Genomes of higher eukaryotes have surprisingly long first introns and in some cases, the first introns have been shown to have higher conservation relative to other introns. However, the functional relevance of conserved regions in the first introns is poorly understood. Leveraging the recent ENCODE data, here we assess potential regulatory roles of conserved regions in the first intron of human genes.We first show that relative to other downstream introns, the first introns are enriched for blocks of highly conserved sequences. We also found that the first introns are enriched for several chromatin marks indicative of active regulatory regions and this enrichment of regulatory marks is correlated with enrichment of conserved blocks in the first intron; the enrichments of conservation and regulatory marks in first intron are not entirely explained by a general, albeit variable, bias for certain marks toward the 5’ end of introns. Interestingly, conservation as well as proportions of active regulatory chromatin marks in the first intron of a gene correlates positively with the numbers of exons in the gene but the correlation is significantly weakened in second introns and negligible beyond the second intron. The first intron conservation is also positively correlated with the gene’s expression level in several human tissues. Finally, a gene-wise analysis shows significant enrichments of active chromatin marks in conserved regions of first introns, relative to the conserved regions in other introns of the same gene.Taken together, our analyses strongly suggest that first introns are enriched for active transcriptional regulatory signals under purifying selection.The online version of this article (doi:10.1186/1471-2164-15-526) contains supplementary material, which is available to authorized users. Drosophila, and C. elegans[C. elegans  and Drosophila  to humans , while the fraction of non-coding DNA including introns greatly varies up to several folds [Recent complete sequencing projects have confirmed that almost all eukaryotes have introns –6. Diffe elegans, 7, 8. T elegans, as well elegans–14. Notial folds , 16, somal folds , 18.D. melanogaster and D. simulans, Haddrill et al. [The mere existence of introns in the eukaryotic genome is intriguing, given the cost of transcription and the cost of maintaining a splicing regulatory system that ultimately eliminates the introns from the functional product of the gene. In particular, whether introns have evolved under selective constraints, and the extent thereof, are not entirely clear; while some studies suggest that introns evolve largely free from selective constraints –22, othel et al.  demonstrl et al.  showed tl et al. , 28, 29,l et al. . Besidesl et al. –33. ThusDrosophila[Tetrahymena, the most conserved introns are found closer to the 5’ end of genes. Furthermore, introns harboring regulatory elements tend to be the first introns [The 5’-most “first” intron differs from the other introns in several aspects in terms of processing, epigenomic marks, length and evolution. First, in terms of processing, despite an overall 5’-to-3’ trend in splicing during transcription, the first intron is removed (on average) somewhat later . Second,osophila, 37. Zheosophila have als introns –40, and, introns . OverallPrevious investigations of potential regulatory role of first intron were mainly performed in Drosophila or plants. The extent to which the previous conclusions hold true in mammals is not known. Moreover, the recent explosion of human epigenomic data via the ENCODE project Table  providesAs a proxy for purifying selection, we compared evolutionary conservation across introns grouped by their position in the transcript structure. Conservation in an intron was estimated as the fraction of intron sites that were conserved based on a PhastCons score threshold. Three different multiple alignments  and different PhastCons score thresholds were used. Our conclusions hold for all choices of alignment and threshold; here we only present the results based on mammalian conservation with PhastCons score threshold of 0.5. The primary focus of our investigation was transcriptional regulatory elements in introns. Therefore, to exclude the possibility of splice site signals biasing the conservation score, especially for short introns, the sequences within 300 bps of the splice junction, which are considered to harbor splicing regulation signals , were exWe found that the median conservation in first introns (3.3%) was significantly higher than all other groups using Wilcoxon rank sum test p < 2.2e-16)  in which a gene is expressed. Genes that expressed in a very few or most tissues have low complexity whereas the genes expressed in an intermediate number of tissues have the highest expression complexity , 62. It Next we assessed the correlation between the regulatory signals and the number of exons. As shown in Figure We next investigated whether or not conservation in first introns is related to the level of gene expressions. For this, the degrees of conservation were plotted against the levels of gene expressions in several human tissues. Gene expression data sets were obtained from RNA-seq atlas constructed by  from 11 Although the analysis thus far was based on all ‘transcripts’, we repeated the analyses by selecting a single representative transcript per gene, resulting in ~16,000 transcripts. As shown in Additional file Our analysis thus far is based on grouped data. Next we assessed for each gene whether the regulatory signals in the first intron preferentially occur within conserved region. We partitioned each first intron into conserved and non-conserved regions, obtained percent coverage by regulatory signals in each part and calculated the log-odds ratio with 95% confidence intervals (see Methods)  each intron was binned into five equal-sized bins, and we estimated for each bin, the fraction of introns in which the highest signal in the intron was in the particular bin. As shown in Additional file Despite an enrichment of signals toward 5’ as well as the fact that the general patterns of enrichment in the first introns also hold for the 5’ flanking regions . Thus we investigated the trends in the first introns compared to that in the second introns when controlled for their distance from the TSS. We categorized first and second introns into bins corresponding to distance of the 5’ end of the intron from the TSS and compared the first and the second intron trends within each bin. Note that because first introns are much longer than the second intron  the presence of active regulatory chromatin marks, 2) higher expression levels of genes, and 3) a greater number of exons within genes. Overall, our results strongly suggest that first introns in human are enriched for evolutionarily selected active transcriptional regulatory signals that are likely to be important for regulating complex gene expression patterns of large multi-domain genes.The precise mechanism by which individual conserved, putative regulatory regions in the first intron, regulate gene expression, as well as other potential functions of conserved regions in the first intron, are unclear. The extreme lengths of mammalian first introns represent another enigma. The evolutionary path leading up to long introns as well as whether the first intron length is under selective constraint are not known. Finally, whether it is beneficial for the regulatory elements to reside within the first intron, as opposed to, say, the upstream region of the gene, or whether evolution is neutral to this outside-inside choice, is another open and interesting question. To ultimately resolve the mystery of introns, these are some of the questions that will need to be addressed.The exon-intron position information of 36,024 Refseq mRNA (KnownGenes) without duplicates was downloaded from the UCSC Table Browser (January 2013). Genomic sequences for each chromosome were obtained from the primary GRCh37/hg19 assembly, and were used for retrieve intron sequences. Very short introns (less the 1 kb in length) were excluded, as well as very long introns (greater than (third quartile + (interquartile range × 1.5)) in length to minimize the outlier effects. To minimize interference from splicing signals in interpreting intronic conservation, we excluded 300 bps from both the 5’ and 3’ ends of each intron . Then rePhastCons scores were used to estimate position-wise sequence conservation . The PhaGenome positions of peaks  for DHSs, TFBSs and chromatin marks measured in the ENCODE Tier-1 cell lines  were downloaded from the ENCODE Project from UCSC genome browser (March 2013). The specific download links are provided in Table 2 (RPKM + 1) to report expression levels of mRNAs for 11 different human tissues  among the variables representing evolutionary conservations of introns or flanking regions, regulatory signals mapped onto introns or flanking regions, and mRNA expression levels. Introns or flanking regions with no conservation or regulatory signal were excluded; we ascertained that this only makes our results more stringent because the fraction of first introns that were eliminated were smaller than those for other introns and including this with “0” value would make the results even stronger.To test whether the regulatory signals and chromatin marks are enriched in conserved regions compared to non-conserved regions in each gene, odds ratio of the signal estimates between the two regions was estimated. Briefly, first introns were divided into two regions, conserved first introns and non-conserved first introns by the criteria of Phastcons ≥ 0.5, and the chromatin signals and regulatory signals described in the main text were overlaid into each region, generating a 2×2 contingency table. Odds ratios were then estimated for each gene from the contingency table. The odds ratio is the ratio of the proportion of regulatory signals in conserved sequences to that in non-conserved sequences. The odds ratios were thus computed using the “Text::NSP::Neasures::2D::odds” Perl module, and the 95% confidence intervals associated with odds ratios were calculated using the formulas, d sites) .Figure S2. Proportions of regulatory chromatin marks in intron ordinal groups in H1-hESC and K562. Figure S3. Correlation between regulatory signals and conservation in first introns in H1-hESC and K562. Figure S4. Correlation between regulatory signals and conservation in the upstream flanking regions in three different cell lines. Figure S5. Relationship between flanking region conservation and the numbers of exons. Figure S6. Relationship between the proportions of regulatory signals in introns of each ordinal position and the numbers of exons. Figure S7. Analysis based on a single representative transcript for each gene. Figure S8. Enrichment of regulatory marks in the first intron in two additional cell lines. Figure S9. Five prime to three prime biases in signal density along the first intron. Figure S10. Excluding spillover of signals s from the promoter. Figure S11. Excluding genes whose first introns overlapped with exons or flanks of another genes. Figure S12. Analyzing the effect of proximity to the TSS. (PPTX 936 KB)Additional file 1: Figure S1: Comparison of conservations in first introns with those in the other introns using an alternative grouping strategy.


