Cordyceps belongs to a genus of acormycete fungi and is known to exhibit various pharmacological effects. The aim of this study was to investigate the effect of Cordyceps species on the proliferation of vascular smooth muscle cells (VSMC) and their underlying molecular mechanism. A cell proliferation assay showed that Cordyceps bassiana ethanol extract (CBEE) significantly inhibited VSMC proliferation. In addition, neointimal formation was significantly reduced by treatment with CBEE in the carotid artery of balloon-injured rats. We also investigated the effects of CBEE on the extracellular signal-regulated kinase (ERK) signal pathway. Western blot analysis revealed increased ERK 1/2 phosphorylation in VSMCs treated with CBEE. Pretreatment with U0126 completely abrogated CBEE-induced ERK 1/2 phosphorylation. In conclusion, CBEE exhibited anti-proliferative properties that affected VSMCs through the ERK1/2 MAPK signaling pathway. Our data may elucidate the inhibitory mechanism of this natural product. Atherosclerosis is a disease characterized by thickening of the inner portion of the artery walls . One of Cordyceps belongs to a genus of acormycete fungi that includes about 400 species [Cordyceps species are C. sinensis and C. militaris, which are have been used as traditional medicine for various diseases in Asian countries. These exhibit various pharmacological effects including anti-inflammatory, antioxidative, antidiabetic, and anticancer properties [ species . RepreseCordyceps species have yet to be addressed. We investigated the anti-proliferative activity of Cordyceps species extracts in VSMC and studied the underlying signal transduction mechanism. Therefore, our study investigates that the molecular mechanism underlying the anti-atherosclerotic effects of Cordyceps species extracts. This pathway might highlight a potential therapeutic intervention for cardiovascular disease.Cordycepin inhibits VSMC proliferation . HoweverCordyceps bassiana ethanol extract (CBEE), Cordyceps militaris butanol extract (CMBE), and Cordyceps pruinosa methanol extract (CPME) were extracted using conventional extraction methods [ methods . The sigCordyceps (175 g) were powdered and extracted at 70 ~ 80 °C with 80 % ethanol, methanol and butanol. The extract was filtered, taken to dryness under reduced pressure and concentrated by a rotary vacuum evaporator . The lyophilized crude extracts were sequentially fractionated with n-hexane (Hexane), ethyl acetate (EtOAc) and n-butanol (BuOH). Each extract was dissolved in dimethyl sulphoxide (DMSO) as a concentration.Solvent extraction was performed as described previously , 7. In b2 flasks in a 37 °C incubator at 5 % CO2.Rat aortic smooth muscle cells (RaoSMCs) were cultured in DMEM supplemented with fetal bovine serum  in 75 cm3 cells per well. The cells were starved with 0.1 % FBS for 24 h and then treated with three kinds of Cordyceps extracts or 10 % serum. After treatment, cell proliferation was measured using a CCK-8 assay kit . The CCK-8 assay kit was treated with DMEM, and then 100 μl was added to each well and incubated for 2 h at 37 °C. The absorbance was measured at 450 nm with a spectrometer.RaoSMCs were plated in triplicate in 96-well plates at 2 × 10Animal experiment conforms to the Animal Care and NIH guidelines were approved by the local animal care committee. Balloon denudation of the common carotid artery endothelium was evoked in four-week old 250 g Male Sprague–Dawley rats  as previously reported . All ratSprague–Dawley rats were sacrificed 3 weeks after the balloon injury. And their carotid arteries were harvested and fixed with 4 % paraformaldehyde. Within 24 h after fixation, each section was embedded in OCT compound , snap frozen in liquid nitrogen, and sectioned in thickness increments of 10-μm carotid artery sections were cut with a microtome and mounted on slides. Sections were stained with hematoxylin and eosin (H&E) and evaluated with light microscopy to assess the histological effects. Normal and neointimal areas were measured using NIH images.The western blot analysis was performed as previously described , 10. Celt-test for unpaired samples between two groups. p < 0.05 was considered statistically significant.All data are presented as mean ± standard deviation (SD) from at least three independent experiments. The statistical significance was determined using the Student’s To confirm the effect of CBEE on VSMC anti-proliferation, cells were cultured for 24 h in complete medium containing 10 % FBS at various concentrations of CBEE (0 ~ 4 μg/ml). As shown in Fig. Cordyceps species, VSMC were cultured for 3 days in complete medium containing 10 % FBS with three Cordyceps species: CBEE, CMBF, and CPME. A cell proliferation assay showed that CBEE significantly inhibited VSMC proliferation   treated group exhibited a significant reduction of neointima formation compare with control (vehicle) group (19) Fig. .Fig. 2EfTo better understand the molecular mechanisms involved in the VSMC anti-proliferative effects of CBEE, we investigated the possible involvement of the MAPK signaling pathway. To determine whether extracellular signal–regulated kinase (ERK) 1/2 activation is involved in CBEE-induced VSMC proliferation, we treated cells with the MEK/ERK inhibitor U0126 for 1h and then stimulated with CBEE for 72 h. Proliferation assays were then performed with the CCK-8 assay. The ERK1/2 inhibitor U0126 significantly blocked CBEE-mediated VSMC anti-proliferation in a dose-dependent manner, as examined by cell counting Fig. .Fig. 3EfOur previous results prompted us to further investigate whether CBEE activates the ERK 1/2 signaling pathway. To address this issue, cell lysates were probed for ERK 1/2 phosphorylation and protein levels. Western blot analysis revealed increased ERK 1/2 phosphorylation in VSMCs treated with 4 μg/mL CBEE after 3, 6, and 24 h, with the maximal increase occurring after 3 h of treatment  ERK 1/2 phosphorylation Fig. .Fig. 5TiOur results clearly demonstrate that CBEE inhibits VSMC proliferation. Notably, CBEE inhibited VSMC proliferation by modulating the ERK1/2 MAPK signaling pathway. To the best of our knowledge, this is the first report connecting molecular mechanisms of CBEE to its anti-proliferative effects on VSMC.Endothelial injury initiates the pathogenesis of various vascular diseases such as atherosclerosis . ChronicCordyceps species on VSMC are not fully understood. We found CBEE, which contained a yellowish colored compound, was reported to have significant anti-proliferative effects on VSMC. However, data on the biological activities of CBEE are limited. One recent report demonstrated that CBEE suppressed the expression of interleukin-12 and phosphorylation of p38 in lipopolysaccharide-activated macrophages without altering cell viability [Cordyceps species extracts (CMBF or CPME) could exhibit inhibitory effect on VSMC. However, major active compounds derived from CBEE and their anti-proliferative or anti-inflammatory properties compare with CMBF or CPME should be clarified in further studies.Recent reports indicate that cordycepin inhibits VSMC proliferation  and atteiability . InteresAccording previous reports, ERK1/2 has distinct functions in the inhibition of cell growth . Jung etCordyceps bassiana, and helps us to understand its protective effects in cardiovascular disease-associated pathophysiology. However, further studies are necessary to identify the major active components of CBEE.In conclusion, CBEE exhibited anti-proliferative properties that affected VSMC through the ERK1/2 MAPK signaling pathway. Our data may elucidate the inhibitory mechanism of this natural product,


To determine the optimal extraction conditions, a number of experimental factors  were studied. The compounds of interest were separated and detected by an HPLC-UV employing a Kinetex XB-C18 column and a mobile phase consisting of acetonitrile and water flowing in a gradient mode. The optimized extraction conditions were: the amount of graphene 15 mg, desorbing solvent (acetonitrile) 5 mL, time desorption 10 min at 40°C, and amount of NaCl 1 g. The detection limit for metazachlor, tebuconazole, λ-cyhalothrin, and chlorpyrifos was 62.5 ng·g−1, and for deltamethrin, it was 500 ng·g−1. The obtained results lead to the conclusion that graphene may be successfully used for the isolation of the five pesticides from rape oil. However, their determination at low concentration levels, as they occur in real oil samples, requires the employment of appropriately highly sensitive analytical methods, as well as a more suitable graphene form .Isolation conditions for five pesticides (metazachlor, tebuconazole,  Due to the potential toxicity, bioaccumulating properties, and wide use of pesticides for supporting and intensification of crops almost all over the world, these substances belong to the most often controlled ones in the environment and in food. Every year, many publications devoted to the development of new methods for pesticide determination in various food matrices appear. However, pesticide residue analysis in high-fat samples is still a challenging analytical task. Edible oil samples belong to such analytically difficult matrices. Due to complex matrices, as well as low concentrations of pesticide residues, frequently originated from various classes, these samples are analyzed using such advanced coupled techniques as LC-MS/MS , 2, LC-Tπ-π interactions between its large delocalized electron system and the aromatic rings of the target molecules In most cases of pesticide analysis involving graphene as an adsorbent, magnetic solid-phase extraction (MSPE) was applied where magnetically modified graphene composites as adsorbents were used. Only few works reported the employment of nonmodified graphene alone or in combination with other sorbents (in the modified QuEChERS method) using dispersive solid-phase extraction. This is the first time when graphene in its nonmodified form, using d-SPE, was studied as the adsorbent for the pesticides from four different groups present in such analytically difficult matrices as edible oil. Graphene may be successfully exploited in the preparation of edible oil samples for determination of pesticides from various groups. However, some improvements should be introduced in such analyses. For example, a more sensitive analytical method  may be used. Additionally, a more suitable modified graphene form, for example, magnetically modified graphene, which will prevent the nanomaterial aggregation, increase its dispersion in solvents, and improve its adsorption properties, would better enable separation of graphene from the isolated analytes in the supernatant using an external magnet.


