Blood samples were taken before any treatment, and the immune response after ex vivo LPS challenge was analyzed, as well as basal expression of PD-L1 on monocytes and levels of sPD-L1 in sera. The patients were followed up for 1 month. Taking into account reinfection and exitus frequency, a significantly poorer evolution was observed in patients from group I. The analysis of HLA-DR expression on monocytes, T cell proliferation and cytokine profile after ex vivo LPS stimulation confirmed an impaired immune response in group I. In addition, these patients showed both, high levels of PD-L1 on monocytes and sPD-L1 in serum, resulting in a down-regulation of the adaptive response. A blocking assay using an anti-PD-1 antibody reverted the impaired response. Our data indicated that SaO2 levels on admission have emerged as a potential signature for immune status, including PD-L1 expression. An anti-PD-1 therapy could restore the T cell response in hypoxemic sepsis patients with SaO2 ≤ 92% and high PD-L1 levels.Sepsis is a pathology in which patients suffer from a proinflammatory response and a dysregulated immune response, including T cell exhaustion. A number of therapeutic strategies to treat human sepsis, which are different from antimicrobial and fluid resuscitation treatments, have failed in clinical trials, and solid biomarkers for sepsis are still lacking. Herein, we classified 85 patients with sepsis into two groups according to their blood oxygen saturation (SaO Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection ; it is ccytokine storm, and secondly adopting an immune depressive phenotype, whereupon they are unable to respond to secondary infections  <90%, a hospital admission threshold of 92% showed be safer  expression , 15. In be safer . How hypbe safer . Therefo2) on admission and analyzed their immunological response. Additionally, ex vivo PHDs inhibition using Dimethyloxaloylglycine (DMOG) were used to stablish causal relationship between both features. Our data open a new window of immunotherapy treatment for patients with sepsis, based on their SaO2 level upon admission.Herein, in order to study the association of hypoxemia with immune alterations, we classified 85 patients with sepsis according to their oxygen saturation  that matched with patients in age, sex and body mass index were collected from the blood donor service of La Paz University Hospital.Eighty-five patients who fulfilled the diagnostic criteria for sepsis according to the Society of Critical Care Medicine and the European Society of Intensive Care Medicine international conferences , 21 wereThe Committee for Human Subjects of La Paz University Hospital approved the study, which was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki. The participants provided written consent for the study.Salmonella abortus was a kind gift from Dr. Galanos (Max Planck Institute of Immunobiology and Epigenetics). Carboxyfluorescein succinimidyl ester (CFSE) for the proliferation assays was purchased from Thermo Fisher. The lymphocyte stimulus pokeweed (PWD) was purchased from Sigma-Aldrich. To inhibit PD-1/PD-L1 interaction, a fully human IgG4 (S228P) anti-PD-1 receptor-blocking monoclonal antibody was used (Bristol-Myers Squibb). All the reagents used for cell cultures were endotoxin-free, as assayed with the Limulus amebocyte lysate test (Cambrex).Roswell Park Memorial Institute (RPMI) medium (Invitrogen) was used for the cell cultures. The following antibodies were used: anti-CD14, anti-HLA-DR, anti-CD3 (Immunostep), and anti-PD-L1 (Miltenyi Biotec). The LPS from The peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Plus gradient  as reported previously , 13.Tumor necrosis factor alpha (TNFα), interleukins (IL)1β, IL6, and IL10 protein levels in the culture supernatants were determined using the Human Inflammatory cytometric bead array (CBA) kit (BD Biosciences).For markers staining, the cells were labeled with: allophycocyanin (APC)-conjugated anti-human CD14, fluorescein isothiocyanate (FITC)-conjugated anti-human HLA-DR, APC-conjugated anti-human CD3 ; and phycoerythrin (PE)-conjugated anti-human PD-L1 . Matched isotype antibodies were used as negative controls. The cells were incubated for 30 min at 4°C in the dark. The data were acquired by flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo vX.0.7 software . Gating strategy is shown in Supplementary Figure Soluble PD-L1 (sPD-L1) on patients with sepsis and HV sera was measured using an enzyme-linked immunosorbent assay .5 PBMCs per well were seeded in a round bottom p96 plate  and stimulated with 2.5 μg/mL of PWD and treated or not with 5 μg/ml of fully human IgG4 (S228P) anti-PD-1 monoclonal antibody (Bristol-Myers Squibb) during 5 days.Proliferation was analyzed by flow cytometry of CFSE-labeled cells, as reported previously . BrieflyMonocytes were isolated from HV peripheral blood by centrifugation on Ficoll-Hypaque Plus (Amersham Biosciences) and adherence, as we have described before .The compThe cells were washed once with PBS and the RNA was isolated using the High Pure RNA Isolation Kit (Roche Diagnostics). The real-time quantitative PCRs were performed using the QuantiMix Easy SYG kit from Biotools and specific primers as described previously , 22–25.t-test for quantitative variables or χ2 test for categorical variables. We studied univariate associations between sPD-L1 and PD-L1 and oxygen saturation, using the Pearson's correlation coefficients. Moreover, we selected membrane PD-L1, sPD-L1, and clinical parameters showing significant differences between normoxemic and hypoxemic groups and fitted a logistic regression model. The severity scores APACHE II was not taking into account in this analysis because it includes SaO2. The statistical significance was set at p < 0.05. The analyses were conducted using Prism 5.0 software (GraphPad) and SPSS version 23 (IBM) software.The number of patients analyzed is indicated in each figure. Descriptive statistics are presented as counts and percentages, or as means with standard deviation as appropriated. Comparisons of subgroups were made using unpaired Student's 2 into two groups . Patients in group I exhibited a number of clinical parameters that match with a poor prognosis . Similarly, the percentage of death was significantly higher in group I than group II,   Figure .2 (group I) showed a patent impaired immune response. Their antigen presentation was affected due to reduced HLA-DR expression on CD14+ cells after ex vivo LPS challenge -treatment before LPS stimulation  takes place, we examined the role of these enzymes in an n Figure . Treatmen Figure . Further2, a blocking assay using a commercial anti-PD-1 antibody was performed. Standard levels of CD3+ cell proliferation were observed in the presence of anti-PD-1 antibody, indicating a patent immune response recovery  exhibited not only an impaired inflammatory response, reduced antigen presentation and diminished adaptive response but also a poor prognosis with higher frequency of reinfection and mortality than those patients over 92% SaO2 (group II).In the current study, we identified on admission those patients who expressed high levels of both PD-L1 and sPD-L1. According to the data presented here, levels of oxygen saturation classified patients with sepsis into two groups that showed statistically different levels of PD-L1 and sPD-L1. In addition, those patients under 92% SaOin vitro data indicated that prolyl hydroxylases inhibition by DMOG reproduced the hallmarks of hypoxemic septic patients. In this regard, the correlation between SaO2 and PD-L1 expression provides a useful tool for stratification of patients with sepsis on admission, indicating those candidates suited for anti-PD-1 immunotherapy, which would prevent a failure of the immune response.The analysis of HIF1α mRNA also revealed increased expression of this transcription factor in the group I of patients. We have previously described a crucial role for HIF1α in the control of hallmarks of sepsis evolution such as downregulation of proinflammatory cytokine production, PD-L1 expression on circulating monocytes, and subsequently, impaired T cell proliferation or adaptive response in the reinfection context , 15. Mor2, an easy-to-measure parameter, provides useful information about the expression of PD-L1 on monocytes from septic patients. Moreover, our results indicate that immunotherapy with anti-PD-1 improves the adaptive response in those patients with low SaO2 . Note that patients from this group also generated high severity scores (APACHE II and qSOFA) and expressed high levels of PD-L1 in their circulating monocytes, as well as showed elevated concentration of sPD-L1 in sera; hence, the lowest rate of T cell proliferation was corrected by blocking PD-L1/PD-1 crosstalk, as referenced before.Recently, blockage of PD-L1/PD-1 crosstalk using an anti-PD1 antibody has meant a revolutionary treatment for many types of tumors, such as melanoma, lung, and renal cancers , 36, 37.2 as a useful marker not only to predict sepsis evolution but also to identify potential immune-exhausted patients susceptible to personalized immunotherapy. Incorporating this indicator into routine emergency protocols for patients with sepsis might enhance their outcome.The present study proposes SaOJA-O collected, analyzed, and interpreted the data. CM-E, CC-C, and AM-Q, recruited the patients and provided clinical data. VT, KM-H, EL-G, and RL-R processed blood samples and data. EL-G, PG-C, and LA supervised statistical analysis and revised the manuscript. EL-C devised and designed the study, interpreted the data and wrote the manuscript. All authors read and approved the final 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.