IDO1 and CXCL9) was associated with MPDL3280A response in melanoma but not NSCLC or RCC. On treatment, responding tumors showed increased infiltration of Th1-dominant immune infiltrate and evidence of adaptive PD-L1 up-regulation. In contrast, progressing tumors displayed the following patterns of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression: (1) few/no TILs and absent PD-L1 expression (immunologic ignorance), (2) TILs present with minimal/no PD-L1 expression , or (3) TILs residing solely around the tumor cell mass outer edge (excluded infiltrate), suggesting that resistance to MPDL3280A may be associated with impaired T cell trafficking and/or function.PD-L1 expressed in the tumor microenvironment regulates Th1 immune responses and mediates cancer immune evasion through interactions with PD-1 or B7.1 receptors on activated T cells. MPDL3280A, an engineered human monoclonal antibody, targets PD-L1 and inhibits its function. To identify immunologic predictive and pharmacodynamic biomarkers of MPDL3280A treatment, we performed a comprehensive analysis of tumors and blood samples collected at baseline and/or on treatment from ≈280 patients with locally advanced or metastatic solid tumors, including NSCLC, RCC, melanoma and bladder cancer. Regardless of tumor type, clinical responses were characterized by PD-L1 expression, the presence of markers of T cell activation (Th1 gene signature and CTLA4), and the absence of fractalkine at baseline in the tumor microenvironment. Elevated baseline expression of IFN-γ and IFN-γ-inducible genes  increased in all patients following MPDL3280A treatment, representing a pharmacodynamic measurement of PD-L1 inhibition. In addition, analysis of PBMC showed an increase in T cell activation, as measured by IFN-γ and granzymes A and B gene expression in responders following MPDL3280A treatment, consistent with the observations in responding tumors. Baseline soluble PD-L1 was not associated with response. Some indication-specific biomarkers, such as plasma VEGF, decreased in responders with RCC but not with other indications. In NSCLC, a decrease in tumor burden markers, CA-125 and CEA, was associated with response. Similarly, IL-6 and IL-8 were differentially expressed on treatment in responders vs non-responders. Additionally, responders exhibited a decrease in circulating tumor DNA (ctDNA) in plasma, suggesting that ctDNA may be used to monitor MPDL3280A clinical activity in NSCLC.In conclusion, these data provide general and indication-specific mechanistic insights into immune checkpoint inhibition, potential mechanisms of response and resistance, as well as identification of potential predictive and pharmacodynamic biomarkers of anti-PD-L1/PD-1 clinical activity across multiple tumor types.


Motor neuron disease (MND) is a neurodegenerative disease of mid-life, with average survival of 3-5 years after diagnosis. The only approved treatment, riluzole, is only moderately effective and so there is a great need for novel therapeutics. TAR DNA binding protein 43 (TDP-43) has held a particular centrality in MND research since its discovery as the principal protein component of the characteristic protein aggregates found in the disease. Subsequent work has investigated its role as an RNA-binding protein and regulator of approximately 3000 RNA transcripts. In the majority of disease cases, both familial and sporadic, normally nuclear TDP-43 is sequestered in the cytoplasm in protein aggregates. Using differentiated motor neuron-like cells (NSC-34) and primary motor neurons, we have treated cells with the disease-relevant ER stressor tunicamycin. Overnight treatment with a low concentration of tunicamycin resulted in the formation of aggregates immunoreactive for endogenous TDP-43, as visualised by immunocytochemistry and Western blotting of the RIPA-insoluble fraction. We have found that these aggregates do not co-stain for TIAR, a well-known marker of stress granules. However, using the oxidative stressor sodium arsenite, we are able to induce formation of stress granules, though they do not co-stain for TDP-43. The tunicamycin treatment also led to a moderate decrease in cell viability, as shown by MTT and Trypan Blue Exclusion assays. In this study, we have recapitulated a known pathological process (ER stress) of MND in vitro, which resulted in the formation of TDP-43 aggregates. The signalling pathways driving TDP-43 aggregation are unknown, hence work detailing these mechanisms is likely to present wide scope for therapeutic intervention.


Biomarkers in easily accessible compartments like peripheral blood that can predict disease progression in idiopathic pulmonary fibrosis (IPF) would be clinically useful regarding clinical trial participation or treatment decisions for patients. In this study, we used unbiased proteomics to identify relevant disease progression biomarkers in IPF.Plasma from IPF patients was measured using an 1129 analyte slow off-rate modified aptamer (SOMAmer) array, and patient outcomes were followed over the next 80 weeks. Receiver operating characteristic (ROC) curves evaluated sensitivity and specificity for levels of each biomarker and estimated area under the curve (AUC) when prognostic biomarker thresholds were used to predict disease progression. Both logistic and Cox regression models advised biomarker selection for a composite disease progression index; index biomarkers were weighted via expected progression-free days lost during follow-up with a biomarker on the unfavorable side of the threshold.A six-analyte index, scaled 0 to 11, composed of markers of immune function, proteolysis and angiogenesis  predicted better progression-free survival in IPF with a ROC AUC of 0.91. An index score ≥ 3 (group ≥ 2) was strongly associated with IPF progression after adjustment for age, gender, smoking status, immunomodulation, forced vital capacity % predicted and diffusing capacity for carbon monoxide % predicted .This index, derived from the largest proteomic analysis of IPF plasma samples to date, could be useful for clinical decision making in IPF, and the identified analytes suggest biological processes that may promote disease progression. CO) or 10% in forced vital capacity (FVC)]  , 7 over y (FVC)] , 8, 9.Many groups have reported on biomarker analysis in IPF using approaches such as genomic or transcriptomic profiling, microbiome analyses and candidate biomarker analyses in blood, bronchoalveolar lavage fluid and lung tissue specimens –14. BlooSimilarly, studies have explored the contribution of angiogenesis to fibrogenesis with mixed results . Some huIn terms of protease imbalances in IPF, several studies have identified alterations and these have been the subjects of several review articles. Alterations in metalloproteases are the most commonly noted , 35, butCOMET (Correlating Outcome Measures to Estimate Time to progression in IPF) ; howeveneumonia , 53, sugpatients . Lower ll models , suggestCath-S is a single chain, non-glycosylated cysteine protease ubiquitously distributed in the lysosome. It is expressed mainly in lymphatic tissues and is characterized as a key enzyme in class II-mediated antigen presentation . LGMN is-/- murine models demonstrate elevated expression of type 1 collagen and alpha-smooth muscle actin, with abnormal accumulation of autophagosomes in macrophages and impaired clearance of damaged mitochondria [Inhibition of Cath S can promote autophagy and apoptosis . Thus, lchondria . Intereschondria . Thus, iLGMN was identified as diagnostic and prognostic liver fibrosis biomarker . LGMN-deProtease imbalances have been noted in IPF , 35. FouVEGF is a potent and specific endothelial cell mitogen that regulates blood and lymphatic vessel development and homeostasis. The VEGF receptor family consists of three members, VEGFR1 (FLT1), VEGFR2 (KDR/FLK1) and VEGFR3 (FLT4) . Among t® validation on another cohort, we chose to address sensitivity and specificity of the index using bootstrap analysis. The index performs well across all 100 different bootstrap analyses performed  in a very small volume. Comparable proteomic assays would include mass spectrometry and ELISA platforms. Mass spectrometry is somewhat cumbersome and requires improved reproducibility and cost before it