Botrytis cinerea in Arabidopsis thaliana and Camelina sativa overexpressing DEWAX and in Arabidopsis dewax mutant. Compared to wild type (WT) leaves, Arabidopsis DEWAX OX and dewax leaves were more and less permeable to toluidine blue dye, respectively. The ROS levels increased in DEWAX OX leaves, but decreased in dewax relative to WT leaves. Compared to WT, DEWAX OX was more resistant, while dewax was more sensitive to B. cinerea; however, defense responses to Pseudomonas syringae pv. tomato DC3000:GFP were inversely modulated. Microarray and RT-PCR analyses indicated that the expression of defense-related genes was upregulated in DEWAX OX, but downregulated in dewax relative to WT. Transactivation assay showed that DEWAX upregulated the expression of PDF1.2a, IGMT1, and PRX37. Chromatin immunoprecipitation assay revealed that DEWAX directly interacts with the GCC-box motifs of PDF1.2a promoter. In addition, ectopic expression of DEWAX increased the tolerance to B. cinerea in C. sativa. Taken together, we suggest that increased ROS accumulation and DEWAX-mediated upregulation of defense-related genes are closely associated with enhanced resistance to B. cinerea in Arabidopsis and C. sativa.The cuticle of land plants is the first physical barrier to protect their aerial parts from biotic and abiotic stresses. DEWAX, an AP2/ERF-type transcription factor, negatively regulates cuticular wax biosynthesis. In this study, we investigated the resistance to  Among tynthesis . The APE and ECR . HoweverCamelina sativa, which belongs to the Brassicaceae family, has recently gained increasing interest for its potential non-food usages, including in cosmetics, lubricants, and as biofuels (C. sativa has a short life cycle (100–120 days), and can be grown on marginal agricultural lands owing to its characteristics, such as tolerance to cold stress, relatively good growth under nutrient-poor soil conditions, and high levels of polyunsaturated fatty acids in the seed oils . To understand the molecular mechanisms underlying the defense responses of Arabidopsis and C. sativa plants overexpressing DEWAX to B. cinerea, further analyses were carried out. Finally, we revealed that DEWAX plays as a transcriptional activator that upregulates the expression of the defense-related genes such as PDF1.2a, IGMT1, and PRX37 via binding to their gene promoters. The results suggest that DEWAX-mediated upregulation of defense-related genes in addition to an increase in ROS levels is important in the enhanced tolerance to B. cinerea in Arabidopsis and C. sativa overexpressing DEWAX.In this study, we observed that DEWAX overexpression lines (OX1 and OX2) and dewax knockout mutant were described in A. thaliana (ecotype Columbia-0) seeds were surface-sterilized with 75% (v/v) ethanol and 0.05% (v/v) Triton X-100, washed with 100% ethanol, dried on filter paper, and germinated on half MS  at 25 ± 3°C under a 16-h-light/8-h-dark cycle , and 6-week-old transgenic C. sativa lines (T1 generation) using the NucleoSpin RNA Plant Extraction Kit (Macherey-Nagel) according to the instructions of the manufacturer. The total RNA was reverse-transcribed with GoScriptTM Reverse Transcription System (Promega), and the cDNA was used in RT-PCR analysis. RT-PCR was performed using the Access QuickTM RT-PCR system  with gene-specific primers (Supplementary Table Arabidopsis EIF4 (At3g13920) and C. sativa ACTIN11 genes  in a final volume of 20 μL. PCR was performed according to the manufacturer’s protocols using gene specific primers described in Supplementary Table PP2A (At1g13320) and CsACT11 genes were used to determine the RNA quality and quantity.Total RNA was isolated from 4-week-old Col-0, 11 genes  were useArabidopsis rosette leaves were stained at room temperature for 8 min without shaking using freshly prepared staining solution containing 0.05% toluidine blue and 0.01% Tween-20. After staining, leaves were rinsed five times with distilled water and photographed.Four-week-old in situ detection of hydrogen peroxide, Arabidopsis leaves were immersed in 10 mM MES (pH 6.5) containing 0.1% 3,3′-diaminobenzidine tetrahydrochloride , gently vacuum-infiltrated, and incubated for 30 min as described in Arabidopsis leaves were incubated in 10% MS salt solution containing 20 μM 2′,7′-dichlorofluorescein diacetate  and 0.1% Tween-20 in the dark for 30 min. DCF fluorescence in aliquots taken from the incubation solution was measured using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek) at 488 and 525 nm. Leaf fresh weights were used to normalize the values (2O2) content, 5-week-old leaves (200 mg) were incubated in 1.5 mL of 5 mM morpholineethanesulfonic acid (MES)-KOH buffer (pH 6.7) for 7 h  and was cultured on 2× V8 tomato agar . Harvesting and inoculation of conidia were carried out according to Arabidopsis leaves were laid out on 3MM paper with water and inoculated with 5-μl droplets of conidial suspension (5 × 105 spores/mL), kept under a sealed transparent cover to maintain high humidity and transferred to a growth chamber . C. sativa plants were sprayed with B. cinerea conidial suspension (2 × 105 spores/mL). After inoculation, the plants were transferred to growth chambers  and covered with a sealed clear lid to maintain low light intensity and high humidity. Quantification of B. cinerea biomass in C. sativa was carried out according to Pto DC3000 strain harboring pDSK-GFPuv  containing 0.05% Silwet L-77  for few seconds. After inoculation, the plants were covered with a clear lid and transferred to a growth room . Inoculated plants were photographed under ultraviolet light to determine PtoDC3000:GFP proliferation using GFP signals and bacterial populations were measured by serial dilution assays (A n assays . Colony DEWAX-F (603 bp for full-length), DEWAX-N , and DEWAX-C  DNA fragments were amplified via PCR using DEWAX cDNA and DEWAX-specific primers (Supplementary Table SmaI and SacI-digested PCR fragments were cloned into the pGBKT7 vector (BD Biosciences Clontech). The recombinant vector  were transformed into yeast strain Y190  according to the manufacturer’s instructions (BD Biosciences Clontech). Transformants were selected on selective medium (SD-Trp) supplemented with 25 mM 3-amino-1, 2, 4-aminotriazole (3-AT). Transformants were cultured on selective medium (SD-Trp-His) for 1 day at 30°C and filter-lift assays were performed for 16 h at 37°C, as described in For the transcactivation assay in yeast, SalI and SpeI sites of the GAL4-LUC binary vector. The recombinant pPZP212 vector , IGMT (∼1.5 kb), PRX37 (∼1.2 kb) promoter regions were amplified using gene-specific primers and cloned into the 2 vector  harborinThe reporter and effector constructs were isolated using a Qiagen Plasmid Midi Kit and co-introduced into tobacco protoplasts . After iArabidopsis actin7 (At5g09810) was used to control cDNA quantity and quality.Chromatin immunoprecipitation (ChIP) assay was performed using 35S:MYC-DEWAX transgenic plants, which were previously reported . GenomicC. sativa plants overexpressing Arabidopsis DEWAX, the full-length DEWAX gene was amplified from cDNA converted from RNA isolated from 3-week-old leaves with DEWAX F1 and DEWAX R1 primers  BASTA herbicide.onstruct  was inocp method . T1 tranC. sativa plants (T1 generation) using extraction buffer  and subjected to genomic DNA PCR analysis with CaMV35S promoter F and DEWAX CR1 primers . When hydrogen peroxide content was spectrophotometrically measured after leaves of WT, dewax, and DEWAX OX lines were preadapted in incubation buffer, the levels of hydrogen peroxide were approximately twofold higher in DEWAX OX1 and OX2 leaves than in WT leaves, but no remarkable differences were detected between WT and dewax leaves .To investigate the cuticle permeability of WT, S levels , ROS assdewax, and two DEWAX OX lines to the necrotrophic pathogen B. cinerea and the hemibiotrophic bacterial pathogen Pto DC3000, first, we examined visible disease symptoms 72 h after inoculation of B. cinerea on the adaxial surface of the leaves. In a spot inoculation assay, significantly larger lesions were observed on the leaves of dewax mutant, while smaller lesions were formed on DEWAX OX as compared with WT leaves , indicating that DEWAX OX shows enhanced resistance, and dewax enhanced susceptibility, to B. cinerea. On the other hand, compared to WT, DEWAX OX was more susceptible to Pto DC3000:GFP, while dewax was more resistant to this pathogen . These results indicate that DEWAX is involved in the disease resistance responses of Arabidopsis and plays a positive role in the defense to fungal pathogens, but a negative role in the defense to bacterial pathogens.To investigate the response of WT, Arabidopsis, comparative transcriptome analysis of WT and DEWAX OX was conducted (http://www.ebi.ac.uk/arrayexpress). Interestingly, the expression of genes involved in pathogen defense and ROS production was clearly upregulated in the stems of DEWAX OX as compared to those of WT  and peroxidase C2 precursor (PRX37 and PRX38) were increased in DEWAX OX1 and OX2 leaves, while no significant downregulation was observed in dewax relative to WT. No noticeable differences in the expression of At1g67810 and At4g16260, encoding SULFUR (Stimulate CpNifS Cysteine Desulfurase Activity) and a β-1,3-glucanase precursor, respectively, were observed in dewax, and DEWAX OX1 and OX2 leaves .DEWAX has been reported to function as a transcriptional repressor in cuticular wax biosynthesis . However1 vector  and usedDEWAX were cloned downstream of the GAL4-binding domain in the pGBKT7 vector. Yeast Y190 cells, which possess the HIS3 and lacZ genes driven by the promoter including GAL4-responsive elements, were transformed with the constructed vectors. Yeast cells that were able to survived on tryptophan-deficient selective medium (SD/-Trp) were further selected on tryptophan- and histidine-deficient medium (SD/-Trp-His) supplemented with 10 mM 3-amino-1, 2, 