Based on the data obtained, we have suggested a mechanism of the observed reduction process in terms of the hydrolysis of the glass wafer with subsequent interaction of the leached alkali and alkali earth cations and silicate anions with graphene oxide, resulting in elimination of the oxygen-containing groups from the latter one. The proposed approach can be efficiently used for low-cost bulk-quantity production of graphene and graphene-based materials for a wide field of applications.This paper reports a facile and green method for conversion of graphene oxide (GO) into graphene by low-temperature heating (80 °C) in the presence of a glass wafer. Compared to conventional GO chemical reduction methods, the presented approach is easy-scalable, operationally simple, and based on the use of a non-toxic recyclable deoxygenation agent. The efficiency of the proposed method is further expanded by the fact that it can be applied for reducing both GO suspensions and large-scale thin films formed on various substrates prior to the reduction process. The quality of the obtained reduced graphene oxide (rGO) strongly depends on the type of the used glass wafer, and, particularly, magnesium silicate glass can provide rGO with the C/O ratio of 7.4 and conductivity of up to 33000 S*cm It seems to have a wide field of applications in different technologies, including fabrication of transparent electrodes, supercapasitors and nanoelectronic devices, gas sensing and catalysis, biochemistry and microbiology9.Graphene continues to inspire interest in various fields of science due to its outstanding physical and chemical properties10, growth of graphene on the surface of transition metals by chemical vapor deposition (CVD)11, and reduction of graphene oxide (GO)12. Among these methods, the reduction proved to be an effective approach to produce graphene with an optimal quality at relatively low cost14.However, the preparation of graphene in large scales remains to be a challenging task. Several strategies have been developed to overcome this challenge, such as epitaxial growth of graphene on silicon carbide (SiC)15, ultraviolet16 and laser17 irradiation of GO films, electrochemical18 and chemical19 treatment of graphene oxide suspensions and films. Compared to other techniques, the chemical reduction of GO offers great ease of large-scale production of rGO in various forms such as suspensions, rGO paper or thin films on various substrates.Numerous approaches are applied to achieve conversion of graphene oxide to graphene, for instance, high-temperature annealing in a reducing enviroment20, dimetilhydrazine21, hydroquinone22 or sodium borohydride23 are typically employed in chemical reduction of GO. However, these reducing agents are highly toxic, unrecyclable, highly unstable, or generate hazardous by-products. Moreover, strong acidic or alkaline conditions are commonly required in these reduction processes thus limiting their applicability for preparing graphene-containing polymer composites24 when the reduction process must be carried out simultaneously with introducing graphene into the polymer matrix.Various chemicals, namely, hydrazine monohydrate25 or L-ascorbic acid26, results in a drastic increase in time required for the reduction of GO, and usually takes place in hot solutions. Furthermore, effective reduction of GO by these chemicals commonly requires addition of ammonia that limits the subsequent use of the obtained rGO suspensions. As such, the development of new chemical methods able to provide rapid and efficient reduction of graphene oxide under mild experimental conditions with the use of a reusable deoxygenation agent is of a high interest nowadays.On the other hand, the use of mild reducing agents, such as green molecules presented by different types of sugars (glucose or sucrose)In this paper we report a new facile method to convert GO to graphene under low-temperature heating by using sodium, alkali-barium and magnesium silicate glass wafers as reducing agents. The method can be applied for both GO aqueous suspensions and large-scale GO films formed on various substrates prior to reducing. A possible model of the GO reduction using different glass wafers is proposed. Overall, we demonstrate that reduction of GO via low-temperature heating in the presence of glass wafers appears to be a green, efficient and easily scalable method that is based on the use of recyclable non-toxic reducing agent and can be effectively employed for low-cost bulk-quantity production of graphene and graphene-based derivatives.27. The removal of the hydrophilic functional groups is further evidenced by the aggregation of rGO sheets as a result of π-π stacking interactions. The rate of the aggregation rises from the rGO_S-gl to rGO_Mg-gl sample. This fact suggests more complete elimination of oxygen-containing functional groups and larger areas of the graphene network in the latter one.Figure 28. However, it also can arise from optical transitions between π and π* states in the nanometer-size sp2 clusters remained in the structure of GO after its oxidation29. Upon the reduction, the 230 nm absorption peak progressively shifts towards higher wavelengths, and overall absorption in the range up to near-infrared (NIR) region rises significantly due to the restoration of sp2-conjugated graphene network. As seen, the UV-Vis spectra of rGO_Mg-gl and rGO_AB-gl are almost similar with the peak of the π-π* transition lying at 265–268 nm, which is a characteristic feature of the high-degree GO reduction30. At the same time, the position of the main absorption peak (at 247 nm) and highly non-linear character of the absorption in the visible and NIR regions in the rGO_S-gl spectrum signifies incomplete elimination of the oxygen functionalities in the case of using sodium silicate glass as a reducing agent.Figure 32. Particularly, the broadband absorption feature at 3000–3700 cm−1 originating from the set of overlapping bands of O-H stretching in water molecules, hydroxyls and carboxyls is observed. Additionally, distinguishable bands at 1720 cm−1, 1620 cm−1, 1415 cm−1, 1225 cm−1 and 1040 cm−1 are presented. These absorption lines correspond to the stretching and bending vibrations of the carbonyl/carboxyl groups, water molecules, basal-plane hydroxyls, epoxides and edge-located hydroxyls, respectively. The distinguishable features at 980 cm−1 and 1280 cm−1 are related to the presence of the five-membered ring lactols32 and ethers33.Figure −1 that corresponds to C=C vibrations within the recuperated conjugated aromatic structure and vanishing of the absorption bands at 3000–3700 cm−1 indicates successful deoxygenation of the rGOs. However, the IR spectrum of the rGO-Sgl sample still exhibits noticeable absorption bands of epoxides (1225 cm−1), edge-located hydroxyls (1040 cm−1) and carboxyls/carbonyls (1720 cm−1). This suggests retention of some amount these functionalities after the reduction. At the same time, the IR spectra for both rGO_AB-gl and rGO_Mg-gl samples demonstrate nearly complete elimination of all oxygen-containing functionalities. The only absorption feature that can be distinguished is related to edge-located hydroxyl groups (phenols). The preservation of these groups is due to their high resistivity to elimination34.The emergence of the prominent absorption band at 1580 cmFigure 35 (peak C-V). The peaks 284.6 eV and 284.9 eV are related to sp2-bonded carbons of perfect graphene lattice (peak C=C) and to carbon atoms being partially sp3-hybridized due to strong graphene network distortion caused by attachment of oxygen-containing groups (peak C-C), respectively36. Note that the C=C peak is asymmetric due to the natural asymmetry inherent for C1s XPS spectra of highly sp2-conjugated graphene-like structure observed in highly reduced GO films38. Other three peaks located at 286.7 eV, 288.2 eV and 288.9 eV correspond to hydroxyl and epoxide groups (C-OH and C-O-C), carbonyl groups (C=O) and carboxyl groups (COOH), respectively39. Three main O1s components  of 7.8 Å. This value is consistent with the published data41. Additionally, the less intense broadened peaks at 2θ = 43° and 2θ = 77.7° characteristic of the (10) and (11) reflections can be observed. These peaks arise from the 2D diffraction from the planar carbon network of GO flakes. The intensity ratio between the crystalline reflections of type (00.l) and lattice reflections of type (hk) indicates that the GO flakes have a lamellar structure, and their lateral size is larger than several micrometers42. Further analysis of the SEM images of arrays of GO flakes on the silicon wafer and laser diffraction measurements of the GO aqueous suspension  XRD-pattern diffraction peak has shifted to higher angles due to the decrease in the rGO interlayer spacing, which proves elimination of the oxygen-containing groups. However, this peak positions are different for different types of the glass wafers. The interlayer spacing has been determined to be 4.1 Å for rGO_S-gl, 3.9 Å for rGO_AB-gl, and 4.5 Å for rGO-Mg-gl. These values are noticeably lower than the GO interlayer distance, which confirms elimination of interlayer water and oxygen-containing groups. On the other hand, these values are larger than both the graphite interlayer spacing of 3.4 Å and that of 3.7 Å published for the reduced graphene oxide45 and suggest that an average lateral size of coherent scattering regions (CSR) corresponding to the defect-free regions in rGO_AB-gl and rGO_Mg-gl is 200 nm.The diffraction patterns of the rGO_AB-gl and rGO_Mg-gl samples also contain distinguishable asymmetric (10.l) and (11.l) reflections which are superposition of reflections of the (hk.l) and (hk) types. The shapes and positions of these diffraction features coincide with those in microcrystalline graphite−1 related to in-plane stretching of the graphene lattice and D peak around 1349 cm−1 caused by the lattice disorder, e.g., edges of the sp2 clusters and boundaries of the flakes47. After reduction, the frequency of the D band and G band in Raman spectra of all rGOs is equal to that of GO. The intensity ratio of these bands (ID/IG) is commonly used to evaluate the stacking order and defect density in the obtained graphene samples12. Upon the reduction, the ID/IG ratio does not significantly change and remains within the range of 1.1, although commonly applied chemical reduction procedures lead to the significant rise of the ID/IG ratio49. This evidences that the used reduction procedure does not cause considerable structural disorder if alkali-barium silicate glass or magnesium silicate glass are applied. The absence of the observable decrease in ID/IG ratio is related to the high number of layers in the studied films that are about 500 nm in thick.Figure 47 in the obtained rGO_AB-gl and rGO_Mg-gl samples, is also shown by the obtained TEM images  along with metasilicate (SiO32−) and ortosilicate (SiO44−) anions from the glass surfaces into the suspension59. In the process, the pH value rises from 3.4 to 8–8.5. Beside the formation of alkali and alkaline-earth silicates, the leached silicate anions may also interact with the oxygen-containing functional groups of GO. In this case intermediates are formed, composed by the metasilicate or ortosilicate anion that is attached to the GO layer by the oxygen-bridge bond originated from rearrangement of chemical bonds in epoxides , or magnesium sulphate (rGO_MgSO4), or sodium hydroxide (rGO_NaOH), or sodium silicate (rGO_Sil), or a combination of sodium silicate and magnesium sulphate (rGO_Sil-Mg). The degree of reduction and chemical composition determined for the resulting rGOs is indicative of the role of each component  in reducing GO with glass wafers. The low-temperature (80 °C) heating