reaches mainstream clinical applicability. ELISA platforms are highly sensitive and useful for identifying single to multiple targets. However, it is now clear that these assays cannot be multiplexed above approximately 70 analytes without multiple measurements secondary to cross reactivity , 70.This study is only capable of describing associations between the selected analytes and disease progression. As discussed above, we feel these analytes may be indicative of pathobiologic processes that could be aberrant in IPF, but it is also possible that these analytes are changed for reasons unrelated to disease. Unraveling this question will require future mechanistic work in animal models and using IPF tissues and cells.Together, our results demonstrate a 6-analyte SOMAmer panel measuring circulating markers of immune function; proteolysis and angiogenesis can be used to create a simple index with excellent sensitivity and specificity for predicting progression-free survival in IPF. Use of this index on easily accessible plasma should be further validated to offer a simple way of predicting IPF patient outcomes. In addition, this analysis serves to highlight aberrant biological pathways associated with IPF disease progression, providing rationale for increased study of these biological pathways in lung fibrosis.S1 FileTable A) Immunodulation Therapy in the COMET Cohort. Fig A. Kaplan-Meier curves showing progression free survival for IPF patients with baseline biomarker levels above or below the identified thresholds (A) Carbonic Anhydrase XIII, (B) Granulin(GRN) and (C) Nascent polypeptide-associated complex subunit alpha (NACA). RMST indicates the comparison of restricted mean survival time (i.e. area under the K-M curves) between the two groups. Fig B. ICOS is shed by activated T cells. A million CD4 positive splenocytes were stimulated with CD3 + CD28 then  or TH1  for 24 hr. Cell free supernatants were collected and concentrated using Amicon Ultra Centrifugal filters . Equal amounts of protein from each sample were separated on a 4–20% gradient SDS-polyacrylamide gel and transferred to a PVDF membrane . PVDF membrane was probed with rabbit monoclonal ICOS (Abcam). Fig C. Periostin levels correlate on SOMAscan and ELISA. The same plasma samples were run on a periostin ELISA developed by Abbot Pharmaceuticals and were compared to measures of periostin made by SOMAmer. The correlation was significant, Spearman r = 0.44; p<0.0001.This file contains the following: (DOCX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


We propose a novel mechanism to construct a tunable and ultracompact refractive index sensor by using the Fano resonance in metal-graphene hybrid nanostructure. Plasmon modes in graphene nanoribbons and waveguide resonance modes in the slits of metal strip array coexist in this system. Strong interference between the two different modes occurs when they are spectrally overlapped, resulting in a Fano-type asymmetrically spectral lineshape which can be used for detecting the variations of ambient refractive index. The proposed sensor has a relatively high figure of merit (FOM) over 20 and its sensing performance shows a good tolerance to roughness. In addition to the wide range measurement enabled by the electrical tuning of graphene plasmon modes, such ultracompact system also provides an angle-independent operation and therefore, it can efficiently work for the detection of gas, liquid, or solids. Such optical nanostructure may also be applied to diverse fields such as temperature/pressure metering, medical detection, and mechanical precision measurement. Exhibiting an asymmetric profile of spectral lineshape, Fano resonance (FR) was first experimentally observed by BeutlerFano-type transmission/reflection based on localized surface plasmon resonance (LSPR)45611In this paper, we propose an ultracompact FR-based metal-graphene hybrid nanostructure for detecting the refractive index variations of surrounding dielectrics. The sensor operates at mid-infrared wavelengths and its performance is electrically tunable and angle-insensitive. A schematic representation of the metal-graphene hybrid nanostructure is depicted in WM), period (p), heights of the upper (h1) and and bottom (h2) silver strips are set as 358 nm, 410 nm, 180 nm, and 40 nm, respectively. To achieve optimal transmission performance, other structural parameters are d1 = 55 nm, d2 = 35 nm, t = 20 nmWe first consider the metallic nanostructure without introducing the graphene ribbons. As mentioned before, TM-polarized normal incidence can excite two different resonant modes in metal strip array, the coupled SP mode at strip surface and the WR mode in the slits of strips, at rather different frequenciesεg = 2.5 + iσg/ε0ωΔ, where ε0 is the permittivity in vacuum, ω is the circular frequency of incident light, and Δ is the thickness of graphene (Δ = 0.5 nm). Under the conditions of low temperature and high doping (i.e. Ef represents the Fermi energy of graphene), the graphene conductivity is dominated by intraband transition and thereby can be simplified as e is the electron charge, τ is the relaxation time. In our considered wavelength range, the relatively low photon energies (compared with Ef) means that the graphene conductivity obtained from the simplified semiclassical model should agree well with that calculated from random phase approximation (RPA) which can give the accurate expression of the graphene dielectric constantIt has been demonstratedWG) of the graphene ribbons is set as 42 nm, which is achievable via current fabrication technologyWhen graphene ribbons are placed inside the slits of the metal strip array, GSPs may be excited by the WR and then interact with the WR. In particular, the spectral overlap of these two plasmon resonances may result in a Fano-type lineshape. To realize such a spectral overlap, the width  of GSPs, another component of wave vector is produced in the graphene nanoribbons by the oscillation modes of free carriers with restricted motions: kx ≈ mπ/WG, where m represents the number of nodes.To reveal the physical mechanism for the excitation of graphene surface plasmons (GSPs), we study the dispersion relation of the GSPs. Assume that the graphene ribbons have a finite width of The thickness of the graphene ribbons is too small and are neglected in the following derivation for the dispersion relationship of GSP in a graphene ribbon. According to the Maxwell’s Equationsρext and the current densities the macroscopic quantities ω is the circular frequency of incident light, μ0 and ε represent permeability and permittivity of the dielectrics. As a characteristic of SPP, the electromagnetic field has a form of β represents the propagating constant on graphene and ky indicates the exponential amplitude decay along y direction. xE component is neccessary for the exctiation of GSPP, and we assume a TM-polarized incidence to the considered system along y direction. where Ex, Ey, Ez, Hx and Hz are included in Ex, Ey and Hx. This is the result that the charge distribution on graphene, which is regarded as graphene surface charge, changes the characteristic of the electromagnetic field near the graphene layer. For example, the non-homogeneous electron distribution on graphene along z-axis produces the Ez component of the electromagnetic field. However, Hy remains zero because it is independent of the surface charge. Then combining It should be noted that Ez can be written asSince Combining y < 0 andfor the half space with y > 0. ks ≡ ky,s is the wave vector component perpendicular to the interface of the two dielectric media. According to boundary relations, when crossing an interface, the tangential component of electric field is continuous while the transverse component of magnetic field is affected by the surface current as the following equation:for the half space with Note thatg). By applying the boundary relations to and the surface conductivity σ is provided by the graphene sheet  and kx = mπ/WG.where Such a complicated structure is experimentally achievable. How to cite this article: Pan, M. et al. Tunable angle-independent refractive index sensor based on Fano resonance in integrated metal and graphene nanoribbons. Sci. Rep.6, 29984; doi: 10.1038/srep29984 (2016).


