± 0.005 nm, which is well-matched with the Moiré pattern wavelength of the twist angle 12° calculated using the Equation (1)a” is the length of graphene’s lattice constant and θ is the twist angle [The SAED pattern obtained from the tBLG, designed with a 12° twist angle, demonstrates that the pick and place approach in the present work allows accurate control of the interlayer twist angle a. The wast angle ,34.To further investigate the property change of the tMLG according to the number of layers, we stacked the graphene layers one by one with the same twist angles. As shown in 2 V−1 s−1, 30°: 1561 cm2 V−1 s−1) have higher value than mono-layer (1298 cm2 V−1 s−1), while the AB-staked bi-layer (567 cm2 V−1 s−1) has a relatively low hole mobility. In addition, the tBLG devices with 3°, 12° and 18° angles were fabricated, and their hole mobilities were calculated as 1513, 1841, and 1814 cm2 V−1 s−1, respectively (We fabricated the back gate field-effect transistors (GFETs) of the tBLGs to evaluate the twist-angle-dependent electrical properties of the tBLGs. ectively . When coIn this study, uniform tBLG and tMLG were fabricated with a designed interlayer twist angle by the pattern pick and place method. Using the pick and place method, tBLG can be fabricated by directly stacking as-grown graphene, without the exposure to supporting materials or solution, and thereby minimizing the interlayer residues that adversely affect the properties of tBLG. We also demonstrated that, in the multilayer structure with more than two layers, the middle graphene layer interacted with the upper and lower graphene layers separately, and blocked the interaction between the upper and lower graphene. Thus, we can consider the twisted multi-layer graphene as a structure in which several tBLGs are not coupled but stacked. In addition, a tBLG structure with a large twist angle was confirmed to improve the charge carrier mobility of the graphene device. This method may enable further research on the complicated multilayer structure of 2-dimensional materials with a controlled interlayer twist angle, as well as tBLG and tMLG.