of the GO aqueous suspension in the presence of the quartz wafer does not result in the deoxygenation of graphene oxide, as shown by UV-Vis, FTIR and XPS spectra Figure . The tre39, the rGO obtained in alkaline solution with pH~8 has the C/O ratio of 2.9 which is significantly lower than those even in the rGO_S-gl samples. On the other hand, the reduction degree of rGO_Sil (C/O = 4.99) is very close to that in rGO_S-gl (C/O = 4.2), and an increase in concentration of carbonyl groups is observed upon the reduction with both sodium silicate glass and sodium silicate powder. Moreover, the formation of nanoscale holes in the rGO_Sil sample is also demonstrated by the obtained TEM image  was oxidized by using concentrated H2SO4, KMnO4, NaNO3, and H2O2 solutions. The resulting mixture was centrifuged (3500 rpm for 1 hour), and the supernatant was decanted away. The material remaining after this was additionally centrifuged (1500 rpm for 10 min) to obtain aqueous GO suspension as a supernatant. In the process of synthesis, sonication was excluded to prevent damaging of graphene oxide flakes and obtain suspensions with the utmost size of GO flakes .Graphene oxide was synthesized by the Hummers methodTo prepare GO films for the subsequent reduction, 200 μL of GO aqueous suspension 0.003 wt % in concentration was drop-casted on silicon and quartz wafers and dried overnight at room temperature.Three types of glass wafers with different chemical compositions were used as possible reducing agents for GO deoxygenation: sodium silicate glass containing only sodium oxide, magnesium silicate glass containing only sodium oxide and magnesium oxide, and alkali-barium silicate glass, as one of the most common glass types, containing various alkali and alkaline earth oxides. The chemical compositions of the used glass wafers and their price are presented in Table The reduction of GO aqueous suspensions was performed as follows: a piece of sodium silicate glass , alkali-barium glass  or magnesium silicate glass  was immersed into GO aqueous suspension (40 mL) 0.01 wt% in concentration with subsequent stirring of the suspension at 80 °C for 5 hours in a fluoroplastic flask. The obtained rGO suspensions were copiously washed by centrifuging (centrifuge Sigma 3–30KS) at 26,200 rpm (60.600 g) and rinsing the obtained sediment with de-ionized water. The described purification procedure was repeated five times. The obtained rGO samples were denoted as rGO_S-gl (reduced by sodium silicate glass), rGO_AB-gl  and rGO_Mg-gl (reduced by magnesium silicate glass). The quantity of graphene, obtained from the 0.5 g piece of glass wafer  with actual size of 15 × 7.5 × 1.0 mm was determined to be about 50 mg. Magnesium silicate glass wafer was applied for the reduction of graphene oxide suspension up to 7 times with successful conversion of GO into rGO during 5 cycles. As a result, the maximum quantity of the produced graphene from a single glass wafer in the applied conditions was determined to be about 0.25 g.To analyze applicability of the studied method for reducing GO films on substrates, the GO films on quartz or silicon substrates were put into a fluoroplastic flask filled with de-ionized water (40 mL); after that, a piece of magnesium silicate glass was added, and the flask was heated at 80 °C for 5 hours. After the reduction, substrates with the rGO film were carefully withdrawn from the solution, washed several times with de-ionized water, and dried overnight at room temperature.For better understanding of the processes that lie behind the observed deoxygenation of GO, a series of control experiments was carried out. Namely, GO aqueous suspensions were heated at 80 °C during 5 hours in the presence of the quartz wafer, after adding 0.01 mol. of magnesium sulphate powder (obtained from Acros Organics Company), or 150 µL of NaOH solution , or 0.7 mmol. of sodium silicate powder (obtained from Acros Organics Company), or 0.8 mmol. of sodium silicate together with 0.8 mmol. of magnesium sulphate. The obtained samples were washed according to the aforementioned procedure.To provide the correct alignment and deconvolution of the XPS spectra of GO and whole series of the studied rGOs, an additional rGO sample denoted as rGO_HT was prepared by annealing the GO film at 600 °C during 2 hours.The pH values of the solutions were determined with a Fisher Scientific Accumet Basic AB15 pH meter. The UV-vis absorption spectra of the GO and rGO samples were collected with a Shimadzu-2450 spectrophotometer. Fourier transform infrared spectroscopy was performed on the Infralum-08 FTIR spectrometer equipped with the attenuation of total reflectance attachment. X-ray photoelectron spectroscopy (XPS) measurements were carried out on a Thermo Fisher ESCALAB 250Xi XPS system with a monochromatic Al Kα X-ray source (1486.6 eV). The spectra were calibrated with respect to the Au 4f7/2 line (84.0 eV). A surface charging revealed for low-conducting GO (and some rGOs) was taken into account by the aligning their XPS spectra with respect to the C1s line position (284.6 eV) for a well-conductive rGO-HT sample ].The X-ray diffraction (XRD) analysis was carried out using a Bruker Smart Apex Duo installation with a CuKα source and Apex 2D detector. The sample for diffraction measurements was fixed with nitrocellulose lacquer at the end of a cactus needle. Diffraction patterns were measured at various angles between the normal to the detector surface and the X-ray direction, the 2D data being subsequently recalculated to the 2θ configuration. The obtained diffraction patterns were analyzed using the DIFFRAC.EVA (Bruker Cor.) software based on the data from Powder Diffraction File ICCD PDF-2 release [JCPDS-International Centre for Diffraction Data  images were acquired with a Jeol JEM-2100F microscope . Samples for TEM were prepared by deposition of aqueous GO and rGO suspensions 7·10−4 wt % in concentration onto conventional lacey carbon films. SEM images were collected with a JSM-7001F, Jeol microscope. Monolayer films for SEM imaging were prepared by the Langmuir−Blodgett method according to the procedures published elsewhereElectrical conductivity measurements of the GO and rGO samples were performed on the base of two-electrode system. The GO and rGO films were deposited by the drop-casting method onto the surface of quartz substrates with two comb Au electrodes 80 nm thick separated by 500 µm. The electrode comb consisted of 8 pairs of the electrode bars Figure .Supplementary Information


ASIP locus which led to an ectopic expression of ASIP mRNAin cattle. In this study, we detected the indel of the L1-BT element atg. ASIP gene is expressed widely in the tissue of cattle, includingadipose and muscle tissue. Hence, it has been suggested that ASIP plays a role in bovine fat metabolism. An inserted L1-BT element was recently identified upstream of the Therefore, another biologicalfunction of ASIP was suggested in the regulation of obesity in mice.The Agouti signaling protein (ASIP) has been described as a secreted protein expressedmainly in the skin and regulated pigmentation in wild mice . Mutations in ASIP is ubiquitously expressed. ASIP mRNA could bedetected in human adipose tissue, skin, heart, testis, ovary, liver, andkidney . In pigs, the expression of ASIP could be found in liver and muscle. Norris and Whan (2008) detected ASIP mRNA inthe liver, kidney, skin, heart, and spleen of sheep. Several bovine studies hadreported on ASIP mRNA expression in adipose tissue, M. longissimus, skin, heart,testis, ovary, and kidney . Studies on ASIP in farmanimals mainly focused on coat color . BothGirardot et al. (2005) and Royo et al. (2005) found that there wasno variant in coding exons of ASIP in different breeds of cattle with differentcoat color. A full-length element  inserted at 5ASIP gene was identified to give rise to an overexpression ofASIP in the skin of Normande cattle . Girardot et al. (2006) concluded thatthe insertion of the L1-BT element may be the reason for the brindle coat colorof the Normande cattle. The authors also presumed that the overexpression ofASIP caused by L1-BT element insertion may influence the meat production incattle, as mice with ectopic expression of ASIP were obese.Liu et al. (2018) reported that in bulls of an ASIP mRNA was observed due to the L1-BTelement insertion at ASIP in 17 bulls, but no association with fat-related traitswas found. However, there were significant correlations between the ASIP mRNA levelof subcutaneous fat and traits related to fat deposition in bulls withoutan L1-BT element . Up to now, there is no research yet for L1-BT elementinsertion at the ASIP locus in Chinese cattle populations. Also, associationanalyses between single nucleotide polymorphism (SNP) of bovine ASIP and carcasstraits and fatty acid composition in cattle have not been reported.Therefore, in this study we scanned for the L1-BT element and further SNPs atthe ASIP locus in a population consisting of 363 Chinese Simmental steers. Herewe focused on the traits related to fat deposition  and fatty acid composition, which can provide importantinformation for meat production and quality in cattle. Carcass traitslike liver weight and thigh meat thickness were also analyzed, as ASIP, asecreted protein, may circulate and act in the different tissues and partsof the body. The correlation between SNPs and traits was determined toillustrate the potential effect of ASIP in cattle, especially for meatproduction and meat quality.In human and farm animals, 22.1There were 363 Chinese Simmental steers involved in this study. Animals wererandomly selected from 15 cattle farms in the Wulagai administrative district of InnerMongolia in China. Steers were slaughtered at 28 months of age at the InnerMongolia Baolongshan cattle farm . Carcass compositiontraits, meat quality traits, and fatty acid composition traits were measuredat the Chinese Academy of Agricultural Sciences meat laboratory as describedpreviously by Fang et al. (2017). Fatty acid content was extracted from M. longissimusand expressed as grams per 100 g fresh tissue. Blood samples  were collected from the jugular vein with anticoagulant  and stored at 2.2http://primer3.ut.ee/, last access: 10 December 2018) and synthesized by GENEWIZ,Inc. . Primer pairs of the ASIP L1-BT element and ASIP 5ASIP. Bothprimer pairs shared the same forward primer which was located upstream ofL1-BT element insertion. The reverse primer of the ASIP L1-BT element was designedat genomic ASIP 5ASIP L1-BT element and the ASIP 5ASIP L1-BT element andASIP 5ASIP L1-BT element and the ASIP 5ASIP L1-BT element and ASIP 5DNA was extracted from leucocytes from whole blood samples (10 mL from eachsteer) using a TIANamp Blood DNA Kit  and followingthe manufacturer's protocol. The purity and concentration of the genomic DNAwere determined using a NanoDrop ND-2000 ultraviolet spectrophotometer, and the quality was verified by agarosegel electrophoresis. Primers (Table 1) were designed using Primer 3web using SPSS 13.0for Windows. The fixed model wasThe genotype frequency and allelic frequency of each SNP were calculatedaccording to genotyping results. The polymorphism information component (PIC)was determined by Botstein's methods . The Hardy–Weinbergequilibrium of the polymorphisms was tested with the chi-squared . The percentageof Ho in three SNPs was higher than that of He. The PIC value ofg. Values of genotypic frequencies, allelic frequencies, PIC, Ho (genehomozygosity), and He (gene heterozygosity) were calculated to illustrate thegenetic diversity of 3.3ASIP, a heavier liver weight. Insertions ofdifferent sizes upstream of first coding exon are the cause forintracisternal A-particle yellow . Both the homozygote andheterozygote of the L1-BT