Understanding the underlying mechanisms involved in graphene growth via chemical vapour deposition (CVD) is critical for precise control of the characteristics of graphene. Despite much effort, the actual processes behind graphene synthesis still remain to be elucidated in a large number of aspects. Herein, we report the evolution of graphene properties during in-plane growth of graphene from reduced graphene oxide (RGO) on copper (Cu) via methane CVD. While graphene is laterally grown from RGO flakes on Cu foils up to a few hundred nanometres during CVD process, it shows appreciable improvement in structural quality. The monotonous enhancement of the structural quality of the graphene with increasing length of the graphene growth from RGO suggests that seeded CVD growth of graphene from RGO on Cu surface is accompanied by the restoration of graphitic structure. The finding provides insight into graphene growth and defect reconstruction useful for the production of tailored carbon nanostructures with required properties. The growth of graphene on copper (Cu) substrates via chemical vapour deposition (CVD)1345789101178910111213142) surfaces serve as templates for the new growth of defective graphene during ethanol CVDCVD growth of graphene on Cu is a surface-mediated process131415161718171722Here we report the variation of graphene properties during lateral growth of graphene from RGO flakes on polycrystalline Cu foils by methane CVD. A combined microscopic and spectroscopic study correlated the growth length of CVD-grown graphene from RGO, reflecting the stages of in-plane graphene growth, with the corresponded structural quality of the graphene. The correlation demonstrated that graphene exhibited substantial enhancement in structural quality while it was laterally grown from RGO flakes on Cu surfaces up to a few hundred nanometres by the CVD process. The monotonous improvement of the structural quality of the graphene with increasing extended length of the graphene grown from RGO suggested that seeded CVD growth of graphene from RGO as low-quality seeds on Cu substrates was accompanied by the restoration of graphitic structure.RGO  were directly measured using a scanning electron microscope (SEM). The GO flakes, instead of RGO flakes, were prepared on Cu foils before CVD because they were naturally reduced  upon hea131626RGO and GGCN on the same Cu surface with the increase of CVD growth time was examined and compared. The GRGO growth length was defined as the distance between the GRGO growth front and the corresponding RGO edge  substrate , RGO (red circle), GGCN (blue hexagon) and GGCN edges (green pentagon) were measured at several positions on the sample. Thick RGO  and thin CVD-grown graphene (purplish area) on the SiO2/Si (pink area) were clearly discernible in an optical microscope image37382/Si substrate was indicated by a green-dotted line in RGO  of GRGO varied widely from 0.55 to 0.80 . The ID/IG values of GRGO were mostly larger than those of GGCN and GGCN edges, whereas these values were substantially smaller than those of RGO, indicating that the structural quality of GRGO was lower than that of GGCN and GGCN edges, but it was higher than that of RGO. Similar to the distribution of the ID/IG values, the intensity ratio of the 2D peak to the G peak (I2D/IG) of GRGO also scattered over relatively wide ranges compared with those of RGO, GGCN and GGCN edges  substantially smaller those of high-quality graphene monolayerD/IG and I2D/IG of GRGO has yet to be specified.Graphene edge structures can govern the structural properties of subsequently grown graphene from the graphene edge structures2930SiO2/Si, . EspeciaCN edges . The I2DRGO during seeded CVD growth has been specified, little is known about the evolution of the structural quality of graphene subsequently grown from the initially-formed GRGO during the later stages of CVD growth. To understand the evolution of the structural quality of GRGO with the increase of growth time, we measured Raman spectra on graphene samples in a direction away from the RGO flakes because the growth front of GRGO moved with time in such a direction during the CVD process, as already demonstrated in RGO, the graphene samples were grown on the Cu with GO flakes by CVD for 900 s. Large-scale characterization424344RGO and GGCN on the Cu completely coalesced into a single large-area RGO and graphene hybrid film after CVD for 900 s, suggesting the scalability in this study  compared with the thin CVD-grown graphene specimens (GRGO and GGCN)  . For a pN)  and width (Γ2D) do not show a significant variation until the defect density increases up to the certain value2D considerably decreases when the defect concentration further increases over the certain value, while Γ2D strongly increases. As shown in D/IG increased until the position moved from x1 to x4, however, it decreased when the position further moved from x4 to x8 (black square). In addition, I2D/IG  patterns varied from fully amorphous diffraction rings to the inner diffraction spots exhibiting streaking. This TEM observation suggested that graphene showed improvement in the structural quality while it was grown from RGO on the Cu by CVD for 100 sTransmission electron microscopy (TEM) is complementary to Raman spectroscopy, ideally suited for more detailed microanalysis of graphene atomic structure and its derivatives2223RGO and GGCN coalesce into a single large-area film as a RGO-graphene hybrid film  was added to a round-bottom flask  containing H2SO4  while stirring in an ice bath. KMnO4 (22.5 g) was added slowly to keep the reaction temperature of the suspension below 20 °C. Next, the flask was placed in the oil bath at 30 °C. The oil bath was removed completely at the end of the 2 days. After air cooling, diluted H2SO4  was added slowly to the flask, and the stirring was maintained for 2 h. To this mixture, H2O2  was added slowly and then the colour of the suspension turned dark brown to yellow, and the stirring was continued for 2 h. The obtained graphite oxide was purified with distilled water repeatedly by centrifugation. GO sheets were exfoliated from graphite oxide by ultra-sonication. The products were re-dispersed in distilled water.GO was arranged by the exfoliation and oxidation of natural graphite flakes (Sigma-Aldrich) according to the modification of Hummers method. A mixture of natural graphite flake (5.0 g) and NaNOAfter exposure to UV, a surface of Cu foil turns into hydrophilic surfaces and it was dipped in mixture of toluene (100 ml) and (3-aminopropyl)triethoxysilane  to ensure a uniform GO distribution on the Cu foil. The 3-APTES treated Cu foil was rinsed sequentially with ethanol after toluene and dried in a 60 °C atmosphere for 20 min. At the end of the process, the Cu foil was immersed in the GO solution (1.5 mg/1 ml) for 1 min and dried at room temperature.2) was introduced. The growth of graphene was then performed at 1060 °C for a certain time under a gas mixture of 2 s.c.c.m. methane gas (CH4), diluted in 300 s.c.c.m. argon gas (Ar) and 60 s.c.c.m. of H2. Finally, as prepared, the sample was cooled down to room temperature with Ar and H2 after turning off the flow of CH4. After all the process was over the Cu foil was removed from the furnace for further characterization. For Raman spectroscopy and its correlated studies, the graphene samples on the Cu foil were transferred to SiO2/Si wafers (Si(100) covered by 300-nm-thick SiO2) using a poly(methyl methacrylate) (PMMA) assisted process. Briefly, the PMMA dissolved in chlorobenzene was spin-coated onto the graphene samples at 2,000 r.p.m. for 30 s. The PMMA-coated samples were placed in a Cu etchant  to remove the Cu foil. After complete etching of Cu foil, the PMMA-coated samples were scooped out of the etchant using the SiO2/Si substrates. Finally, the PMMA layer was then removed with acetone and the surface was further rinsed several times with deionized water.Following GO deposition onto a Cu foil , the Cu foil was loaded into a CVD furnace and heated up to 1000 °C under a pressure of 1.0 Torr while 100 s.c.c.m. hydrogen gas (H−1 (using a grating with 600 grooves mm−1). The intensity of each Raman peak was extracted from the maximum value without any data processing over the corresponding spectral range .Spatially resolved Raman spectroscopy was performed using a Raman microscope  at Korea Basic Science Institute, equipped with a piezoelectric sample scanner. The wavelength of the excitation laser was 473 nm, and the power of the laser was kept below 0.3 mW without noticeable sample heating. The laser spot size was approximately 0.32 μm with a 100× objective lens . The spectral resolution was 2.0 cmHow to cite this article: Chang, S.-J. et al. Graphene growth from reduced graphene oxide by chemical vapour deposition: seeded growth accompanied by restoration. Sci. Rep.6, 22653; doi: 10.1038/srep22653 (2016).