4-aminotriazole (3-AT) and subsequently subjected to an X-gal filter-lifting assay. Yeast cells transformed with the F or N construct survived on SD/-Trp-His medium supplemented with 10 mM 3-AT, and generated blue colonies by the β-galactosidase activities in X-gal lift assay. However, yeast cells transformed with the pGBKT7 vector or the C construct did not survive on the selective medium, and showed no β-galactosidase activity. These results indicate that DEWAX is able to act as a transcriptional activator, and amino acid residues 1 to 106 of DEWAX are involved in transcriptional activation .In addition, the full-length , N-terminal , and C-terminal  regions of PDF1.2a, IGMT1, and PRX37, their promoter regions were transcriptionally ligated to the luciferase gene (LUC) in the Gal4-LUC binary vector, and used as reporter constructs . p35S and p35S-DEWAX as effector constructs were used in transactivation assay of tobacco protoplasts  and β-glucuronidase (GUS) activities were measured, and the LUC activity was normalized to the GUS activity. When DEWAX was expressed, LUC activity was upregulated in the protoplasts transformed with PDF1.2a, IGMT1, and PRX37 reporter constructs by approximately 2.3, 4.2, and 6.3-fold, respectively, but was downregulated in the protoplasts transformed with CER1 by approximately twofold relative to the control co-transformed with p35S .To examine whether DEWAX is able to activate the expression of the putative target genes, toplasts . After tPDF1.2a promoter region using 35S:MYC-DEWAX transgenic plants . This observation indicates that DEWAX activates the expression of PDF1.2a gene by direct interaction with its promoter region.We further investigated whether DEWAX directly interacts with the consensus GCC-box motifs of c plants . QuantitDEWAX overexpression in Arabidopsis led to strong resistance to B. cinerea, we next evaluated how overexpression of DEWAX in a Brassica crop, C. sativa, affects disease resistance. The 35SP-DEWAX construct harboring DEWAX gene under the control of CaMV 35S promoter was used in the transformation of C. sativa . Transgenic plants were screened through spraying of 0.03% BASTA herbicide . Genomic DNA PCR analysis of non-transgenic (NT) and 16 transgenic plants (TO) using gene-specific primers . To measure B. cinerea biomass, genomic DNA was isolated from the leaves 4 days after inoculation with B. cinerea and subjected to real-time quantitative PCR using B. cinerea-specific cutinase A-specific gene primers (Supplementary Table C. sativa GSK3/shaggy-like (iASK) gene was used for normalization. Compared to NT plants, all TO lines tested had lower B. cinerea biomass . Subsequently quantitative RT-PCR analysis showed that the levels of C. sativa PDF increased by 2- to 3.5-fold in DEWAX OX lines compared with NT . These results indicated that overexpression of Arabidopsis DEWAX confers tolerance to B. cinerea in transgenic C. sativa.Next, we examined the defense responses of NT and TO plants to DEWAX increased cuticle permeability and ROS accumulation in Arabidopsis. DEWAX is a transcriptional activator that upregulates the expression of the defense-related genes, PDF1.2a, IGMT1, and PRX37, by binding to their gene promoters. In particular, DEWAX directly interacts with the GCC motifs in the PDF1.2a promoter. Overexpression of DEWAX conferred enhanced tolerance to necrotrophic fungal pathogen, B. cinerea in Arabidopsis and C. sativa. Therefore, convergent line of evidence supports that increased ROS levels as well as upregulation of defense-related genes might be related to the resistance to B. cinerea in Arabidopsis and C. sativa overexpressing DEWAX.Plant immune responses are essential for not only growth and development, but also productivity and yields of crops. In this study, we investigated a plant defense mechanism related to the cuticle, which is the first defensive barrier of the aerial parts of plants. We revealed that overexpression of OCTADECANOID-RESPONSIVE ARABIDOPSIS (ORA)59 and ERF96 with the “EDLL” motif to the two GCC motifs in the PDF1.2 promoter region, is essential for the expression of PDF1.2 , which is consistent with the results in the Arabidopsis mutants, bdg and lacs2  and ethephon, a form of liquid ethylene (PDF1.2 depended on ERF1 and ORA59 transcription factors, which act as integrators of JA and ethylene signaling pathways. Overexpression of ERF1 and ORA59 caused increased resistance, and ora59 mutant enhanced susceptibility to B. cinerea (DEWAX were elevated neither by JA nor by ethephon1, although DEWAX is classified with ERF1 and ORA59 into the group IX of Arabidopsis AP2/ERF gene family (Arabidopsis defense responses to herbivores and pathogens are regulated by circadian rhythms (PDF1.2 by the diurnally controlled DEWAX may be important in circadian clock-mediated plant innate immunity. In addition, JA and salicylic acid are known to play a role as an antagonist in plant innate immunity (PDF1.2 gene in DEWAX-overexpressing Arabidopsis indicates that JA-associated defense responses are activated, and this may cause the suppression of SA-related signaling. This hypothesis supports that DEWAX-overexpressing plants are more resistant to the necrotropic fungal pathogen, B. cinerea, but are more susceptible to the semi-biotropic bacterial pathogen, Pto DC3000 compared with WT.The expression of ethylene . The JA- cinerea . Howevere family . Based o rhythms , it woulimmunity . The indC. sativa is considered to be resistant to many diseases, it is reportedly sensitive to some fungal and bacterial pathogens causing wilting, root rot, clubroot, white rust, and downy mildew diseases (C. sativa overexpressing the antimicrobial peptide cecropin P1 exhibits enhanced resistance to Erwinia carotovora and Fusarium sporotrichioides. In this study, the Arabidopsis dewax mutant showed increased resistance to Pto DC3000, whereas overexpression of DEWAX conferred elevated resistance to B. cinerea in Arabidopsis and C. sativa, suggesting the potential for developing Brassica lines resistant to various diseases. In conclusion, our study revealed that DEWAX is a transcriptional activator that upregulates the expression of defense-related genes, and overexpression of DEWAX enhances the resistance to B. cinerea in Arabidopsis and C. sativa. We provide that DEWAX-mediated defense mechanisms may be applicable in the improvement of crops with enhanced resistance to fungal and bacterial pathogens.Although diseases . ZakharcSJ, YG, and MS conceived and designed research. SJ, YG, and HC conducted experiments. SJ, JP, and MS analyzed data. SJ, JP, and MS wrote the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Graphene layers were grown by chemical vapor deposition (CVD) on copper substrates. Single and layer-by-layer graphene stacks were fabricated combining graphene transfer techniques and metal oxides grown by magnetron sputtering. The electrochemical properties of the samples were analyzed and the results suggest an improvement in the performance of the device with the increase in the number of graphene layers. Furthermore, deposition of transition metal oxides within the stack of graphene layers further improves the areal capacitance of the device up to 4.55 mF/cm2, for the case of a three-layer stack. Such high values are interpreted as a result of the copper oxide grown between the copper substrate and the graphene layer. The electrodes present good stability for the first 850 cycles before degradation.The combination of graphene with transition metal oxides can result in very promising hybrid materials for use in energy storage applications thanks to its intriguing properties, i.e., highly tunable surface area, outstanding electrical conductivity, good chemical stability, and excellent mechanical behavior. In the present work, we evaluate the performance of graphene/metal oxide (WOThe online version of this article (10.1186/s11671-017-2385-1) contains supplementary material, which is available to authorized users. Recently, electrochemical energy storage devices such as supercapacitors are becoming the most popular apparatus as power supplies in a wide variety of applications, from portable electronic devices, such as cell phones and laptops, to hybrid electric vehicles . SupercaThis is a result of the different energy storage mechanism between the two devices. In contrast to batteries, where ions are stored through chemical bonding to the electrode materials, in supercapacitors, an electrostatic storage of the energy takes place through the separation of charge in a Helmholtz double layer . AdditioHence, it is considered that supercapacitors have the potential to replace or complement batteries in energy storage applications. Research in this direction focuses in the development of novel electrodes that can exhibit superior characteristics. Similar to Li ion batteries, carbon-based materials are preferred due to their low environmental impact, chemical stability, high conductivity, and low cost .2-hybridized carbon atoms, has some very exciting properties which make it very attractive for being used as an electrode in this kind of applications. We highlight its light weight, high electrical and thermal conductivity, highly tunable surface area (up to 2675 m2/g), strong mechanical strength (~ 1 TPa), and chemical stability [2 and a corresponding specific capacitance of around 550 F/g when the entire surface area is fully utilized. At present, it is given much attention to three-dimensional graphene materials, such as graphene nanowalls and nanofoams, which can deliver high energy density and power density, in the order of 13 Wh kg−1and 8 kW kg−1, respectively [Graphene, an emerging nanomaterial which consists of all spup to 267 m2/g, stectively .Further, planar graphene films present the benefit of a homogeneous growth and good coupling to the metallic substrate, resultant of the mixture of covalent and ionic bonding on the graphene/copper interface , which