element indel were detected in bulls of theMontbéliarde breed . Albrecht et al. (2012) foundheterozygote insertion of this element, which gave rise to an ectopicexpression of ASIP in different tissues of Japanese Black and Charolais cattle.In our study, three genotypes of the L1-BT element indel were also detected. Itis obvious that homozygosity of L1-BT element insertion is not a lethalfactor in cattle. Liu et al. (2018) reported that steers with a heterozygote of the L1-BT element indel had anextremely high ASIP mRNA level in various tissues, but it was not associatedwith any traits related to fat deposition. In the current study, there wasno significant difference between GL and GG genotype Chinese Simmentalsteers in fat-related traits. However, steers with the LL genotype had lowerperirenal fat weight, thinner back fat, and less carcass fat coveragepercentage compared with other genotypes , it can be presumed thatin this Chinese Simmental population overexpression of ASIP in steers withthe LL genotype showed a negative association with traits related to fatdeposition. In a previous study, we found that a heterozygous insertion ofL1-BT led to an ectopic expression of ASIP mRNA but not protein. Hence, further experiments should focus on the expression pattern andeffect of ASIP in cattle with the LL genotype.In mice, there are five dominant mutations causing ectopic expression ofASIP locus. Among them, indel of the L1-BT element at g. ASIP could be found (data not shown). Likewise,Girardot et al. (2005) and Royo et al. (2005) did not find any SNPof bovine ASIP in coding exons. This suggested that the coding region of bovineASIP is highly conserved. The analysis of haplotypes is an important component ofgenetic association analysis. A haplotype consists of a group of linked SNPmarkers, and it may increase the coverage value of genotype over single SNPanalysis . The associationanalysis between haplotype combinations and traits of Chinese Simmentalsteers indicated that the H1H2 genotype was positively correlated to carcass andmeat quality traits, e.g., with a higher carcass weight and thicker back fat.But the H3H3 genotype had a negative association with fat-related traits such as a lower marbling score and perirenal fat weight. Moreover, linoleic acidand In this study, we detected the L1-BT element indel and three SNPs at the bovine5ASIP showed significantcorrelations with fat-related traits in Chinese Simmental steers. Thissuggests that ASIP is involved in fat deposition at different stages ofthe growth and fattening of beef cattle, such as intramuscular fat, back fat,and perirenal fat deposition. The haplotype combinations of H1H2 and H3H3may be molecular markers for traits related to fat deposition and meatquality . However, further studies arenecessary to unravel the mechanism of how the ASIP gene affects meat production andquality in cattle.In summary, both polymorphisms and haplotype analysis of


Using molecular dynamics (MD) simulations, the frictional properties of the interface between graphene nanoflake and single crystalline diamond substrate have been investigated. The equilibrium distance between the graphene nanoflake and the diamond substrate has been evaluated at different temperatures. This study considered the effects of temperature and relative sliding angle between graphene and diamond. The equilibrium distance between graphene and the diamond substrate was between 3.34 Å at 0 K and 3.42 Å at 600 K, and it was close to the interlayer distance of graphite which was 3.35 Å. The friction force between graphene nanoflakes and the diamond substrate exhibited periodic stick-slip motion which is similar to the friction force within a graphene–Au interface. The friction coefficient of the graphene–single crystalline diamond interface was between 0.0042 and 0.0244, depending on the sliding direction and the temperature. Generally, the friction coefficient was lowest when a graphene flake was sliding along its armchair direction and the highest when it was sliding along its zigzag direction. The friction coefficient increased by up to 20% when the temperature rose from 300 K to 600 K, hence a contribution from temperature cannot be neglected. The findings in this study validate the super-lubricity between graphene and diamond and will shed light on understanding the mechanical behavior of graphene nanodevices when using single crystalline diamond as the substrate. On the r copper . Researcr copper ,20,21. For graphene-based electronic devices, it is critical to understand the mechanics of the graphene nanoflake–diamond interface when lateral frictional forces are present in order to design a stable nanoelectronic device. Therefore, researchers have been conducting extensive experiments using atomic force microscopy (AFM) and friction force microscopy (FFM) to explore the frictional behavior of interfaces between graphene and different types of substrate materials ,24,25,26In order to obtain a more realistic understanding of the interfacial friction behavior, molecular dynamics (MD) simulations can be used because of the difficulty of performing experiments at such a small length scale ,27. BoneInspired by previous experiments, in this work, a comprehensive evaluation of anisotropic frictional behavior of graphene nanoflake-diamond interface has been studied by considering the effects of chirality and temperature. Based on the fact that electronic properties of graphene nanoribbon changes for different chirality, it is important to consider the entire range from armchair to zigzag graphene when studying the interface properties. Understanding the graphene flake–diamond interface properties is essential for the fabrication of graphene-based electronic transistors .3-hybridization) with the diamond substrate. Therefore, van der Waals forces governed the interaction between the graphene nanoflake and the diamond substrate [In order to obtain the friction coefficient of the interface between the graphene nanoflake and the diamond substrate, a molecular model of a monolayer graphene nanoflake that is sliding over a single crystal diamond substrate has been created. Molecular dynamics (MD) simulations were conducted using LAMMPS . The sizubstrate . The atoubstrate . These aThe graphene nanoflake moved over the C(100) surface of the diamond substrate, and the details of the diamond substrate are shown in Since graphene’s properties depend significantly on the chiral angle, the graphene nanoflakes with different chiral angles, as shown in Molecular dynamics simulations were performed using LAMMPS  in the NThe definition of each term that appears in Equation (1) can be found in Zhang et al. . The upp results  and has  results ,39,40,41 results . This diNormal force over the graphene layer was applied by decreasing the distance between graphene and diamond. After obtaining the thermodynamic equilibrium distance in each case, the normal force was almost zero, which means the graphene layer and the diamond substrate started to repel each other when having a distance less than the equilibrium distance. When sliding the graphene nanoflake along the z direction, the force needed to maintain the sliding motion in the z direction was the friction force between graphene and diamond. The variations of force in the x and z direction with respect to time were plotted, and the details of the simulation results are discussed in the following sections.The equilibrium distance between graphene and diamond was reached so that the net force on graphene fluctuated around zero. It is necessary to find the equilibrium configuration for each geometry in order to later apply normal load on the graphene in the MD simulations to then calculate the interfacial friction force. In order to find the equilibrium distance between the graphene and the diamond, the graphene nanoflake was first placed 7 Å above the center of a square 4-layer diamond substrate which was 10.0 nm × 10.0 nm, as shown in The MD simulations were performed for the interface of the rectangular graphene nanoflake of 2.0 nm × 4.0 nm sliding over the surface of a rectangular diamond substrate as shown in In order to understand the effects of temperature on the interfacial friction coefficient, simulations were performed at different temperatures. Firstly, simulations at absolute zero degree Kelvin were performed to serve as a reference to be compared with the results at other temperatures. When the graphene nanoflake was sliding along its armchair direction, the normal force under the distance of 2.6 Å at 0 K temperature was around 55 nN and exhibited periodicity according to Friction forces were plotted with respect to the sliding distances over the diamond surface in The magnitude of the fluctuation of the frictional force is taken as the frictional force of the graphene–diamond interface. The friction force versus the corresponding normal force is summarized in As temperature may have a significant influence on the friction coefficient, the MD simulations were extended to 300 K, 400 K, 500 K, and 600 K. In The friction coefficient between graphene and diamond was within the range of ~0.004 to ~0.023 at 300 K depending on chirality. For all the cases that were simulated, the friction coefficients were within ~0.004 to ~0.024. The interfacial friction coefficient always had the smallest value when the angle between graphene and the diamond was 0 degrees, which is the armchair direction of the graphene nanoflake. In comparison, the interfacial friction coefficient has the largest value when the sliding direction is along the zigzag direction of the graphene nanoflake, which has a 30 degrees angle between graphene and the diamond. The interfacial friction coefficients of all the simulations accompanied with the calculated friction angles for each case are summarized in Contrary to the findings of Dienwiebel and Frenken , we founThe friction coefficients of the graphene–diamond interface were compared with relevant previous studies, as shown in The increasing friction coefficient from armchair direction to zigzag direction is considered to be due to more intense interactions between graphene and the diamond substrate. In order to verify this hypothesis, we introduce the term Proximity (P) to describe the intensity of the interaction between the graphene flake and the diamond substrate as the summation of the reciprocal of the pairwise distance  surface of a single crystalline diamond substrate. Relaxation simulations were first performed in order to find the equilibrium distance between graphene and diamond at different temperatures. The equilibrium distance between graphene and diamond was close to 3.35 Å, which is the interlayer distance of graphite. The friction forces exhibited periodic stick-slip motion with respect to the sliding distance, and it was due to the periodic lattice arrangement of both graphene and diamond. The simulation was first performed under 0 K and then extended to other temperatures—300 K, 400 K, 500 K, and 600 K. The friction coefficient was the lowest when graphene was sliding along its armchair direction and the highest when it was sliding along the zigzag direction. Moreover, the interfacial friction coefficient can increase by 20% with a rise in temperature from 300 K to 600 K. In summary, the friction coefficient ranged from 0.0042 to 0.0244 depending on the chirality angle and the temperature. Compared to the graphene–N-coated 52100 steel interface, the friction coefficient of the armchair graphene–diamond interface was lower, while the friction coefficient of the zigzag graphene–diamond interface was comparable. The above findings can help to understand the behavior of graphene-based nanodevices sitting on single crystalline diamond substrate.