Arabidopsis TPP riboswitch as an elaborated k-turn. The Escherichia coli TPP riboswitch contains a related k-junction, and analysis of >11 000 sequences shows that the structure is common to these riboswitches. The k-junction exhibits all the key features of an N1-class k-turn, including the standard cross-strand hydrogen bonds. The third helix of the junction is coaxially aligned with the C  helix, while the k-turn loop forms the turn into the NC  helix. Analysis of ligand binding by ITC and global folding by gel electrophoresis demonstrates the importance of the k-turn nucleotides. Clearly the basic elements of k-turn structure are structurally well suited to generate a three-way helical junction, retaining all the key features and interactions of the k-turn.The k-junction is a structural motif in RNA comprising a three-way helical junction based upon kink turn (k-turn) architecture. A computer program written to examine relative helical orientation identified the three-way junction of the  The kink turn (k-turn) is an extremely widespread structural motif that generates a tight kink in duplex RNA ,2, thereThe standard k-turn comprises duplex RNA with a three-nucleotide bulge followed by G•A and A•G pairs . The nucHaloarcula marismortui Kt-7 or the human U4 snRNA k-turn. Non-standard simple k-turns have a substitution in one of the G•A pairs. For example, in Kt-23 sequences of 30S ribosomal subunits of different species the 2n position has a frequency U>C>G>A, although examples analysed can form normal k-turn structures despite the departure from the standard sequence  riboswitches contain a hitherto unsuspected motif that is a k-turn that has been elaborated into a three-helix junction. This nevertheless retains all the key interactions found in a simple k-turn. We term this structure the k-junction, adding another branch to the scheme shown in Supplementary Materials. Visual analysis of structures and the computational results was performed in PyMOL 1.5.3. The software was implemented on a standard desktop workstation running Windows 7. RNA-containing PDB files were downloaded from the Protein Data Bank (pdb.org) and stored on the local hard drive. Non-RNA atoms were removed from these files and only files containing at least ten complete nucleotides were retained.RNA structures were analysed using software written in Python 2.7 together with the BIOPYTHON package. The scripts are available for download in All standard Watson–Crick base pairs, including the G–U wobble pair were identified, and for each structure minimal helical segments consisting of two base pairs in close proximity and suitable relative orientation were identified, and abstracted to sets of four vectors. The first vector defines the location of the helix segment, while vectors 2–4 define the orientation of the helix segment and are orthonormal. The second vector defines the main helix axis, the third the direction of the Watson–Crick hydrogen bonds projected to the plane perpendicular to the main helix axis, and the fourth is defined by being orthonormal to the second and third vectors. Therefore, these vectors define a coordinate system, with its origin located at this particular base pair and its coordinate axes aligned with the main features of the helix segment.The k-turn motif search pattern was defined by calculating the relative coordinates of helix segments from the C and NC helices of known k-turn structures. To do this, helix segments from the NC helix were expressed in terms of the coordinate system defined by a helix segment from the C-helix and the relative location and orientation was recorded. For each structure the identified helix segments were checked against pairs matching the relative location and orientation of helix segments from the search pattern. Positively scoring helix segments were written to output files. Helix segments from each were saved as residues consisting of pseudo-atoms into a PDB file. These structures were then analysed by visual inspection.Arabidopsis thaliana TPP riboswitch (3D2G) and E. coli TPP riboswitch (2DGI) were used to define secondary structure masks for the sequence alignments. All sequence composition and covariation analysis was calculated using a modified version of Jalview which was kindly provided by Dr James Procter (University of Dundee).A full alignment of 11 197 TPP riboswitch sequences taken from the Rfam database , accessi2, 50 μg/ml T7 RNA polymerase, 1 U/ml inorganic pyrophosphatase (Sigma), for 3 to 3.5 h at 37°C. The products were purified by gel electrophoresis under denaturing conditions. RNA was extracted from gel slices by electroelution and exchanged five times into 40 mM K-Hepes (pH 7.5), 100 mM KCl, 10 mM MgCl2 for ITC analysis, or 25 mM Tris, 192 mM glycine (pH 8.3), 1 mM MgCl2 for analysis by gel electrophoresis. Concentrations were measured by absorbance at 260 nm using extinction coefficients calculated from the nucleotide composition and a hypochromic effect correction factor.Transcription templates were prepared by PCR amplification from a plasmid-borne gene for the TPP riboswitch using primers hybridizing to the T7 promoter and the 3′-end of the riboswitch. RNA was transcribed from 75 μg/ml DNA template in 30 mM Tris–HCl (pH 8.0), 10 mM DTT, 0.1% Triton X-100, 0.1 mM spermidine–HCl, 4 mM each NTP , 40 mM MgClE. coli TPP riboswitch was:The sequence (written 5′ to 3′) of the unmodified GGACUCGGGGUGCCCUUCUGCGUGAAGGCUGAGAAAUACCCGUAUCACCUGAUCUGGAUAAUGCCAGCGUAGGGAAGUUCE. coli TPP riboswitch and variants were performed by isothermal titration calorimetry (ITC) at 303.15 K as described by Kulshina et al., 1 mM MgCl2 with or without TPP for 5 min at 20°C before addition of glycerol to 25% and loading onto the gel. Electrophoresis was performed either at 4 W for 2 h or 3 W for 2.5 h without or with (respectively) TPP added to the running buffer, such that the temperature remained below 20°C. The contents of anode and cathode chambers were mixed to prevent depletion of Mg2+ ions. After electrophoresis RNA was visualized by UV shadowing.The electrophoretic mobility of TPP riboswitch RNA and sequence variants was analysed in 10% polyacrylamide gels in 25 mM Tris, 192 mM glycine (pH 8.3), 1 mM MgClA. thaliana TPP riboswitch (PDB code 3D2G) contained two helices with a strong structural similarity to the k-turn geometry.We wrote a computer program in Python 2.7 to analyse relative orientation of segments of double helix in coordinate files of RNA structures downloaded from the PDB. A search pattern was defined by calculating the relative coordinates of helix segments from the C and NC helix of known k-turn structures. This structural criterion was then applied to other RNA structures to search for sections in which two helical segments had a relative orientation that was similar to those of a conventional k-turn. For all helix segments we searched for nearby candidate helical segments that matched the search pattern. Using this approach we observed that the A. thaliana was solved at 2.25Å resolution by Ban and colleagues •Hoogsteen (A) pairs just as found in a standard k-turn structure, although the G1b•A1n pair is less buckled than is normally observed in a standard k-turn. Unlike a simple k-turn, an additional adenine is inserted between the G1b and A2b. This is directed away from the helix, re-entering the major groove of the NC helix and making a triple-base interaction with the U5b•A5n base pair via the Hoogsteen edge of A5n.The secondary structure and conformation of the junction are shown in junction . This hajunction , beginnijunction . Both G•Upon an initial inspection it seemed that the UUU sequence that precedes the 1b position should be the loop of the k-turn, where the central U is stacked upon the end of the NC helix in the manner of a normal L2 position. Nevertheless, further analysis of the detailed interactions in the core (see below) led us to extend the formal definition of the loop sequence in the 5′ direction to include the preceding C, so that we now define the loop as CUUU, labelled L1, L1.1, L2 and L3 sequentially, whereby L1, L2 and L3 are in positions equivalent to those in the simple k-turn see B. In conThus all the global features of a k-turn occur in the TPP junction, and the structure can be superimposed with a standard k-turn such as Kt-7 in the ribosome, using the backbone atoms for nucleotides –1n, L1, 2b, 1n, 2n D with anA. thaliana TPP riboswitch k-junction to see if corresponding interactions could be identified.The core of the k-junction and key hydrogen bonding interactions are shown in H. marismortui Kt-58. This k-turn has a two-nucleotide AG loop opposed by a single A nucleotide loop; the A is stacked onto the NC helix, thus taking the normal role of L2, and the preceding base paired nucleotide provides the O2′ that donates its proton to A1n (O2′ of the cytosine designated as L1 (see above) is hydrogen bonded to A1n N1 (O-N distance 2.7Å), and is thus performing the role of L1 in a simple k-turn E. Althoun to A1n .The nucleotide 5′ to L1 is designated –1b, and that to which it is base paired on the opposite strand is –1n. In a simple k-turn the O2′ of –1n donates a hydrogen bond to either N3 or N1 of the conserved A2b nucleobase . This –1A. thaliana k-junction is a standard k-turn structure, with all the key hydrogen bonding interactions.Thus once we have set the component nucleotides in the correct structural