sThe recent advances in the design and optimization of higher efficiency electrodes has promoted the combination of graphene and graphene oxide films with different metal and metal oxide composites –19, likeOn this design, graphene contributes, apart from its storage capacity, as a platform which permits the strong coupling and a good electric contact between the metallic nanoparticles and the current collector. Previous studies have revealed the beneficial role of graphene as a coupler between the current collector and carbon nanotubes .2O3 and MnO2, they show capacitances up to 380 F/g [On other research work, single-layer graphene electrodes have been measured to exhibit a specific double-layer capacitance of up to 135 F/g, while when combined with other compounds such as Fe 380 F/g , 22.3 and CeOx were sputtered.In the present work, we have manufactured graphene/metal oxide nanocomposites made from a single layer or three stacked layers of graphene/metal oxide nanocomposites, combining graphene transfer and magnetron sputtering techniques. On top of each graphene layer, different metal oxide particles of WOx in the whole manuscript as we have not characterized the grown particles. Although the sputtering process was performed with a CeO2 target, the formed particles should appear suboxidized because of the possible loss of oxygen during the sputtering process, but they are mainly formed by CeO2, which is the most stable form of cerium oxide. Compared to monolayer graphene, stacks of graphene films have more electrode/electrolyte interfaces, which is beneficial to the absorption/desorption of electrolyte ions and provide more electrical pathways for electrolyte ions during charging and discharging processes. The deposition of metal oxide particles enhances the specific capacitance of ultrathin layers at a relatively low mass loading [x particles have demonstrated high capacitance, in the order of 119 mF/cm2, when combined with nickel foam [3 films, a recent work has reported electrodes made with WO3 rods presenting a capacitance of 266 F/g [Cerium oxide is mentioned as CeO loading . In prevkel foam . Conside 266 F/g . Both naThe use of the same experimental conditions in the preparation of the two hybrid materials provides us with the opportunity to directly compare the electrochemical performance of the electrodes.To better interpret our results, we take into consideration the contribution of the native copper oxide layer in the overall capacitance of the electrode.2 pieces, cleaned in ultrasound bath of isopropanol and acetone, 10 min each, to remove impurities and loaded in the chamber. First, we apply a hydrogen plasma etching to remove the native copper oxide from the copper surface. Radio frequency (RF) plasma is generated by applying 100 W at 20 Pa pressure, under 20 sccm hydrogen flow. The plasma etching lasts 10 min. Then, the sample is forwarded in a quartz tube (coupled to the plasma chamber) surrounded by an oven. The oven is heated at 1040 °C and the gases are introduced in the tube. A mixture of methane and hydrogen is introduced (5/20 sccm of methane/hydrogen) during 20 min at 15 Pa, resulting in the complete coverage of the copper foil by single-layer graphene. Then, the sample is let to cool down in room temperature in high vacuum (3 × 10−4 Pa) before being removed from the chamber. Then, the sample is placed in another reactor to deposit the metal oxide particles. The metal oxide particles were deposited on the graphene layer by pulsed reactive magnetron sputtering , using, each time, the corresponding target (W or Ce). To prepare three stacked layers of graphene/metal oxide nanocomposites, we used graphene transfer method with a polymer coating support [3 to etch away the copper. The remaining graphene/metal oxide layer was then transferred over another layer of the same nanocomposite enabling the preparation of the stacked material. After the transfer process, the PMMA was removed by rinsing with acetone. The preparation process of the composite is presented in the schematic drawing of Fig. Continuous graphene films were grown by CVD following the growth recipes described in our previous work . We repo support . Polymetα line of 1486.6 eV energy and 350 W). Depth profile measurements of chemical composition by XPS were obtained by sputtering the surface with an Ar+ ion source (4 keV energy). All these measurements were made at ultra high vacuum (UHV) conditions, between 7 × 10−7 and 3 × 10−6 Pa.Samples were characterized by Raman spectroscopy (Jobin-Yvon LabRam HR 800), scanning electron microscopy (SEM) (JEOL JSM7100F), and transmission electron microscopy (TEM) (Bioscan Gatan JEOL 1010). X-ray photoelectron spectroscopy (XPS) measurements were performed in a PHI 5500 Multitechnique System  with a monochromatic X-ray source (Al K4 solved in ethylene carbonate (EC) and diethyl-carbonate (DEC) mixed in 1:1 volumetric proportions) electrolytes. A glass fiber filter served as separator (Whatman glassy-fiber GF/A). Figure 2 glove box (< 1 ppm O2 and < 1 ppm H2O) by sandwiching an organic electrolyte-soaked separator between two graphene/MeO electrodes. To study the supercapacitance behavior of the devices, we first performed cyclic voltammetry (CV) measurements at different scan rates and with a voltage window of 1.8 V.The electrochemical properties of the samples were analyzed using a Swagelok cell and organic  and the 2D peak FWHM (~ 40 cm−1) confirm that graphene is a single layer. The spectrum was obtained after transferring the graphene film over a SiO2 substrate in order to eliminate the noise resulting from the copper foil luminescence [The short sputtering deposition of the MeO nanoparticles aims in avoiding the damage of the graphene layer. Longer sputtering periods could result in the damaging of the graphene, since the sputtering is performed in an argon/oxygen plasma. Figure ilm Fig. . We obseilm Fig. , the I2nescence .Fig. 2MoXPS provided information considering the copper oxide formation after plasma annealing and graphene growth. The measurements were performed on copper substrates with and without graphene grown on top in order to show that the graphene presence favors the formation of the copper oxide layer. The native copper oxide layer was reduced by plasma annealing in all samples . After each pickling process, we obtain information on the chemical composition at the most depth. The first two spectra (black and red line) have the same intensity. The rest of the spectra have a lower intensity. Defining the x. The specific capacitance, Cs, was calculated by the equation,Cs is the specific capacitance in farad per gram, m is the mass of the active material in grams, ΔV is the voltage window in volts, and qa and qc are the anodic and cathodic charges in coulomb, respectively.In Fig. Ci, was calculated using the relation,A is the area of active material dipped in the electrolyte . A similar behavior, resulting although in a smaller capacitance increase, is observed for the Gr/WO3 electrodes. Their capacity increases from 2.69 to 4.15 mF/cm2 when two more layers of Gr/WO3 are added over the first layer (+ 54%).The as-grown graphene film presents an interfacial capacitance 3 electrodes, reveal an agreement in the percentage increase with respect to the Gr/MnO2 hybrid structure.Similar percentage increase is expected when more graphene/metal oxide layers are added, as the surface area will increase proportionally while the interlayer distance may also allow multilayer ion absorption. In Fig. −4 W/cm2 in the case of three layers of Gr/CeOx electrodes, a value of the same order of magnitude as that of other electrodes, with similar architecture, combining graphene with MnO2 particles [−8 W-h/cm2, a value which is two orders of magnitude lower than the values given for the case of Gr/MnO2-based electrodes.To demonstrate the overall performance of the supercapacitors, we illustrate a Ragone plot with the energy density and power density of the various electrodes Fig. . We obsearticles . Althoug2, while in our work, it is measured to be 0.87 mF/cm2. In our work, the graphene layer was deposited on a copper foil, which was used as a current collector, making transfer of graphene unnecessary. We consider that the formation of copper oxides in the graphene/copper interface, resulting from the copper oxidation, affects the total capacitance of the system. Additionally, we know that the presence of graphene favors the growth of a copper oxide layer of some tenths of nanometer, as it has been observed by us and also reported by other authors [2O) layers. Their “brighter” appearance is the result of the higher backscattering of electrons on copper oxide than in the case of naked copper.We observe that the capacitance of the sample with single-layer graphene is much higher, about nine times, than those mentioned elsewhere . In the  authors , 29. Alt authors . By delact is the total capacitance that we measure, cox the capacitance of the copper oxide, and cg the graphene quantum capacitance. Although, as it has been evaluated by experimental observations, graphene presents a negative capacitance when it is decorated with metallic adatoms. These adatoms act as resonant impurities and form nearly dispersionless resonant impurity bands near the charge neutrality point (CNP). Resonant impurities quench the kinetic energy and drive the electrons to the regime dominated by the Coulomb energy with negative compressibility. If we consider a negative quantum capacitance of the graphene [2), we will be able calculate the copper oxide capacitance (11.1 mF/cm2) that corresponds to a copper oxide thickness of approximately tenths of a nanometer [Therefore, to better interpret our results, we should consider that each electrode consists of two capacitors, the graphene film and the copper oxide film, in series, contributing to the total capacitance, asgraphene  with a vgraphene  (0.1 mF/anometer , in agreanometer , conside3 and three-layer Gr/CeOx. The electrodes consisting of Gr/CeOx have a better charge/discharge efficiency during more cycles as shown in Fig. Finally, we present results considering the stability in the device performance. All electrodes present a capacitance retention of between 70 and 90% during the first 850 cycles, as we can see in Fig. x hybrid and in Fig. 3 hybrid. One-layer Gr/CeOx needs 1.7 s approximately for a charge/discharge cycle when charged by 400 mA/cm2. When two more layers were added over the first one, this period increased to ~ 4.7 s. Measurements performed on a single-layer graphene showed similar charge/discharge time as in the case of the single-layer Gr/CeOx electrodes. Similar results were obtained in the case of WO3 particles, where charge-discharge time was 1.9 s for one layer and 5.5 s for three layers. This demonstrates the higher power density that the CeOx hybrid is performing. The electrochemical results of the study are listed in Table The galvanostatic charge/discharge curves reveal that when more layers of metal oxide/graphene are added, more time is needed for the charging and discharging process. This is visualized in Fig. x have a slightly higher charge/discharge efficiency than those in which Gr is combined with WO3. Considering stability, all devices maintain their initial performance for more than 800 cycles. The charge/discharge period increases about 2.5 times with the addition of two more layers over the first one.Layer-by-layer evaluation of graphene electrodes combined with different metal oxides has been performed. The deposition of metal oxide particles over the graphene increases the total capacitance of the hybrid material, as metal oxide particles contribute with an additional pseudocapacitance. An increase has been also observed when more layers of Gr/metal oxide were added over the first layer. The devices in which Gr is combined with CeO


Moreover, the changes in both the thermal and electrical conductivities with grain size change are greater than those of typical semiconducting thermoelectric materials.Understanding the influence of grain boundaries (GBs) on the electrical and thermal transport properties of graphene films is essentially important for electronic, optoelectronic and thermoelectric applications. Here we report a segregation–adsorption chemical vapour deposition method to grow well-stitched high-quality monolayer graphene films with a tunable uniform grain size from ∼200 nm to ∼1 μm, by using a Pt substrate with medium carbon solubility, which enables the determination of the scaling laws of thermal and electrical conductivities as a function of grain size. We found that the thermal conductivity of graphene films dramatically decreases with decreasing grain size by a small thermal boundary conductance of ∼3.8 × 10 Understanding the effect of grain boundaries on the electrical and thermal transport properties of graphene is of both fundamental and technological importance. Here, the authors fabricate graphene films with controlled grain size, and determine the scaling laws of thermal and electrical conductivities. Graphene has attracted increasing interest because of the extraordinary properties of its defect-free pristine form, such as the highest known carrier mobility, record thermal conductivity and extremely high mechanical strength1245678910111256812121314151617181920212223From the point of view of crystal growth, it is equally difficult to reduce and increase nucleation density on metals by CVD to fabricate large-size single-crystal graphene and polycrystalline graphene with nano-sized grains, respectively, while keeping monolayer growth of graphene. For surface adsorption growth on the commonly used Cu with a low carbon solubility, a high-concentration carbon source and/or defective substrates have usually been used to obtain a high domain density, however, these conditions led to the formation of multi-layer graphene domains102527285It has been theoretically predicted that electrical transport in graphene could be markedly altered by electron scattering at GBs14410111275209 W m−2 K−1, while the electrical conductivity slowly decreases with an extraordinarily small GB transport gap of ∼0.01 eV and GB resistivity of ∼0.3 kΩ μm. Moreover, both the thermal and electrical conductivities of graphene change more significantly with grain size change than that of typical thermoelectric materials3334Here we have developed a segregation–adsorption CVD (SACVD) method to achieve a great increase in the nucleation density of graphene (by segregation) and monolayer growth (by surface adsorption) simultaneously, by using a Pt substrate with medium carbon solubility. As a result, we can easily grow well-stitched high-quality monolayer graphene films with a tunable uniform grain size from ∼200 nm to ∼1 μm, which have never been achieved before by the present conventional CVD methods based on either surface adsorption45678910111213272728−2 were obtained 5We obtained histograms of grain sizes by measuring more than 100 grains for each sample . The mea2/Si holey substrates  to make sure that the suspended area is intact. The SEM image shows that most area of the substrate is covered by graphene without visible cracks  of the graphene films was calculated by κ=χ(1/2hπ)(δw/δP)−1, where δw is the shift of 2D peak position due to the change of heating power δP on the sample, χ is the 2D peak temperature coefficient, and h is the thickness of the graphene film.Confocal micro-Raman spectroscopy is an efficient method for measuring the thermal conductivity of suspended graphene. Its value is extracted from the dependence of the Raman G or 2D peak frequency on the excitation laser power37e cracks . Figure ID/IG increase sharply while the G peak intensity decreases dramatically when the laser power is larger than 1.2 mW  and the extracted temperature coefficient (0.039 cm−1 K−1), we estimated that the temperature at the GBs of the graphene film with ∼200 nm grains reached 650 K when the laser power was 1.2 mW. Combined with the Raman spectra evolution, the physical origins of the D and G peak12n 1.2 mW . The 2D n 1.2 mW . Howeverecreased . These pκ) as a function of grain size (lg). It is clear that the thermal conductivity increases exponentially from ∼610 to ∼5,230 W m−1 K−1 when the grain size is increased from ∼200 nm to ∼10 μm. In fact, the graphene films with grain size larger than ∼5 μm (the size of the suspended area) all show a similar thermal conductivity of ∼5,200 W m−1 K−1 , which is similar to the value reported for pristine graphene made by mechanical exfoliationleff−1= lph–ph−1+lg−1, where lph–ph denotes the phonon–phonon scattering length and lg is the scattering length due to the boundaries κ−1) versus the inverse of grain size (lg−1) can be well fit by κ−1=κg−1+(lgG)−1, where κg is the thermal conductivity within the grain  and G is the boundary conductance9 W m−2 K−1, which is consistent with the theoretical value obtained using non-equilibrium Green's functions (3–8 × 109 W m−2 K−1) ]} , R is the universal gas constant, T is the absolute temperature, lg is the grain size and c is the correction value. The fitting gives σ0≈2.85 × 106 S m−1 and Ea≈0.01 eV. Note that the GB transport gap extracted here is dramatically smaller than the theoretically predicted value for asymmetric GBs (0.3–1.4 eV) ]} , where σeV) ref. . Using tG+ρGB/lg 13, whereBs 0.3–1. eV (ref.lg lg−1). Note that the thermal conductivity change rate (Δκ κ−1) increases linearly with grain size change rate (σ σ−1) increases exponentially with grain size change rate 47483334nL, mL) and  that belong to the left and right crystalline domains, respectively. If only one matching vector fulfills the criterion (n–m)=3q , then the GB is of class-II type. Otherwise it belongs to class-I. For class-II GB, there is significant misalignment of the allowed momentum–energy manifolds corresponding to the two crystalline domains of graphene, which introduces a transport gap  that depends exclusively on the periodicity1414The GBs in graphene can be approximated as linear periodic arrays of dislocations9 W m−2 K−1, while the electrical conductivity slowly decreases with an extraordinarily small GB transport gap of ∼0.01 eV and GB resistivity of ∼0.3 kΩ μm. Moreover, the changes in both the thermal and electrical conductivities with grain size change are greater than those of typical semiconducting thermoelectric materials. These findings provide valuable information for tuning the thermal and electrical properties of graphene for electronic, optoelectronic and thermoelectric applications through grain size engineering.In conclusion, we report a SACVD method to grow well-stitched high-quality monolayer graphene films with a tunable uniform grain size from ∼200 nm to ∼1 μm, by using a Pt substrate with medium carbon solubility. Using these materials, we determined the scaling laws of the thermal and electrical conductivities of graphene films as a function of grain size. It was found that the thermal conductivity of polycrystalline graphene films dramatically decreases with decreasing grain size by a small thermal boundary conductance of ∼3.8 × 10A typical procedure for the SACVD growth of graphene films with grain sizes <1 μm includes four steps: surface growth, etching, segregation and surface growth. Before growth, a piece of Pt foil  was rinsed with acetone and ethanol in sequence for 1 h each, loaded into a fused-silica tube (inner diameter: 22 mm), heated to a certain temperature under the protection of hydrogen, and then annealed for 10 min to remove any residual carbon or organic substances. The first step: surface growth was started with the substrate being held for a certain time under a mixture of methane and hydrogen. In the second step the methane and hydrogen flows were turned off, and pure argon (700 sccm) was introduced to the system for 20 min to etch the graphene grown on the Pt in the