The miniaturization trend leads to the development of a graphene based nanoelectromechanical (NEM) switch to fulfill the high demand in low power device applications. In this article, we highlight the finite element (FEM) simulation of the graphene-based NEM switches of fixed-fixed ends design with beam structures which are perforated and intact. Pull-in and pull-out characteristics are analyzed by using the FEM approach provided by IntelliSuite software, version 8.8.5.1. The FEM results are consistent with the published experimental data. This analysis shows the possibility of achieving a low pull-in voltage that is below 2 V for a ratio below 15:0.03:0.7 value for the graphene beam length, thickness, and air gap thickness, respectively. The introduction of perforation in the graphene beam-based NEM switch further achieved the pull-in voltage as low as 1.5 V for a 250 nm hole length, 100 nm distance between each hole, and 12-number of hole column. Then, a von Mises stress analysis is conducted to investigate the mechanical stability of the intact and perforated graphene-based NEM switch. This analysis shows that a longer and thinner graphene beam reduced the von Mises stress. The introduction of perforation concept further reduced the von Mises stress at the graphene beam end and the beam center by approximately ~20–35% and ~10–20%, respectively. These theoretical results, performed by FEM simulation, are expected to expedite improvements in the working parameter and dimension for low voltage and better mechanical stability operation of graphene-based NEM switch device fabrication. The success of the semiconductor industry depends on reliable performance and scalable manufacturing processes. As the size of semiconductor devices miniaturize to a few tens of nanometers and the demand for more applications keeps increasing, another strategy will be needed. A nanoelectromechanical (NEM) switch is one of the promising devices to solve the problems of high power consumption in complementary metal-oxide semiconductor (CMOS) circuits ,2. The N2/V·s, high Young’s modulus of 1 TPa, superior current density capacity of 108 A/cm2, the ultra-thin thickness of 0.335 nm and low resistivity of 1 µΩ·cm [Graphene is one of the suggested 2D materials for high-performance NEM switch application because of its superior properties namely high electron mobility excess of 200,000 cm 1 µΩ·cm ,10,11,12 1 µΩ·cm . An opti 1 µΩ·cm ,15,16. O 1 µΩ·cm ,18. Ther 1 µΩ·cm . HoweverIn this work, we emphasize on the actuation pull-in voltage and von Mises stress reduction for a graphene-based NEM switch by using 3D finite element (FEM) modeling and simulation of a multilayer graphene beam with and without the perforated structure of different geometrical dimensions. We concentrate on optimizing the design of the graphene-based NEM switch in-line with the experimental work . In addi3- and sp2-hybridized carbon atoms. Besides, this NCG film is polycrystalline in nature [L, thickness t, and air gap thickness g because of their influence on the mechanical and electrical properties of the switch. The length and thickness of the graphene beam were varied from 0.8 µm to 1.5 µm and 3.0 nm to 9.0 nm, respectively. The air gap thickness was varied from 50 nm to 130 nm. It is worth to mention that the surface adhesion effect between graphene beam and bottom electrode contact is neglected as the influence of surface effect such as van der Waals force becomes insignificant as the device size increases. It is known to be dominant when the air gap thickness, g < 20 nm [HL, hole width HW, distance between two holes DL and number of hole column CN. This perforation concept is expected to reduce the biaxial residual stress and the graphene beam stiffness, thus contributing to the reduction of graphene beam buckling-effect to increase the switch lifetime and reduced actuation pull-in voltage [This section concisely explains the geometry of NEM switch and its operation principles. Firstly, the switching operation of multilayer graphene-based NEM switch was studied based on the previous experiment work . The muln nature . We defin nature . Based on nature . Afterwa < 20 nm . The opt voltage . The schThe operation of graphene-based NEM switch depends on the distance and corresponding electrostatic force between the bottom electrode and suspended graphene beam. The voltage between the gold bottom electrode and the graphene beam is increased until it reaches a critical point known as pull-in voltage. At this stage, the electrostatic force overwhelms the elastic restoring force of the deformed graphene beam to accelerate towards the bottom actuation electrode, resulting in closing the device which leads to a sharp rise in the current flow through the device . As the x, y, and z). Therefore, we need to set our model boundary conditions to get the accurate results. There are two different boundary conditions used in our IntelliSuite software analysis namely the fixed boundary condition and the contact pair boundary condition. The fixed boundary condition was set at the both ends of graphene beam, and the bottom surface of the model geometry. While, the contact pair boundary condition was set at the top of the bottom electrode surface and the bottom of the graphene beam surface. In general, we used the Face pair type of the contact pair to find the pull-in voltage, where the suspended graphene beam deflection will be stop at a specific spatial coordinate value. For the electrostatic force domain, the following equation is solved for the electromechanical problem in TEM module by IntelliSuite software for each discretized element.σ is the surface charge density, G is the green function, and da′ is incremental in the surface area. The following equations were used by IntelliSuite software to solve the problem for the mechanical domain.In our simulation work, the FEM-based CAD tool IntelliSuite   under theleF. Parameter, q, in the first equation is the vector containing the displacements of the mechanical structure. This equation also known as the equilibrium equation of the mesh. While, the vector u in the second and third equations are the displacement of the electrostatic mesh nodes. The third equation is the electrostatic system which depending on the vector, u, displacement. Finally, the electro-mechanical coupling appears through this deformed mesh.The first equation is the purely mechanical system which considering the electrostatic forces, g is the air gap between the graphene beam and bottom actuation electrode, K is the beam spring constant, ɛ0 is the vacuum permittivity of 8.853 × 1012 F·m−1, L and bW are the graphene beam length and width, respectively. Besides, cW is the contact width of the bottom electrode, E is the Young’s modulus of graphene, and t is the graphene thickness which is thoroughly related to the number of graphene layer. The derivation of this pull-in voltage equation can be found in [The actuation pull-in voltage empirical equation for a fixed-fixed end graphene beam NEM switch is given by ,28:(3)Vpσ, is defined as the applied force per unit original under deformed cross-sectional area of the perforated graphene. While strain, ɛ, is the engineering strain defined as the change of graphene length during ‘on-state’ mode per unit of the initial graphene length during ‘off-state’ mode. The applied force upon an object is based on second law of motion given by [ρ is density of the beam defined as beam mass per unit volume of the beam. While a is the acceleration of the beam. We can assume that the perforated graphene beam volume is defined as the effective volume of graphene beam after deducting the volume of holes.The actuation pull-in voltage equation of perforated graphene beam NEM switch is also specified by Equation (3) but with modification of the beam spring constant because of the changes in its Young’s modulus which corresponding to the beam volume and mass. The typical Young’s modulus, stress and strain equations of a beam structure are given by :(4)E=σε,given by :F = ma, First, the device dimensions mentioned in In addition, we conducted an FEM scaling analysis on these validated models, as tabulated in The results are summarized in HL), hole width (HW), distance between two holes (DL), and number of hole column (CN) are tabulated in Here, we evaluate the effect of perforation in the graphene beam on the actuation pull-in voltage of the NEM switch. The devices dimensions such as hole length  and (4) based on the perforated NEM switch dimensions in However, the variation of vmσ is given by [zσ, yσ, and xσ are the stress in the z, y, and x Cartesian coordinate system, respectively. The mechanical stability of the graphene-based NEM switch can potentially be enhanced by optimizing the dimensions of the switch. The maximum von Mises stress employed across the graphene beam length was compared in order to demonstrate the mechanical stability of the graphene-based NEM switch quantitatively [One of the methods used to estimate the yield of ductile material such as metals is the von Mises stress analysis . Failuregiven by ,33:(6)σvtatively . In addiL and t on the von Mises stress. L and t, respectively. The maximum von Mises stress was achieved at the graphene beam ends when t and L was increased and decreased, respectively, due mainly to the increased electrostatic force between the gold bottom electrode and graphene beam. Based on We carried out another FEM scaling analysis by using IntelliSuite software to justify the influence of L and t were adopted from our previous optimized intact graphene beam analysis. We further extend our FEM scaling analysis to elucidate the effect of the perforation concept in the graphene beam on the von Mises stress and compared it with the intact graphene beam structure analysis. The value of v, of 0.17 and a Young’s modulus, E, of 860 GPa. The free-space environment was used in the model of the NEM switch. We used free tetrahedral triangular mesh elements to mesh the switch model to simplify computational complexity. The mesh density was varied adaptively to analyze the graphene beam structural displacement. In this analysis, the graphene beam and the gold electrode was positioned at the top and the bottom, respectively. The applied voltage was swept at the graphene beam and 0 V of fixed bias was applied to the gold bottom electrode to analyze the electrical field distribution at different voltages, while other boundaries are electrically insulated. Besides, the voltage potential (V) and the electrical field (E) in the vacuum condition can be acquired by solving the Poisson’s