reference frame we see that in most respects the E. coli solved by Serganov and coworkers •G(sugar) base pair, the 1b nucleotide is an adenine so that the 1b•1n pair is a trans A(Hoogsteen)•A(Hoogsteen) base pair that is achieved by placing the A1b in a syn conformation. This contrasts with the 2b•2n base pair of Thelohania solenopsae Kt-23 for example, which is A(Hoogsteen)•A(sugar)  that do not interact elsewhere within the same molecule. The loop is identified as the CGU sequence (i.e. L1 through L3), but the 5′ C and G are base paired with the opposing CG sequence again making a continuously stacked C-T helix.The observation of the k-junction as the major structural component prompted us to examine carefully the structure of the corresponding TPP riboswitch from oworkers  at 2.05Åoworkers A. We finA(sugar) . Like thA. thaliana riboswitch, has all the critical features of a k-turn. Like the A. thaliana riboswitch, there is an additional hydrogen bond between G-2n O2′ and A3b O2′ (O–O distance = 2.8Å).The standard k-turn cross-strand hydrogen bonds accepted by the conserved adenine nucleobases are present; CL1 O2′ to A1n (O-N distance 2.5Å) and the G-1n O2′ to A2b (O–N distance 2.9Å) B. The acE. coli TPP riboswitch using two distinct methods. We used gel electrophoresis under non-denaturing conditions to follow changes in the global conformation of the riboswitch RNA. In addition, we have studied the binding of TPP to the RNA using isothermal titration calorimetry (ITC); Ferré D’Amaré et al.  leads to a lower affinity of TPP binding by a single order of magnitude.Studied by ITC, the unmodified riboswitch sequence exhibits an exothermic reaction on binding of TPP . The titd for the natural-sequence riboswitch, 2+ ions were present. In agreement with previous observations . The k-E. coli sequence) in 16.89%. The analysis also shows that 21.53% of sequences have an A•A pair at the 2b•2n position. The 3b•3n CG base pair is strongly conserved at 99.10%. An analysis of the full riboswitch is presented in Supplementary Figure S1. This reveals that the most conserved nucleotides are those contributing to the ligand binding pocket, and those of the k-junction.How general is the k-junction in RNA structure? Analysis of 11 056 TPP riboswitch sequences suggests that the essential features of the k-junction is preserved in this large sample . The mosH. marismortui in the J4,5 region. Interestingly, this was previously identified as a k-turn . There is a standard G1b•A1n base pair, but a trans A2b(Hoogsteen)•A2n(Hoogsteen) pair. In contrast to the riboswitch k-junctions there are no nucleotides separating G1b and A2b, and the G1b•A1n pair is significantly buckled. The loop is clearly identified as the preceding GUA sequence and designated L1 through L3 with their usual structural functions. The C helix begins with the uridine immediately 5′ to GL1. The structure of the non-bulged strand of the NC helix does not map linearly onto the sequence. In terms of the sequence the nucleotides run 5′ to 3′ as G3n A1n A2n U; the final uridine is base paired with AL2, requiring an S-shaped turn to form in the backbone. A similarly ‘out of sequence’ non-bulged strand is found in the NC helix of the complex k-turn Kt-11 of T.thermophilus, and we may therefore regard this as a complex k-junction. As previously, we designate the nucleotides in terms of their role in the structure, so that A1n is paired with G1b for example. With the components of the k-junction assigned we find that all the standard hydrogen bonding interactions are present. L1O2′ donates a proton to A1n N1 (O–N distance = 2.7Å), and A-1n O2′ to A2b N1 (O–N distance = 2.7Å). Thus this is another N1 class structure, and the A2b N6 to A2n N7 distance is 3.5Å. Like the simple k-turns, the L3 O2′ to L1/L2 phosphate proS O is found in this structure (O–O distance = 2.7Å), and like the riboswitch k-junctions one further hydrogen bond forms between G-2n O2′ and A3b O2′ (O–O distance = 3.3Å).The k-junction is not restricted to the TPP riboswitches, and we have found further examples in other RNA species. A k-junction is found the large ribosomal subunit of a k-turn , but cloH.marismortui 50S ribosomal subunit, at J94/99 . Although the T helix is only partially defined by the available structure this is plainly a three-helix junction that is similar to those of the TPP riboswitch. The three-base GCC loop can be defined according to function as L1 (stacked onto the C helix), L1.1 and L2 (stacked onto the NC helix). The NC helix begins with a standard trans G(sugar)•A(Hoogsteen) pair at the 1b•1n position. Once again this is almost planar, in contrast to those of the simple k-turns. A1n N1 accepts the standard hydrogen bond from L1 O2′ (O–N distance = 2.7 Å). The 2b•2n position is a trans A(Hoogsteen)•A(sugar) pair connected by a single hydrogen bond. A2b N1 accepts a hydrogen bond from –1n O2′ (O–N distance = 2.9 Å). Thus both the standard cross-strand hydrogen bonds are present, and once again this is an N1-class structure.The importance of the k-junction motif is further underlined by finding a second example in the H. marismortui ribosome. Given that the k-junctions are not immediately easy to recognize, and indeed have been overlooked in a number of structures, it is likely that they are relatively widespread and that more examples remain to be discovered.Our analysis shows that the basic elements of k-turn structure are well suited to accommodate a three-way helical junction, preserving all the key features and interactions of the k-turn. This has been extensively exploited in the TPP riboswitches, but clearly occurs more widely as demonstrated by the two examples found in the Supplementary Data are available at NAR Online.


An enhanced Raman scattering from a thin layer of adenine molecules deposited on graphene substrate was detected. The value of enhancement depends on the photon energy of the exciting light. The benzene ring in the structure of adenine molecule suggests π-stacking of adenine molecule on top of graphene. So, it is proposed that the enhancement in the adenine Raman signal is explained by the resonance electron transfer from the Fermi level of graphene to the lowest unoccupied molecular orbital (LUMO) level of adenine. Non-invasive sensing of biological molecules, especially deoxyribonucleic acid (DNA) and its constituents, by means of label-free optical spectroscopy can open new prospects in biomedical analysis. Recently, we described a possibility for fluorescent detection of low concentrated DNA solution infiltrated into globular photonic crystal . Since tMaximal theoretical enhancement of the Raman signal caused by the surface of nanostructured noble metals can reach tens of orders of magnitude . Such siApplication of graphene as a substrate for SERS has renovated the interest in the use of surface-enhanced spectroscopy in optical sensing. It has been established that the Raman signal from some organic molecules deposited on graphene can be enhanced but usually less than in the abovementioned plasmonic case. The effect itself is quite novel for graphene and depends both on the type, spatial orientation of the tested molecules, strength of their interaction with graphene, molecular energy levels, and graphene properties , 9. GrapIn the present work, we report about up to 12 times enhancement of the Raman signal from an adenine layer on graphene in comparison with adenine deposited on quartz substrate and discuss the reasons for such effect.We used wide-area commercial graphene sheets prepared by chemical vapor deposition and transferred on a silicon substrate covered with a 200-nm-thick silica layer. The adenine was dissolved in distilled water at a low concentration (0.1 mg per ml). This solution was deposited on graphene samples as microliter droplets. Hydrophobicity of the graphene surface and the low concentration of adenine provided homogeneous drying of the small droplets in the form of thin films  energies of the adenine molecule with respect to the vacuum level taken as a reference point. Experimentally, it can be done, for example, by derivation of the real and imaginary values of the permittivity of solid adenine films in the deep ultraviolet spectral range. It is done, for example, in article [It can be speculated that the light with photon energy 2.41 eV (514 nm) fits better to the energy gap than the photons with energy of 2.54 eV (488 nm). The complication here is in the identification of the exact LUMO and highest occupied  article  by means article , 23. Sim article . There, If we suppose that the graphene Fermi level is situated near −4.6 eV , we can Voltage-dependent shift of the graphene’s Fermi level could be another way to control the resonance conditions for electron transfer to adenine on graphene. Our first attempts in this direction were not very successful because graphene suffers from hysteresis of electrical characteristics in atmospheric conditions . It compEnhanced Raman scattering from thin adenine layers deposited on graphene was detected. It was accompanied by quenching of the background fluorescence of adenine by the underlying graphene layer. The enhancement of the Raman signal depends on the photon energy of the exciting light in a manner which is in agreement with the assumption that a resonant electron transfer from the Fermi level of graphene to the LUMO level of adenine molecule takes place.