first step into the bulk. In the third step, a small amount of hydrogen was introduced into the system to mix the argon flow, initiating segregation of the carbon to form small graphene domains on the Pt surface. In the fourth step a low flow rate of methane (0.1 sccm) was introduced into the system while maintaining the hydrogen and argon flows, to cause surface growth of the graphene domains produced in step 3 to form a continuous polycrystalline film. The detailed experimental conditions were given in the main text and 2 (290 nm) substrates using a improved bubbling transfer methodTo investigate the structure of the graphene formed at different stages, the Pt foil was quickly pulled out of the high-temperature zone after SACVD growth. The furnace was then shut down and the methane flow was stopped after the furnace temperature had decreased to 600 °C. Finally, the Pt foil was taken out and characterized by SEM . The small graphene domains and polycrystalline films were transferred onto Si/SiO2/Si substrate and thermal conductivity of the suspended graphene in our experiments show the similar values with those of graphene with similar grain size reported in the literatures443 W m−1 K−1, close to the reported value (5.3 × 103 W m−1 K−1) , and the integration time at each position was 10 s. The temperature rise was determined from the shift of the Raman 2D peak. The sheet resistances of the graphene films were measured by a four-probe method (RTS-9) at room temperature. These two methods have been widely used in the literatures12133844The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Ma, T. et al. Tailoring the thermal and electrical transport properties of graphene films by grain size engineering. Nat. Commun.8, 14486 doi: 10.1038/ncomms14486 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary References


It is found that the contact resistances between all metals and graphene are remarkably reduced after annealing, indicating that not only chemically adsorbed metal (Pd) but also physically adsorbed metals (Ag and Pt) readily form end-contacts at intrinsic defect locations in graphene. In order to further improve the contact properties between Ag, Pd, and Pt metals and graphene, a novel method in which self-aligned He+ bombardment to induce exotic defects in graphene and subsequent thermal annealing to form end-contacts was proposed. By using this method, the contact resistance is reduced significantly by 15.1% and 40.1% for Ag/graphene and Pd/graphene contacts with He+ bombardment compared to their counterparts without He+ bombardment. For the Pt/graphene contact, the contact resistance is, however, not reduced as anticipated with He+ bombardment and this might be ascribed to either inappropriate He+ bombardment dose, or inapplicable method of He+ bombardment in reducing contact resistance for Pt/graphene contact. The joint efforts of as-formed end-contacts and excess created defects in graphene are discussed as the cause responsible for the reduction of contact resistance.Low contact resistance between graphene and metals is of paramount importance to fabricate high performance graphene-based devices. In this paper, the impact of both defects induced by helium ion (He There has been plentiful studies on the properties of graphene-metal (G-M) contact [−7 Ω·cm2. Similar to this plasma treatment, ultraviolet/ozone (UVO) was used to clean the graphene surface after lithography which reduced contact resistance to 7 × 10−7 Ω·cm2 and 568 Ω·μm respectively [c over a wide range cannot be easily realized, or even the Rc will be degraded under some circumstances. Alternatively, the graphene/metals end-contacts, initially disclosed in a theoretical work [c dramatically. The utilization of end-contacts in reducing Rc is also confirmed by experimental efforts [c were demonstrated by introducing holes (~2.2 × 10−9 Ω·cm2) [c can be reduced using end-contacts, the approach to introducing various patterns in the contact area of graphene is too complicated to be implemented in practical applications, since in these cases the lithography and subsequent dry etching is usually needed in order to shape the holes, zigzag edges, or line cuts [c. In this regard, a simple while efficient method featuring the end-contacts concept to reduce Rc is of great importance and interest. In this work, a novel method to form end-contacts between graphene and various metals  is explored. In this method, defects are introduced to graphene by light He+ bombardment and thereafter end-contacts are formed between graphene and metals. Since the thermal annealing treatment is a common yet effective way to reduce Rc [c with and without He+ bombardment is also investigated in this work.In the past decade, graphene has attracted worldwide attention as a miracle material with unique electrical and physical properties ,2,3. Graformance ,16,17,18 contact ,20,21 an contact  and Robi contact  proposedectively ,25. Nevecal work , is clai efforts ,28. In t9 Ω·cm2) , or lineine cuts ,28. Moreeduce Rc ,30, the −1 respectively are observed and the ratio of 2D/G peaks is higher than 2 indicating that the transferred graphene is single layer.Thermal chemical vapor deposition (CVD) grown graphene ,32 was tx) isolation layer is deposited between PR and graphene as a hardmask by Atomic Layer Deposition (ALD) [x and underneath graphene (cf. 3PO4 (1:3) at 40 °C to remove the unwanted AlOx layer in these contact windows  were deposited directly on graphene by e-beam evaporation  is used to extract the contact resistance between graphene and metals. The fabrication of TLM test structures is schematically shown in on (ALD)   consists of graphene sheet resistance (RS), G-M contact resistance (RGM), metal wire resistance (RW), and probe-pad contact resistance (RPP) as seen in Equation (1).For the extraction of G-M contact resistance, four probe configuration illustrated in S relates to the length of graphene diffusion strip (lG) between two G-M contacts and the value of RW depends on the length of metal wires between pads and G-M contact windows (lW). Benefitting from the four point probe configuration in TLM method, RPP can be ignored. The slope in T which is the sum of RS, 2RGM, and RW. Normally, Rw is small which can be omitted in Equation (1) if metal wires are thick and/or short. However, since in this work the deposited metal wires are thin i.e., 50-nm-thick and long, Rw cannot be omitted which is also a disturbing factor for the linear plotting of RT vs. lG. In order to get a perfect linear plotting, it is a must to subtract Rw from RT. The sheet resistances of metal wires are extracted to be 0.96, 3.72 and 4 Ω/□ for Ag/Au, Pd/Au and Pt/Au, respectively. As a result, the values of Rw can be calculated according to lW and the width of metal wires and be subtracted from RT. After the Rw subtraction, perfect linear relationships between RT and lG for all G-M contacts are accomplished as shown in Among them, R+ bombardment, the linear plots of RT vs. lG before annealing (a–c) and after annealing (d–f) are shown in G, 10 data points are used and the linear fitting is performed using concatenate fit method. The perfect linear fittings for all G-M contacts are evident demonstrating the validity of employed method to extract Rc in this work. The interception of the fitted red line with RT (Y-axis) yields the value of 2RGM. Since the width of the graphene diffusion strip is 15 μm, Rc is therefore calculated by RGM × 15 μm which is also offered in each corresponding plot. For all three G-M contacts, it is clearly observed that Rc values are reduced significantly after annealing which confirms the effectiveness of the annealing in improving G-M contacts [For the contacts between Ag, Pd, Pt, and graphene without Hecontacts .c with different metal work functions [c data in c values for different metals  and filled red squares depict the mean Rc values with error bar. As seen, albeit the metal work function alters from ~4.26 to ~5.65 eV for different metals, the Rc value remains nearly invariable, manifesting that Rc is barely—or even not—relevant to metal work functions before annealing. This observation is also in accordance with previous finds of E. Watanabe’s et al. [Except the plot of Runctions ,36 in Fis et al.  in whichs et al. .c values for all G-M contact before and after annealing shown in c value is reduced significantly after annealing since it is a chemically adsorbed metal. The intriguing point in this work is why physically adsorbed metals like Pt and Ag also show remarkably reduced Rc values after annealing, i.e., from 247.43 and 285.07 to 180.75 and 227.81 Ω·μm for Pt/graphene and Ag/graphene contacts, respectively. In order to clarify this point, elaborate characterizations of graphene in G-M contact windows before and after annealing are implemented by Raman scattering spectrum. The contact windows are opened using aqua regia (HCl:HNO3 = 3:1) to remove metals thus exposing the graphene under metals [−1 for the graphene after annealing in comparison to the graphene before annealing. Note that the Raman spectra of the graphene before annealing resemble those of as-transferred graphene on SiO2  [S and rA are length scales that determine the region where the D band scattering takes place. rS determines the radius of the structurally disordered area and rA is the radius of the area surrounding the point defects in which the D band scattering takes place. CA is a measure of the maximum possible value of the ID/IG ratio in graphene. CS is the value of the ID/IG ratio in the highly disordered limit [D is the mean defect distance and the defect density is proportional to 1/LD2. In accordance with the empirical data in [A = (4.2 ± 0.1), CS = (0.87 ± 0.05), rA = (3.00 ± 0.03) nm, and rS = (1.00 ± 0.04) nm, the ID/IG as a function of LD for the graphene with and without He+ bombardment is plotted in D/IG increases from 0.032 for the un-bombarded graphene to 0.143 for the