equation as given by [We used the exact model in COMSOL Multiphysics version 5.2   to invesgiven by :−∇·(ε·∇Vz-direction at the different applied voltages. The electrical field strength was extremely concentrated at the graphene beam edges and hole edges. These outputs reveal that the downward component of the electrostatic force stand-in on the hole edges and graphene beam edges, leading to the higher von Mises stress at the hole edge compared to the von Mises stress at the beam center.Besides, L, t, g, HL, HW, DL, and CN. These analyses suggested an actuation pull-in voltage of 1.5 V can be achieved by the following dimension: graphene beam width of 0.3 µm with a length of 1.5 µm, a thickness of 3.0 nm, air gap thickness of 70 nm, hole length of 250 nm, hole width of 25 nm, distance of 100 nm between two holes, and 12 numbers of hole column of the graphene-based NEM switch. This simulated actuation pull-in voltage value of 1.5 V was comparable with the conventional semiconductor switch in the CMOS circuit that typically operated at 2 V. More efforts are still needed to further reduce the actuation voltage and enhance the mechanical stability of the graphene-based NEM switch. Parameters like air damping effect, Casimir effect, and van der Waals force are to be considered for the future NEM switch analysis.In this article, we have studied the characteristics of electromechanical switching and mechanical stability of an intact and perforated graphene-based NEM switch by FEM simulations. In order to estimate their effect towards actuation pull-in voltage and von Mises stress, we had scaled the device dimensions namely


Corrosion and corrosion-induced damage have resulted mostly in malfunctions and sometimes even in failures of metallic structures, including oil and gas pipelines. In this study, new high-performance composite coatings were developed by incorporating nanoparticles in the polymer resins with applications to oil and gas pipelines. The graphene nanoplatelets under different concentrations were used to prepare the epoxy-based nanocomposites and were then evaluated through mechanical and electrical tests. The integration of high-speed disk and ultrasonication were adopted as the dispersion technique to overcome nanoparticle agglomeration. Electron microscopy techniques were used to investigate the agglomeration. The new composites were qualitatively and quantitatively evaluated in terms of contact angle, surface roughness, adhesion to the substrate, corrosion resistance, and abrasion resistance. The results suggested that the composite with 0.5~1.0 wt.% of the graphene nanofillers led to the largest improvement in both mechanical and electrochemical properties. Distribution of nanoparticles in the matrix was observed using scanning electron microscopy and surface roughness using atomic force microscopy. Large agglomeration that was observed at the higher concentrations mainly resulted in the reduction of corrosion resistance and abrasion resistance. The United States consists of over 2.6 million miles of gas and oil pipelines. As the major pipeline system, the onshore transmission metallic pipelines for gas and hazardous liquids demand a high standard of safety. Corrosion is one of the leading causes of failures of metallic pipelines in the United States and worldwide ,2.The report  revealedPolymeric coating is widely used to provide a protective film for the metal that can prevent the direct contact between the substrate and the corrosive environment . The agg2/Vs at room temperature), thermal conductivity (5000 W m−1 K−1), and Young’s modulus of 1 TPa [Graphene, a two-dimensional carbon nanomaterial with the building block of natural graphite, is currently the strongest known material . It is aof 1 TPa ,37,38,39Much research has been conducted on the implementation of graphene for multifunctional coatings ,22,23,24Therefore, the objectives of this study aim to evaluate the effect of incorporating GNPs into epoxy adhesive as additions to develop high-performance coatings for oil and gas pipeline applications. High-speed disk and ultrasonication were introduced in the dispersion process. Both electrical and mechanical properties were evaluated with various concentrations of nanocarbon additives. The effects of incorporating nanofillers into polymer matrix were experimentally evaluated by the following tests: (i) electrochemical impedance spectroscopy (EIS) test, (ii) Taber abraser test, (iii) pull-off strength test, and (iv) water contact angle test. In addition, we also evaluated nanofiller reinforcement in the polymer matrix by using scanning electron microscopy (SEM) and surface roughness using atomic force microscopy.This section describes the experimental methods to characterize the effectiveness of incorporating nanomaterials into the coating where the epoxy is selected as a prime as a representative. The detailed information of the materials, dispersion methods, and the procedure of the experiments are described below.2/g with an average thickness of 8–12 nm. EPON™ Resin 828 resin and Epikure 3175  were used as the baseline prime matrix to mix with the nanoparticles. EPON™ Resin 828 is a bisphenol A/epichlorohydrin derived liquid epoxy resin and can obtain good mechanical, adhesive, dielectric, and chemical resistance properties when crosslinked with appropriate curing agents. EPIIKURE™ Curing Agent 3175  was used as the curing agent of EPON™ Resin 828. Q-panel steel panels  with surface roughness of 0.5~1.14 μm were used as substrates for the prepared coatings.Graphene nanoplatelets without any modification were selected from commercially-available products  where the average specific surface areas of the graphene are 500–700 mAs schematically illustrated in As such, the new developed nanofiller composite coatings were then coated on the standard Q-panel steel substrates with a dimension of 76 × 152 × 0.8 mm, and three samples per group were used for the characterization, as presented below. The panels were used without any modification and were cleaned with acetone before the coatings were applied. Single layer coatings were applied and cured at room temperature for 24 h. All the coated samples were allowed to completely dry for a few days before testing. Elcometer 415 thickness gauge were used to measure the thickness of dried coating films, and an average thickness of 110 ± 5 μm were obtained for the tested samples. For simplicity, the test samples were labeled in terms of graphene contents, unless stated otherwise. For instance, 0.5%G-Epoxy denotes the composite reinforced with 0.5 wt.% graphene, while 3.0%G-Epoxy is the composite with 3.0 wt.% graphene. The neat epoxy samples were used as the controlling reference.The EIS is often used to characterize the electrochemical behavior of a coating. The EIS tests of the coated samples were herein conducted using the Gamry Reference 600 potentiostat/Galvanostat/ZRA  with counter and reference electrodes that were immersed in electrolyte solution and were then evaluated based on the interfacial properties between the substrate and conductive electrolyte solution. Specifically, a 30-mm diameter O-ring glass tube with a silicon joint was clamped on the test panels. A saturated calomel electrode reference electrode was used for the reference electrode; while a platinum mesh was employed as the counter electrode and the test panel was worked as the working electrode. The glass tube was filled with 1.0% NaCl solution at all times during the test. The test was carried out by applying both DC potential and small superimposed AC excitation to a working electrode which was immersed in the conductive solution. After AC and the potential measurements were collected over a very wide range of frequency excitation, the collected data of current and potential of the electrochemical cell were converted into the impedance vs. frequency curve. The impedance-frequency plot analysis produced useful information about the coating and dielectric properties.Moreover, the ASTM standard B117-Salt Spray Test using Q-Fog CCT chamber  at NDSU were conducted as an accelerated durability test of the test samples for 200 h to evaluate their overall long-term performance.2. The abrasion resistance was then calculated through the mass loss in a specified number of abrasion cycles with the selected loads.Abrasion resistance of a coating is usually evaluated by either the Falling abrasive method (ASTM D 968) or the Taber abraser method (ASTM D 4060). In this study, the Taber abraser method was selected due to the fact that such a method provides a better representation of the wear resistance on a wide range of materials by selecting different abrading wheels and applied loads. Each specimen was loaded for a total of 1000 cycles under an applied load of 1000 g in accordance with ASTM D 4060 and the rotational speed of 72 rpm with two CS-10 abrading wheels. With such an applied load and the rotation, the abrading wheel generated a circular abrasion mark with an approximate area of 30 cmAdhesive bonds of the coating to its metal substrate were characterized by tensile button testing. Dollies were glued to each specimen, and the pull-off strength was measured during the experiment. Scotch weld 460  was used to glue the dollies, and the test area was abraded with sandpaper (100 grit or finer) to enhance the bonding strength between the dollies and the tested coatings. The test was performed 24 h after the dollies were applied. The test area was isolated with other parts of the coating by die cutting. An adhesion tester was used to apply tension load normal to the test surface. The adhesion strength was measured when the dolly was detached by the tension load.A contact angle test of the composite was conducted to understand the surface modification when adding nanoparticles, based on the ASTM D7334. Images were captured by the camera and were processed by a computer to calculate the angle between the water droplet and the contact surface.Besides that, the scanning electron microscopy (SEM) technique was used to observe the nanoparticles and their distribution in the epoxy matrix. The SEM was carried out by a field emission scanning electron microscope (FE-SEM), with a JSM-7600F Schottky  at 2 KV. Note that 2-D graphene alone is optically active and, thus, can be easily discriminated by microscopes. However, after being dispersed in a polymeric matrix, the discrimination of graphene sheets in the microstructure is a challenging job, as identified by other researchers ,41,42. AaR).Moreover, surface roughness analysis was performed with an atomic force microscopy, a Nanoscope IIIa system. By scanning the surface with a sample-probe, the height changes of the tested samples were recorded to generate three-dimensional images that were used to determine the surface roughness  had a semi-circuit curve in Nyquist, as presented in the second model shown in Long-term performance of the coatings was evaluated using accelerated durability salt spray tests. The test results after the samples had been exposed to salt spray for 200 h are illustrated in As clearly illustrated in The abrasion resistance of a coating is the ability against the removal of materials from its surface by rubbing, scraping, or erosion actions that the real-world coatings often experience and, thus, low abrasion resistance of a coating could lead to low operational durability . The resAs clearly illustrated in Similar to the previous observation in corrosion resistance, the group 3.0%G-Epoxy had no further increase in abrasion resistance see  when addAdhesion between the coating and substrate is another important factor to contribute to the barrier properties for the coating system . The adhAs illustrated in aR, of the nanocomposites by atomic force microscopy, as shown in aR value of the epoxy resin was 29 nm, while the graphene-based composites were only less than 5 nm. The results demonstrated that there was a significant decrease in the surface roughness by adding a small amount of nanofillers.