When multiple statistical tests are conducted, this procedure needs to be adjusted to compensate for the otherwise inflated Type I error. In some instances in tabletop gaming, sometimes it is desired to roll a 20-sided die (or 'd20') twice and take the greater outcome. Here I draw from probability theory and the case of a d20, where the probability of obtaining any specific outcome is Interestingly, this value coincides with the probability of obtaining a specific outcome on a 20-sided dice (or ‘d20’), as1.  This parallels the use of NHST without any correction for multiple comparisons, as it is more likely to get a significant effect due to chance  if many tests are conducted without a correction for multiple comparisons.In scientific research, it is important to consider the issue of conducting multiple statistical tests and the likelihood of spuriously obtaining a ‘significant’ effect. Within a null-hypothesis significance testing (NHST) framework, statistical tests are usually conducted withn die. This approach assumes that each statistical test is wholly independent from each other, and thus is likely to over-estimate the effect related to conducting multiple statistical tests using variations in how the measures are calculated or the use of different, but correlated, measures. Nonetheless, this exploration is based in probability theory and mathematical derivations, rather than computational simulations, and can serve as an comprehensible primer in understanding the relationship between repeated statistical tests and probability distributions.Here I wondered how much the probability of obtaining a 20, on a d20, would increase due to multiple tests–i.e., obtaining at least one 20 across2 and scientists4, have misconceptions about NHST. This is further compounded by critics of NHST, which often over-emphasize the limitations of the approach, e.g., see8—but rather to improve comprehension of the characteristics of NHST.Developing an intuition of statistics and probability distributions is of particular importance as most people, both laymenn die, each withd sides is:The probability that of a specific outcome occurring on each onn rolls of ad-sided die are listed inThe probability of obtaining a specific outcome acrossTo develop some intuition of the effect of multiple die rolls, several simple cases can be considered.d = 2, i.e., a coin, the probability of obtaining at a heads when flipping one coin (n = 1) isn = 2) isFord = 6, i.e., a ‘conventional’ six-sided die, the probability of obtaining any specific outcome isany of multiple dice, the complementary event should again be considered, i.e., the probability of not obtaining that outcome on any of the die. Forn = 1, the probability ofnot obtaining a specific outcome isn = 2, the probability of not obtaining a six on either of the dice isFord = 20, i.e., a 20-sided die, the probability of obtaining any specific outcome isn = 2 dice are rolled, the probability of obtaining at least one 20 isn = 10 dice are rolled, the probability of obtaining at least one 20 is ≈ .4013. Withn = 20 dice, this increases further to ≈ .6415.Foro or greater, on at least one ofn d-sided die:We can also consider a more general problem, the probability of obtaining an outcome ofo on ad = 20 die, acrossn dice.For instance, when rolling a six-sided die, the probability of obtaining a five or higher isWhile it is widely understood that multiple comparisons need to be corrected for, many would underestimate the degree of inflation in Type-I error associated with additional, uncorrected statistical tests. Critically, statistical procedures have been developed to correct for multiple comparisons , see I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The author provides an excellent foundation for developing an intuitive understanding of null hypothesis significant testing. The concept of using a 20 sided die to assist graduate students and new researchers to better understand what exactly is meant by .05 and how multiple comparisons have a dramatic influence on interpretation is an interesting one — and I believe novel. This intuitive approach can also be used to improve understanding for the general public and overall science communication. Although this report includes a mathematical derivation, which may be too advanced for new scholars or science communication, it provides an excellent rationale for the use of 20 sided die. When can also easily imagine that this rationale can be used better understand robustness of the outcomes of studies that may be influenced by experiment-wise error rates, such as in the cases of multiple attempts at replication or multiple trials of a specific experiment. The 20 sided die provides a concrete and real world method of communicating the complexities of multiple comparisons that is far more user-friendly than random number generators and variations of Monte Carlo studies. I am curious as to whether the exactitude in engineering and manufacturing a 20 sided die will result in exactly equal probability of each number appearing. A six sided die is created with right angles and is relatively easy to create an equal probability of landing on each side. Obviously, this makes no difference or changes the point of the paper; yet, it may add error should anyone actually attempt to roll the 20 sided dice multiple times. Just a thought as I am unsure on this issue. I suppose should a 20 sided die found to contain significant error or even bias, then there would be scandal in the Dungeons & Dragons world. The author also deserves credit for establishing high levels of nerd credibility. When Dungeons & Dragons, mathematical derivations, and useful statistical communication methods are combined into a single published scientific paper; the trifecta of nerd credibility has been achieved. Overall, this is a well-written report and the mathematics is correct. I am hopeful that the author continues to elaborate on this concept and develops other uses of a 20 sided die for communication and lesson plans in courses on research design and basic applied statistics. This will also be helpful in explaining experiment wise error rate/multiple comparisons in the cases of science communication as well.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This short report provides a simple and concise illustration of some of the issues surrounding multiple comparisons in statistical testing. I see nothing wrong with the logic or the mathematics, and can see that this would make a valuable contribution as an assigned text on an introductory statistics course.1 The only suggestion I have for material that could be added would be to include a citation to additional material on the topic of multiple-comparisons correction for the interested reader. This would make the piece more valuable as a teaching aid. I'd suggest adding a couple of sentences to the discussion along those lines and citing a review paper on the topic, such as Curran-Everett, 2000The paragraph after table 1 starts 'For intuition,'. This seems an odd phrase to me. Maybe replace with 'Intuitively' or 'For the purposes of familiarity...' or something like that.Just before the general equation the phrase 'across n die' appears on its own, as a separate paragraph. An error? Or is this supposed to be a subtitle? Couple of minor points:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The author provides a novel demonstration of the increasing probability of spurious research results when performing multiple tests: noticing the equivalence of a p = .05 statistical threshold and a d20 die (as used in popular games), the author goes on to describe changes in probabilities when using multiple dice, or when allowing multiple sides of the die to count as a correct result. The aim of this article, as I understand it, is to demonstrate that these changes have a surprisingly large influence on statistical hypothesis testing, and at the same time to provide a hands-on example that many readers might be able to relate to (in the form of the d20 die). The article is well-written and in my opinion fulfills both of these goals. Its rationale and the mathematical derivations seem to be sound and correct. I can easily see this article being used by educators to teach the topic of spurious statistical results and to make the topic more accessible.When the game name "Dungeons & Dragons" is first mentioned in the introduction, it should be followed by a citation of the game's publisher, year of publication, and, if necessary/appropriate, copyright information.A d6 die is described as "regular" in the text. This term seems ambiguous to me and I recommend replacing it with a term such as "cube-shaped," "classical," or "conventional."The current version of the manuscript appears to contain a printing error: before the last paragraph of the "mathematical derivations" section, the sentence fragment "across n die." is printed without context. I recommend the indexing of this article after these minor issues have been addressed, and I hope the author will continue to produce research notes that highlight statistical issues in an approachable manner. I have some minor edits/recommendations to increase the overall clarity and readability of the article:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


We have demonstrated that by tuning the ratio between organic solvents such as N-methyl-2-pyrrolidinone or ortho-dichlorobenzene, and n-octylbenzene molecules, the concentration of exfoliated graphene can be enhanced by 230% as a result of the high affinity of the latter molecules for the basal plane of graphene. The LPE processed graphene dispersions were further deposited onto solid substrates by exploiting a new deposition technique called spin-controlled drop casting, which was shown to produce uniform highly conductive and transparent graphene films.Due to a unique combination of electrical and thermal conductivity, mechanical stiffness, strength and elasticity, graphene became a rising star on the horizon of materials science. This two-dimensional material has found applications in many areas of science ranging from electronics to composites. Making use of different approaches, unfunctionalized and non-oxidized graphene sheets can be produced; among them an inexpensive and scalable method based on liquid-phase exfoliation of graphite (LPE) holds potential for applications in opto-electronics and nanocomposites. Here we have used  The former relies on the formation of covalent bonds between atoms or suitably designed small molecular building blocks to form 2D network by using chemical1921231728via Coulomb repulsion; this can be achieved upon addition of small molecules such as sodium dodecyl sulfate accompanied by alcohols36383940ortho-dichlorobenzene (o-DCB). In particular, we focus our attention on n-octylbenzene (NOTBZ) as dispersion-stabilizing agent due to its total miscibility in the selected solvents and its known capacity to promote the exfoliation yield of grapheneo-DCB during the LPE process on the quality and quantity of produced graphene flakes. We also describe a new deposition technique used for fabrication of uniform graphene films deposited from solution on flat solid surfaces. The set-up includes an integrated heating system to guarantee the fast evaporation of the solvent. More importantly, the as-obtained graphene film shows low sheet resistance and high optical