bombarded one, the LD increases dramatically from 58.05 nm to 27.56 nm. This means that the defect density in the graphene is increased approximately 4.4 times using He+ bombardment.The graphene in G-M contact windows was bombarded by Hetion (2) :(2)IDIGed limit ,40. LD i data in , CA =  and after annealing (d–f) are shown in + bombardment, 10 data points are used and the linear fitting is performed using concatenate fit method. As seen, the effectiveness of annealing in improving the G-M contacts with He+ bombardment is also evident. However, how the He+ bombardment impacts the specific contact resistance is still not clear. In order to provide a direct comparison for the readers, the Rc values for the G-M contacts with/without He+ bombardment are summarized in + bombardment before annealing. It is obvious that except Pd, He+ bombardment leads to an increase in Rc value for the Ag/graphene and Pt/graphene contacts. The defects induced by He+ bombardment should be blamed for the increase of Rc value and this observation also agrees well with the G-M contacts formed by metal sputtering on graphene [c [+ bombardment after annealing. As seen, for Ag/graphene and Pd/graphene contacts with He+ bombardment, the Rc values are reduced dramatically by 15.1% and 40.1% compared to their counterparts without He+ bombardment. Note that the obtained Rc values in this work for all three G-M contacts, i.e., 193.33, 118.28 and 189.15 Ω·µm for Ag, Pd, Pt/graphene contacts respectively, are among the lowest specific contact resistances [+ bombardment after annealing, the Rc value is increased a little bit compared to its counterpart without He+ bombardment and this could be tentatively interpreted as what follows.For Ag, Pd, and Pt/graphene contacts with Hegraphene . The defphene [c . In Figuistances ,37. Nevec value is lower for Ag/graphene and Pd/graphene contacts whereas is higher for Pt/graphene with He+ bombardment after annealing. We think the discussion about the higher or lower of contact resistance after annealing should take both the capability of metals forming end-contacts with defects and carrier scattering induced by defects within graphene into account [optimum) of He+ bombardment where the lowest Rc value (Rcmin) takes place. Four cases happen to the dose of He+ bombardment. (1) If He+ dose is Doptimum, the created defects in graphene can be completely consumed by the formation of end-contacts between metals and graphene. As a result, Rcmin can be obtained; (2) If He+ dose is less than Doptimum, Rc value is reduced but not to Rcmin in that as-formed end-contacts are not saturated; (3) On the other hand, if He+ dose is more than Doptimum but less than Dmax, though the formation of saturated end-contacts leads to the reduction of Rc, excess created defects enhance the carriers scattering which unfortunately degrades Rc concurrently. The joint efforts of as-formed end-contacts and excess created defects render the Rc value not the lowest Rcmin; (4) If He+ dose is more than Dmax, the effort of excess created defects prevails over that of saturated end-contacts and this leads to drastically increased Rc value that is even larger than Rc0 i.e., the Rc value for G-M contacts without He+ bombardment after annealing. Specifically, for the Ag/graphene and Pd/graphene contacts, they belong to cases (2) and (3) where their Rc values are reduced but possibly not to the lowest values Rcmin. It is worth noting that for Pd/graphene contact, the employed dose 2 × 1013 cm−2 is even closer to its Doptimum since its reduction percentage 40.1% is much bigger than 15.1% for Ag/graphene contact. For Pt/graphene contact, it belongs to case (4) where the effort of excess created defects prevails over that of as-formed saturated end-contacts, resulting in even larger Rc value compared to its counterpart without He+ bombardment after annealing. The specific contact resistance as a function of ion bombardment dose is still under investigation and will be revealed as a continuation of this work in the near future.In  account ,42,43,44+ bombardment before and after annealing are also performed by Raman scattering spectrum. The Raman spectra of as-exposed graphene with He+ bombardment  are depicted in −1 for the graphene before annealing in comparison to as-transferred graphene in + bombardment. However, for the graphene with He+ bombardment after annealing, the D peak becomes startlingly conspicuous. If scrutinized, the intensities of D peak for the graphene with He+ bombardment in all G-M contact windows are higher than those for the graphene without He+ bombardment in + bombardment. We believe the key to achieve extremely low specific contact resistance is to introduce defects in graphene thus forming plenty of end-contacts after annealing like other methods to form end-contacts [+ bombardment, but also by the implantation of other ion species. It is a remarkable fact that the optimum dose to achieve the lowest specific contact resistance may vary depending on the ion species used or on the implantation energy.Similarly to the discussions in previous section, the elaborate characterizations of graphene in G-M contact windows with Hecontacts ,27,28. T+ bombardment to induce exotic defects in graphene and subsequent thermal annealing to form end-contacts, was proposed in order to further reduce the specific contact resistance. Achieved results show that the specific contact resistances are reduced significantly by 15.1% and 40.1% for Ag/graphene and Pd/graphene contacts with He+ bombardment compared to their counterparts without He+ bombardment, respectively. For the Pt/graphene contact, the contact resistance is, however, not reduced as anticipated with He+ bombardment and this might be ascribed to either inappropriate He+ bombardment dose, or inapplicable method of He+ bombardment in reducing contact resistance for Pt/graphene contact. The effort of as-formed end-contacts prevailing over that of excess created defects is attributed to the reduction in Rc values for G-M contacts with He+ bombardment after annealing. By manipulating the He+ bombardment dose in conjunction with Ag, Pd, and Pt metals, the lowest Rc value for each G-M contact could be possibly accomplished. It is worth noting that the proposed He+ bombardment and metal depositions share the same lithography mask and this processing simplicity demonstrates that our proposed method is very efficient in improving the contact properties for graphene-based devices in the future.To summarize, the specific contact resistance for three G-M contacts  are all reduced after annealing from 285.07, 267.60, and 247.43 Ω·µm to 227.81, 197.33, and 180.75 Ω·µm, respectively. This indicates that not only chemically adsorbed metal (Pd) but also physically adsorbed metals (Ag and Pt) readily form end-contacts at intrinsic defect locations in graphene. Along with this guideline, a novel method, in which self-aligned He


The characterization of protein interactions has become essential in many fields of life science, especially drug discovery. Microscale thermophoresis (MST) is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. In addition, one of the major advantages of this technique is that no tedious purification step is necessary to access the protein of interest. Here, we describe a protocol using MST to determine the binding affinity of the PD-1/PD-L1 couple, which is involved in tumour escape processes, without purification of the target protein from cell lysates. The method requires the overexpression of fluorescent proteins in CHO-K1 cells and describes the optimal conditions for determining the dissociation constant. The protocol has a variety of potential applications in studying the interactions of these proteins with small molecules and demonstrates that MST is a valuable method for studying the PD-1/PD-L1 pathway. While the immune system attempts to neutralize malignant cells, some of the cells persist in a state of equilibrium between their elimination by the immune system and their proliferation. Indeed, tumour cells have developed immuno-evasion mechanisms that allow them to remain in equilibrium with the host by overexpressing immunoregulatory molecules such as PD-L1 and B7-12.The implications of the immune system for tumour control are well known3. Blocking the interaction between PD-1 and its ligand using an anti-PD-1 antibody enables restoration of this loss of immune function in cancers of the oropharynx4. Thus, some patients may benefit from a more targeted therapeutic approach that blocks the interaction of PD-1 with its ligands4. These observations have led to various clinical trials aiming to block the interaction between PD-1 and PD-L1 using the monoclonal antibodies anti-PD-1 or anti-PD-L1 in several types of cancers 5. Several therapeutic approaches that target the PD-1/PD-L1 blocking pathway are currently being developed.The tumour environment is highly immunosuppressive and locally regulates the anti-tumour response of T lymphocytes infiltrating the tumour. Generally, tumour-infiltrating T lymphocytes express PD-1, and tumour cells express PD-1 ligands (PD-L1 and/or PD-L2)6. A low level of PD-1 is sufficient to ensure the inhibitory role of T-cell activation. In the case of PD-1 deficiency, the administration of PD-L1 does not inhibit the activation of T cells. This lack of inhibition confirms that the inhibitory function of PD-L1 is tightly connected to its interaction with PD-17.PD-1 is a type I transmembrane glycoprotein composed of 288 amino acids. It belongs to the immunoglobulin superfamily, which also includes the CD28 and CTLA-4 molecules. PD-1 consists of an extracellular domain, a transmembrane domain and a cytoplasmic domain. The cytoplasmic domain possesses two tyrosines, one in the immunoreceptor tyrosine-based inhibitory motif (ITIM) and the other in the immunoreceptor tyrosine switch motif (ITSM), which are essential for the inhibitory role of the protein. PD-1 is