(a)Graphene-based composite coatings exhibited enhanced mechanical and electrochemical properties that enabled the accommodation of the needs in wide structural applications, while maintaining high adhesion strength to the substrate.(b)Electrochemical behaviors of the test samples revealed that the nanocomposites with 0.1 to 1.0 wt.% of GNPs offered effective barrier properties for corrosion mitigation in both short- and long-term performances. Due to the agglomeration when increasing the high content of nanoparticles, the samples with 1.5 or 3.0 wt.% of GNPs exhibited some levels of reduction in corrosion resistance.(c)A similar trend was observed in the abrasion resistance. The results demonstrated no significant influence on the water contact angle and adhesion test by incorporating graphene in the epoxy resin. The graphene-based composite coatings with 0.5 to 1.5 wt.% of GNPs provided an over 20% increase in abrasion resistance in terms of low mass loss, as compared to the neat epoxy.(d)Results of the adhesion strength of the composites to the substrate showed that incorporation of the nanoparticles has minor effects on their interfacial bonding, showing a reduction within 10%. Moreover, the contact angle tests and surface roughness of the composite coatings both supported that the graphene particles smoothened the surface texture, as compared to the neat epoxy samples.(e)The composite coatings that were reinforced by graphene nanofillers display promising results in terms of enhanced mechanical and electrical properties, thereby offering the potential for widespread real-world applications, including oil and gas pipelines and bridges. Note that conclusions were mainly drawn from the findings in a short-term manner. Although the accelerated durability tests by 200 h exposure of salt spray confirmed the enhanced performance of the graphene-loaded composite coatings over conventional neat epoxy, it still necessitates the further investigation of the long-term durability tests in future studies.The incorporation of graphene nano-platelets into the polymer matrix were evaluated for their potentials as high-performance coatings. The factors affecting the composite coatings, including nanoparticle contents, and corresponding electrochemical, abrasion resistance, and adhesion properties, were investigated with specific conclusions, as shown below:


Immune checkpoint therapies (ICTs) targeting the programmed cell death-1 (PD1)/programmed cell death ligand-1 (PD-L1) pathway have improved outcomes for patients with non-small cell lung cancer (NSCLC), particularly those with high PD-L1 expression. However, the predictive value of manual PD-L1 scoring is imperfect and alternative measures are needed. We report an automated image analysis solution to determine the predictive and prognostic values of the product of PD-L1+ cell and CD8+ tumor infiltrating lymphocyte (TIL) densities (CD8xPD-L1 signature) in baseline tumor biopsies.Archival or fresh tumor biopsies were analyzed for PD-L1 and CD8 expression by immunohistochemistry. Samples were collected from 163 patients in Study 1108/NCT01693562, a Phase 1/2 trial to evaluate durvalumab across multiple tumor types, including NSCLC, and a separate cohort of 199 non-ICT- patients. Digital images were automatically scored for PD-L1+ and CD8+ cell densities using customized algorithms applied with Developer XD™ 2.7 software.p = 0.00002). The CD8xPD-L1 signature provided greater stratification of OS than high densities of CD8+ cells, high densities of PD-L1+ cells, or manually assessed tumor cell PD-L1 expression ≥25%. The CD8xPD-L1 signature did not stratify OS in non-ICT patients, although a high density of CD8+ cells was associated with higher median OS  in this group.For patients who received durvalumab, median overall survival (OS) was 21.0 months for CD8xPD-L1 signature-positive patients and 7.8 months for signature-negative patients .Actual study start date: August 29, 2012.Primary completion date: June 23, 2017 .The online version of this article (10.1186/s40425-019-0589-x) contains supplementary material, which is available to authorized users. The immune response to cancer is complex and involves a number of cellular proteins that may promote or suppress immune function. Interaction between programmed cell death ligand-1 (PD-L1) and its receptor programmed cell death-1 (PD1), which is expressed primarily on T lymphocytes, exemplifies a major immunosuppressive pathway . PD1 sigPD-L1 expression assessed by immunohistochemistry (IHC) has been at the forefront of predictive biomarkers for ICT. Higher PD-L1 expression on tumor cells (TCs) and/or immune cells has been associated with greater efficacy of anti-PD1/PD-L1 immunotherapies , 6–13. MGiven the limitations of PD-L1 IHC, alternative diagnostic strategies have been studied to find better predictors of response to PD1/PD-L1 blockade. Key among these is tumor mutational burden (TMB), which has recently been shown to have promising predictive potential for anti-PD1/PD-L1 monotherapy and combination therapy with anti-cytotoxic T-lymphocyte-associated protein 4 agents in multiple NSCLC clinical settings –22. LikeRelevant to assessment of the predictive potential of a cancer therapy screening test is the need to assess its prognostic value, especially with respect to standard of care chemotherapy. A recent literature review, and other studies, have found conflicting results of the prognostic value of PD-L1 expression in NSCLC patients generally or those receiving chemotherapy , 29–31. 2 of tumor) multiplied by PD-L1+ cell density using automated image analysis (CD8xPD-L1 signature). We chose a digital approach to quantify CD8+ and PD-L1+ cell densities, as properly validated image analysis solutions have multiple advantages over manual assessment. These include the production of continuous quantitative data, improved reproducibility and avoidance of inter-observer variability, which is an inherent disadvantage of manual assessments of both PD-L1 and CD8 expression [To better identify patients likely to derive clinical benefit from anti-PD-L1 immunotherapy, we explored a biomarker signature consisting of tumoral CD8+ cell density , disease control rate, duration of response, and progression-free survival (PFS) using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 guidelines , as wellPatients in Study 1108 were initially enrolled regardless of tumor PD-L1 expression. Tumor samples from these patients were used to develop an IHC assay to determine PD-L1 expression  . After an = 84) and a test set (n = 79), which were balanced by PD-L1 status (PD-L1 ≥ 25%), ORR, previous lines of therapy, stage, and gender. To understand whether the CD8xPD-L1 signature differed in patients with NSCLC who had not been treated with a checkpoint inhibitor, an additional 199 surgically resected baseline specimens were analyzed from an independent cohort of non-ICT-treated patients who underwent surgery between 2001 and 2005. Patients with advanced-stage NSCLC received cisplatin + gemcitabine (approximately 50% of cases), cisplatin + vinorelbine, cisplatin + paclitaxel, or platinum salt/other drugs. For stage I disease, all patients underwent surgery and rarely received adjuvant chemotherapy. For stage II–IIIA disease, patients underwent surgery followed by adjuvant chemotherapy. For stage IIIB disease, patients received neo-adjuvant chemotherapy or if they did not respond to chemotherapy, they underwent radiotherapy, followed by surgery, followed by adjuvant chemotherapy. Baseline patient characteristics from both sample cohorts are shown in Additional file Baseline tumor specimens with consecutive slides of CD8 and PD-L1 stains were available for 163 patients from Study 1108. These were split between a training set  and for CD8 , both performed on the Ventana BenchMark ULTRA staining platform  , 39. For2). All digital images were manually reviewed to ensure the quality of immunostaining, digital scanning and the precise detection of positive cells by image analysis. In addition, the quality of signal detection for both PD-L1 and CD8 in single- and dual-stain assays was validated as comparable, as previously reported [2 for CD8+ cells and 644 cells/mm2 for PD-L1+ cells (tumor cells + macrophages). The CD8xPD-L1 signature was then defined as the product of CD8+ and PD-L1+ cell densities in the annotated tumor region and a cutoff of 1.54 × 105 cells2/mm4 for signature positivity was applied. The CD8xPD-L1 signature, its two individual components, and PD-L1 expression (TC ≥25%) assessed manually were applied to the training, test and combined set of durvalumab-treated patients from Study 1108, as well as to the independent set of non-IO treated patients.Rule-based methods combined with machine learning were used to segment and classify cells and nuclei of acquired images  using Dereported . For PD-reported . The cutp-values for OS and PFS (≤ 0.05) and on the prevalence of signature-positive patients between 30 and 70%  by the number of all signature-positive patients. This is equivalent to the ORR within the signature-positive subgroup. With the exception of PD-L1 TC ≥25%, the cutoffs for all signatures were defined by optimizing for PPV on the training set, while applying constraints on log-rank test-derived p-value. The p-values for the covariates in the Cox model and the ANOVA were considered significant if ≤0.05. Statistical calculations were performed using R version 3.4.2 with the Survival package 2.41–3 [Multivariate Cox regression analysis –43 was pe 2.41–3  and the e 2.41–3 .p = 0.024) (Table p = 0.012). Likewise, PD-L1 TC ≥25% . PD-L1+ cell density did not provide a significant stratification for OS (p = 0.071).At baseline, 31.0% of samples from patients enrolled in Study 1108 in the training set were CD8xPD-L1 signature-positive, 38.1% had high densities of CD8+ cells, 31.0% had high densities of PD-L1+ cells, and 58.3% had PD-L1 TC ≥25%. The CD8xPD-L1 signature provided the highest PPV (0.42), versus a high density of CD8+ cells (0.34), a high density of PD-L1+ cells (0.39), and PD-L1 TC ≥25% (0.29). For ORR, the CD8xPD-L1 signature was the only tested measure for which the PPVs for positive and negative patients had non-overlapping 95% confidence intervals (CIs): 0.42  for signature-positive patients and 0.09  for signature-negative patients. Additionally, CD8xPD-L1 signature-positive patients had significantly longer median OS  than signature-negative patients . Further, median OS was significantly longer for patients with high CD8+ cell density  than for those with low density  and significantly longer in patients with high PD-L1+ cell density  than in those with low density . Additionally, median OS was numerically longer in patients with PD-L1 TC ≥25%  than in those with PD-L1 TC < 25%  (Table p = 0.000945) and CD8+ cell density   PD-L1 expression in the automated image analysis scores versus the exclusion of immune cell PD-L1 expression in the manual scores. However, any advantage provided by automated image analysis compared to manual PD-L1 scoring in terms of overall predictive value was not clear.The multiparametric Cox analysis of OS showed that the CD8xPD-L1 signature in the test set had better predictive value than its single components, manual PD-L1 status and also the presence of liver metastasis. Additionally, it was the only statistically significant measure in terms of the overall Cox model Table .Table 2Mp = 0.00002) . Likewise, median OS was significantly longer in patients with high PD-L1+ cell density than in those with low density  and was significantly longer in patients with PD-L1 TC ≥25% than in those with PD-L1 < 25%   Fig. a. Patienp ≤ 0.005). However, in the subgroup of patients with liver metastases, CD8xPD-L1 signature-positive patients had significantly longer median OS than CD8xPD-L1 signature-negative patients . In contrast, there was no statistically significant difference in OS between patients with PD-L1 TC ≥25% and those with PD-L1 TC < 25% in the subgroup with liver metastases . In the subgroup of patients without liver metastases, the CD8xPD-L1 signature demonstrated greater stratification for OS  than PD-L1 TC expression . A nested model approach was performed to further test if the CD8xPD-L1 signature would provide added predictive value to a model composed of its individual components. The model consisted of a reduced set of cofactors compared to the Cox models; liver metastasis, CD8+ cell density, and PD-L1+ cell density were compared to an identical model that contained CD8xPD-L1 as an additional cofactor. Other cofactors did not show significant contribution in the Cox models; they were therefore not considered for this comparison. CD8xPD-L1 significantly contributed to the model (p = 0.025).The multiparametric Cox analysis showed that the CD8xPD-L1 signature was significantly and independently associated with OS for patients treated with durvalumab and had improved value in predicting OS compared with its single components, manual PD-L1 status and the presence of liver metastasis  .The CD8xPD-L1 signature was found not to be prognostic in the non-ICT setting. The median OS from the time of surgery for signature-positive patients was 52 months 95% CI, 37–89) versus 56 months 95% CI, 42–69) for signature-negative patients  Fig. . The PD-To date, the search for predictors of patient response to PD1/PD-L1 blockade has focused mainly on tumoral PD-L1 expression assessed manually via IHC, TMB, interferon-γ gene-related signatures, and CD8 analysis. We identified an automated image analysis signature comprised of PD-L1+ and CD8+ cell densities in tumor biopsies that predicts response to durvalumab monotherapy in patients with NSCLC. Multiple characteristics of this CD8xPD-L1 signature showed that it predicts response better than manual PD-L1 IHC scoring (TC ≥25%), which is the current benchmark for profiling patients most likely to respond to anti-PD1 and anti-PD-L1 immunotherapies. We also evaluated the individual components in comparison to manual PD-L1 scoring and further investigated the prognostic value of these measures in patients with NSCLC treated with non-ICT, which further supported the predictive value of the CD8xPD-L1 signature. This signature offers a number of advantages compared with current means of predicting response to anti-PD1 and anti-PD-L1 immunotherapies.We tested the predictive benefit of CD8xPD-L1 signature in tumor samples of NSCLC patients enrolled in a Phase 1/2 study of durvalumab (Study 1108). Following optimization in a training set of samples, in the test set we found that the CD8xPD-L1 signature provided significant stratification for ORR, with non-overlapping 95% CIs between the PPVs for signature-positive and signature-negative patients. Additionally, it was the only statistically significant measure in the overall Cox model in the test set. In the combined set of samples from patients treated with durvalumab, the CD8xPD-L1 signature also demonstrated greater stratification for OS compared to PD-L1+ cell density, CD8+ cell density and manual PD-L1 TC ≥25%. These results were irrespective of liver metastasis status, although patients with liver metastases had significantly shorter median OS than those without. However, even in patients with liver metastasis, CD8xPD-L1 better identified patients with longer survival, as it provided improved stratification in terms of median OS compared with PD-L1 TC ≥25%.After demonstrating the improvement of CD8xPD-L1 over PD-L1+ and CD8+ cell densities individually and manual PD-L1 TC ≥25% in predicting OS in durvalumab-treated patients, we tested the signature in a Cox model. A nested approach showed that the inclusion of CD8xPD-L1 as a covariate in addition to its single components and liver metastasis status resulted in a model statistically different from the same model lacking CD8xPD-L1, further reinforcing the predictive value of the signature.Separately, we applied the CD8xPD-L1 signature to a set of surgically resected NSCLC tumor specimens from patients who received chemotherapy but not ICT to assess its prognostic effect. The signature did not stratify for OS, providing additional support for its utility as a predictive factor for durvalumab. However, we did find that CD8+ cell density alone provided statistically significant stratification of OS in patients not receiving ICT. These findings are consistent with other lines of evidence pointing to the prognostic value of tumoral CD8 status , 35, 46.While the predictive value of the CD8xPD-L1 signature appears promising in this regard, a number of signature-positive patients did not respond to durvalumab. However, this limitation is not unlike the imperfect predictive value attributed to other assays of ICT, namely TMB and interferon-γ gene-related signatures –22, as wAutomated image analysis applied to IHC biomarkers provides potential advantages over manual scoring in the clinical setting. This is especially important in the case of manual IHC assessment of PD-L1 expression, where inter-observer variability in scoring has been reported in multiple studies , 48, 49.Furthermore, automated image analysis becomes of even greater value in the setting of combined IHC markers, where assessment of individual markers across the entire tumor region needs to be coordinated. As we demonstrated here and previously , this isWe successfully developed an automated digital signature based on the product of the densities of CD8+ cells and PD-L1+ cells measured by automated image analysis applied to consecutive IHC-stained lung cancer tissue sections. This signature resulted in significantly greater stratification of survival for patients with NSCLC treated with durvalumab than CD8+ TIL density, PD-L1+ cell density or manually derived PD-L1 expression alone. This demonstrates the predictive value of accounting for both tumor factors (PD-L1) and immune contexture in profiling cancer patients for response to ICT. Liver metastasis was the only other covariate considered to significantly contribute to the model. This study also showed that computational analysis of routine tumor specimens can be practically applied to large sets of clinical trial and non-clinical sample biopsies in a manner with multiple advantages over manually derived means. This approach, therefore, may offer a foundation for the development of companion diagnostic tests of even greater complexity to select patients most likely to respond to ICT targeting the PD1/PD-L1 axis with greater precision than current methods. Nonetheless, the predictive value of the CD8xPD-L1 signature requires verification in additional studies.Additional file 1:Figure S1 Digital image analysis segments CD8+ and PD-L1+ cells in dual-labelled sections of NSCLC. Figure S2. Channel unmixing used to segment CD8+ and PD-L1+ cells in CD8/PD-L1 dual chromogenic immunohistochemistry assay. Figure S3. Optimization of cutoff values for CD8xPD-L1 signature, CD8+ cell density, and PD-L1+ cell density was performed on the training set of samples of durvalumab-treated patients. Figure S4. The predictive value of the CD8xPD-L1 signature compared to its individual components in training sample set. Figure S5. Predictive values of the individual components of the CD8xPD-L1 signature in the combined sample set. Figure S6. Analysis of OS for the CD8xPD-L1 signature and liver metastasis. (DOCX 990 kb)Additional file 2:Table S1. Patient demographics and baseline characteristics for analysed samples. Table S2. Performance of the CD8xPD-L1 signature, its individual components, and PD-L1 TC expression in the combined set of durvalumab-treated and non-ICT-treated patients. Table S3. Multiparametric Cox analysis of signatures in the entire data set including additional PD-L1 readouts. (DOCX 28 kb)