expressed on the surface of CD4+ cells, CD8+ cells, NK cells, B cells, peripheral T cells, dendritic cells and monocytes8. PD-L1 is expressed on B and T lymphocytes, macrophages, mesenchymal cells and dendritic cells9. Unlike other members of this family, PD-L1 is also present on the surface of non-haematopoietic cells such as endothelial, cardiac, pulmonary, pancreatic, muscular and placental cells. The PD-L1 protein was discovered in 1999 as a new member of the B7 molecule family10 and was identified as the first known PD-1 ligand the following year11. PD-L1 and B7–1 both interact via their IgV-like domain11.The PD-1 protein interacts with two known ligands: programmed death ligand 1  and programmed death ligand 2 . The PD-L1 molecule is a type I transmembrane protein with 290 amino acids. It is encoded by the CD274 gene, which is found on chromosome 19 in mice and on chromosome 9 in humans. This protein consists of an extracellular domain, a transmembrane domain and an intracellular domain12. Nivolumab, an antibody targeting the PD-1/PD-L1 pathway, has also recently gained FDA approval based on its significant tumour response in clinical trials for the treatment of melanoma13.Various therapeutic approaches that target the blocking of the PD-1/PD-L1 axis are currently being developed. Blocking receptors on immune effector cells can help eliminate cancer, as described recently with regard to clinical trials evaluating ipilimumab, a blocking antibody of the CTLA-4 receptor, which was accepted as an anti-cancer treatment in 201114. Bristol-Myers Squibb has also recently shown that new PD-L1 inhibitors ((2-methyl-3-biphenylyl) methanol derivatives) under patent can bind to PD-L1 and dissociate the PD-1/PD-L1 complex at stoichiometric concentrations. However, these compounds induce the dimerization of hPD-L1 in solution15.Low-molecular-mass inhibitors have also been studied for their ability to modulate the PD-1/PD-L1 signalling pathway. These inhibitors are based on different types of molecules, such as macrocyclic peptides or oxadiazole derivativesd). Equilibrium binding assays were performed to determine the Kd between murine PD-1 and PD-L1 and yielded a 4 µM dissociation constant16. The cross-species protein interaction of murine PD-L1 and human PD-1 has also been reported in a few studies17. In 2007, the equilibrium binding responses between PD-1 and immobilized PD-L1 or PD-L2 were measured by SPR, and a 0.77 µM dissociation constant was obtained for the binding of PD-1/PD-L118. More recently, PD-1 ligands were once again characterized using SPR-based assays, and the results gave a Kd of 8 µM for the binding of hPD-1 to hPD-L1 and a Kd of 29 µM for the binding of mPD-1 to mPD-L119.The binding interactions of PD-1 with PD-L1 have been studied by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) over the years, but these investigations led to controversy regarding the values of the dissociation constant  with low sample consumptionTo determine the best formulation for analysis of the interaction between a protein and its ligand, several lysis buffers and several buffers for the analysis of thermographs during thermophoresis were studied. The lysis buffer modulates the fluorescence of the labelled protein. As shown for the PD-1-eGFP protein lysis, the use of a buffer without detergents .CHO-K1 cells (ATCC® CCL-61) were cultured in Ham’s F-12 Nutrient Mixture  with 1% PS  and 10% FBS . Cell lines were cultured at 37 °C in a 5% COSeveral lysis buffers, including some containing detergents, were tested for their extraction efficiency , HEPES buffer , SSB buffer , and PBS-T buffer .Various buffer solutions were used to determine which ones led to the best stability and homogeneity within capillaries when processing microscale thermophoresis experiments: MST buffer  or a calibration curve . Different types of additives such as Tween 20, various MgCld constants between a protein and its ligand can be calculated using the saturation binding curve at equilibrium27. The fitting function is derived from the law of mass action:MST experiments were performed on a NanoTemper ® Monolith NT.115 with blue/red filters . Samples were prepared in the different buffers listed in the MST buffer section and loaded into standard/premium treated capillaries. Measurements were performed at 22 °C using 20% MST power with laser off/on times of 5 s and 30 s, respectively, and 60% MST power with laser off/on times of 5 s and 20 s. All experiments were repeated three times for each measurement. Data analyses were performed using the NanoTemper® analysis software. The K[LP]: Bound complex concentration[Ptot]: Total protein concentration[Ltot]: Total ligand concentrationKd: Dissociation constantA pcDNA3.1 plasmid containing an mPD-1-YFP fused protein was purchased from Life Technologies . The pcDNA3.1 Hygro plasmid was also purchased from Life Technologies. The plasmid was digested by two restriction enzymes, HindIII and EcoRI (New-England-Biolabs 2000), to allow the insertion of our mPD-1-YFP construct. The pVITRO eGFP plasmid was purchased from InvivoGen and was used for PCR amplification to obtain the eGFP amplicon. The plasmid containing hPD-L1-eGFP was purchased from GeneCopoeia (CS-GS402L-M10/pReceiver-M10). The plasmid containing the mutant variation of hPD-L1-eGFP was also purchased from GeneCopoeia (CS-GS406L-M10/pReceiver-M10). Both sequences are based on the crystal structure of the PD-1/PD-L1 complex (PDB code: 3BIK) and are described in Supplementary Information Thermo Scientific webtools were used to design two set of primers, which were purchased from Eurogentec for use in pcDNA 3.1 Hygro constructs. The first set of primers, which flanked the PD-1-linker-YFP gene sequence for insertion into the pcDNA 3.1 Hygro plasmid, was as follows: F: 5′-ATGTGGGTCCGGCAGGTA-3′ and R: 5′-TTATCTGAGTCCGGAGCGGTAC-3′. The second set of primers, which was used to switch the YFP with eGFP, was as follows: F: 5′-GTGAGCAAGGGCGAGGAG-3′ and R: TTACTTGTACAGCTCGTCCATGC  were used for the cloning of DNA fragments and the preparation of plasmids because they possess recA1 and endA1 gene mutations that increase the insert stability and the extracted DNA quality. The strains were stored at −80 °C. The pcDNA3.1 Hygro/PD-1-eGFP plasmid was transformed into the E. coli cells and then purified using the Mini Plasmid Prep Kit . The plasmid concentration was then quantified using a NanoDrop (ThermoFisher Scientific), and the concentration was adjusted to 2 µg/µL to simplify further cell transfection.Mix & Go competent CHO-K1 cells were transfected with the Cell Line Nucleofector® Kit T, Program U-023 and the pcDNA 3.1 Hygro plasmid expressing PD-1-eGFP. Two micrograms of plasmid was transfected into one million CHO-K1 cells suspended in 100 µL of Nucleofector solution. Cells were analysed by flow cytometry 48 h after Nucleofection®. Cells were then subjected to an appropriate selective pressure that allowed the growth only of cells containing the DNA insert. After three weeks of selective pressure, the multiparametric analysis of transfected CHO-K1 cells overexpressing mPD-1-eGFP allowed the definition of sub-populations that could be separated from the global population. The population of cells strongly expressing eGFP was analysed by flow cytometry and retained for microscale thermophoresis analysis  were treated when they reached 80% confluency in T75 flasks. The cells were washed with 5 mL of PBS and then trypsinized with 1.5 mL of trypsin to avoid using a cell scraper. After the cells were incubated for 5 min, 3.5 mL of complete culture medium as described in the cell culture section was added to the trypsinized cells. Cells were then centrifuged at 1200 rpm at 4 °C for 5 min. The supernatant was removed, and the cells were washed again with 5 mL of PBS and then centrifuged again at 4 °C for 5 min. Once the PBS supernatant was removed, cells overexpressing mPD-1-eGFP were suspended in 300 µL RIPA buffer. The cells were kept on ice to minimize local overheating. Protease inhibitor cocktail (1%) was also added to the mixture . The lysate was collected by centrifugation at 15,000 rpm at 4 °C for 45 min to remove large aggregates and cell debris. Strong centrifugation is recommended to reduce the background that can occur in an MST experiment. The lysate was divided into aliquots, which were stored at appropriate temperatures for further investigation. The overall schematic process of this protocol from cell culture to MST measurements is described in Supplementary Fig. MST was used to establish the binding affinity between PD-1-eGFP and PD-L1 . Cell lysate was diluted in PBS-T buffer (0.05% Tween 20) to a final concentration where the fluorescent signals of the GFP proteins were similar and above the typical detection limit of the Monolith NT.115 instrument (NanoTemper Technologies). The final concentration of cell lysate was kept constant at 35 nM. For the binding between mPD-1-eGFP and h/mPD-L1, 10 µL of the 54 µM PD-L1 solution was diluted 2:1 in 10 µL buffer to make a 16-sample dilution series extending to 61.7 nM. Another concentration range was prepared using a 1:1 dilution from 24 µM to 0.7 nM with the other parameters remaining the same. Ten microliters of cell lysate was added to 10 µL of each ligand solution.MST was used to establish the binding affinity between PD-L1-eGFP and PD-1 . Cell lysate was diluted in PBS-T buffer (0.05% Tween 20) to a final concentration where the fluorescent signals of the GFP proteins were similar and above the typical detection limit of the Monolith NT.115 instrument (NanoTemper Technologies). The final concentration of cell lysate was kept constant at 35 nM. For the binding between hPD-L1-eGFP with h/mPD-1, 10 µL of the 48 µM h/mPD-1 solution was diluted 1:1 in 10 µL buffer to make a 16-sample dilution series extending to 0.7 nM. Ten microliters of cell lysate was added to 10 µL of each ligand solution.Supplementary Information