transparency.Graphene, a 2D honeycomb lattice of carbon atoms, has been the subject of countless studies since its first experimental isolation by mechanical exfoliation in 20041367910121516o-DCB . To quantify the concentration of graphene after centrifugation, graphene dispersion was mixed with chloroform (CHCl3) and heated up to 50 °C for 30 min and then passed through polytetrafluoroethylene (PTFE) membrane filters (pore size 100 nm). The remaining solvent and NOTBZ molecules were washed several times with diethyl ether and CHCl3. As previously reportedIn order to cast light onto the role of the addition of NOTBZ in the graphene liquid-phase exfoliation, we first prepared dispersions by adding graphite powder in two different solvents most commonly employed for LPE, i.e. NMP and ions see  were chao-DCB. The average concentration of graphene amounted to 85 ± 5 μg mL−1 for NMP, and 100 ± 6 μg mL−1 for o-DCB as portrayed in 29The LPE studies started by analysing the result of two independent blank experiments performed using pure NMP and o-DCB. Among the four solvents used, upon addition of NOTBZ at specific percentage, we found that the concentration of graphene was more pronounced in NMP and o-DCB, and the highest yield was obtained for NMP as displayed in −1 was reached upon addition to the NMP dispersion of 15 volume % of NOTBZ. Surprisingly, at this percentage, the concentration of graphene in each of the solvents even TCB and DMF  is the highest. In the case of o-DCB, the maximum of graphene concentration is 190 μg mL−1 and is also observed upon addition of 15 volume % of NOTBZ. These concentrations correspond to an approximate increase of the exfoliation yield by 230% for NMP and 100% for o-DCB as compared to the blank experiments. It is worth noting that 15 volume % corresponds to a surface of graphene fully covered by NOTBZ molecules as detailed in Figure S2. Interestingly, the exfoliation at 100 volume % of NOTBZ, i.e. in pure NOTBZ, resulted in dramatic decrease of the exfoliation yield. In fact, no graphene was exfoliated, as revealed by TEM analysis. The exfoliation was successfull only when the amount of NOTBZ is below 40%. This phenomenon can be expained by the fact that NOTBZ itself is a bad solvent for liquid-phase exfoliation. While, recent reports have demonstrated that solvents with surface tensions of ca. 40 mJ m−2, e.g. NMP, o-DCB, are the best candidates for carying out LPE experiments, the relatively low surface tension of NOTBZ (ca. 30 mJ m−2) and notably high increase of the LPE yields in the case of NMP/NOTBZ and o-DCB/NOTBZ provided unambigous evidence that NOTBZ molecules can act as dispersion-stabilizing agent.We have then extended our LPE studies to the use of NOTBZ as dispersion-stabilizing agent. At room temperature and ambient pressure, NOTBZ is a liquid which is perfectly miscible with NMP and In order to characterize the exfoliated graphene sheets, qualitative analysis providing more relevant details such as the percentage of single-layer graphene (SLG) flakes, the lateral size of the flakes and the presence/absence of defects is needed. Currently, the most reliable method for the identification of the number of graphene layers in material produced by LPE is based on High-Resolution Transmission Electron Microscopy (HR-TEM). Typically, the analysis of the folded edges of the graphene sheets provides estimates of the number of layers composing the flakes. Moreover, from the TEM analysis, the average lateral size of the graphene sheets can be defined.First, we discuss dispersions prepared in NMP in the absence and/or the presence of NOTBZ molecules at the highest concentration obtained, i.e. the 15%. The TEM micrographs reveal that the majority of dispersions in NMP are composed of a large fraction of monolayer 15% and bilayer 18% graphene sheets folded over themselves with lateral size smaller then 500 nm. In the presence of NOTBZ, graphene flakes were found mainly in a monolayer 35% and in a bilayer 28% form . Also, to-DCB in the presence and/or absence of NOTBZ as dispersion-stabilizing agent at the highest concentration obtained, i.e. the 15%. The TEM micrographs reveal that the majority of dispersions in o-DCB are composed of a large fraction of monolayer 25% and bilayer 22% graphene sheets folded over themselves with lateral size smaller then 500 nm .To complement the characterization result by HR-TEM, Raman spectroscopy was used. In fact, Raman analysis is more qualitative and it has been shown that this technique is sensitive to the changes in thickness composition under different experimental conditions. It has been revealed that Raman spectroscopy offers a simple and fast analysis of graphene sheet thickness with the evolution of 2D peak shape43 for NMP . TherefoNoteworthy, the thickness analysis based on Raman spectroscopy is only quantitative and must be used in combination with HR-TEM results. Commonly, the results based on TEM are more reliable for liquid-phase exfoliated graphene, and the determination of number of layers based on Raman spectra is more suitable for graphene prepared by micromechanical exfoliation and CVD methods. Therefore, the small discrapancy in the flake thicknes analysis between the Raman and TEM is not surprising.2 and quartz) to form uniform films for applications in electronics remains a challenge. The solvents used for LPE have high boiling points, which results in a re-aggregation problem particularly when drop-casting method is used. Moreover, spin-coating of such dispersions is very challenging because as a results of the poor wetting, only few graphene sheets remains on the surface of the substrate, making it an unsuitable technique to form uniform films of graphene. Another well-known phenomenon observed when a drop of graphene dispersion is drying on a surface, is the coffee-ring effect45462 . AFM and SEM were used in order to investigate the change of morphology of these films, when different numbers of deposition (n) are applied. The graphene sheets are forming islands (as observed in n = 9) is needed in order to form a film with more than 95% of surface coverage. A correlation between the coverage and the number of deposition has been extracted from AFM analysis .Despite the increase of the exfoliation yield and the successful production of mono and few layers thick graphene sheets, the deposition of these graphene dispersions on commonly used substrates  regardless the type of solvent or the presence or absence of NOTBZ. In all cases and as expected, the transmittance decreases when the number of deposition or the surface coverage increases. For n = 9, where the sample is uniform with coverage exceeding 95%, the transmittance at 550 nm is lowered as compared to a single deposition and it decreases from ~93 to 72%.Optical transmittance and electrical resistance of graphene films are two important characteristics for many opto-electronic applications requiring transparent electrodes. Therefore, benefiting of the advantages offered by our developed set-up, we prepared on quartz substrates conductive graphene films from graphene dispersions in o-DCB, and were higher in the case of NMP (3200 to 2200 Ω/square) and in the presence of NOTBZ because of the low conductivity of the molecule. However, if more depositions are performed, the film resistance decreases (see o-DCB only) shows a surface coverage close to 100% for 9 depositions with 72% of transmittance and 680 Ω/square. With a surface coverage amounting 100%, the thickness of the graphene film times the sheet resistance value expressed in Ω/square is equal to the volume resistivity in Ω m of the graphene film. In our case the volume resistivity is as low as 1.6 × 10−5 Ω m. Also the sheet resistances and transparency reported in this manuscript are very close to those of graphene deposed by casting-dropping graphene/acetone suspension onto a polystyrene glass ride 3 (~400 Ω/square and 73% at 550 nm)3 (~30 Ω/square and 90% at 550 nm)The measured sheet resistances of the 24 nm thick graphene film led to very high values for films prepared from only three depositions see . This isases see  due to to-DCB. Careful analysis revealed a significant increase in the yield of exfoliation with the volume percentage of NOTBZ until a maximum. In particular, the most effective exfoliation was obtained with 15 volume % of NOTBZ in NMP as a solvent, with a concentration of exfoliated graphene of 250 μg mL−1 corresponding nearly to 230% increase in exfoliation yield when compared to samples prepared in pure NMP (85 μg mL−1). In the case of o-DCB, the most effective exfoliation was observed using also 15 volume % of NOTBZ, which led to ca. 100% increase in the exfoliation yield. Furthermore, a remarkable increase of single-layer graphene (SLG) flakes was observed in some cases. Careful investigation made it possible to correlate the 15% NOTBZ volume to 100% of surface area estimated which was found to be accurate for all studied solvents. TEM analysis of dispersions produced in NMP revealed that the quantity of SLG is improved by 100% when using NOTBZ molecules. In the case of o-DCB, the quantity of SLG was found amounting 25% even after addition of NOTBZ molecules, although a qualitative Raman analysis showed an opposite trend between NMP and o-DCB after adding NOTBZ. Graphene ink produced with our approach was deposed uniformly on SiO2 or quartz substrate by using a new deposition technique relying on a spin-controlled drop casting. These graphene films exhibit sheet resistance as low as 1 kΩ/square and transparency of 70% at 550 nm. By controlling the number of depositions and the concentration of graphene dispersions, film resistance and transparency can be tuned. Our approach opens up new avenues for the technological applications of graphene ink as low-cost electrodes and conducting nanocomposite for electronics.We have demonstrated that it is possible to enhance the yield of graphite exfoliation by mastering a molecule-assisted LPE-based approach to produce high-quality graphene flakes from bulk graphite. To attain a fundamental understanding of the role of molecule-graphene interaction on the yield of graphene LPE, we have carried out a comparative study by using NOTBZ as dispersion-stabilizing agent upon increasing the volume ratio of NOTBZ in two organic solvents, i.e. NMP and p.n. 332461) and ortho-dichlorobenzene  were purchased from Sigma-Aldrich and used as solvent for the exfoliation. N-octylbenzene  and graphite powder (p.n. 332461) were also acquired from Sigma-Aldrich and used without further treatment.N-methyl-2-pyrrolidinone  in two different solvents, i.e. NMP and −1. The laser power was well below 1 mW to avoid damage. Around 50 flakes drop-cast on silicon substrates were analyzed and the shape of the 2D peak and intensity (as height) ratio between D and G peak, I(D)/I(G) were analyzed. In particular, the 2D peak was fitted with a Lorentzian lineshape and the coefficient of determination, R2, was used to discriminate between monolayer (symmetric peak), few-layers graphene (asymmetric 2D peak) and graphitic (2 peaks). Such an approach allowed us to gain qualitative information on the number of layers distribution and the amount of defects. Atomic Force Microscopy (AFM) characterization was carried out by using a Veeco Dimension 3100, running with a Nanoscope IV controller. The analysis of AFM images and the extraction of the topographical profiles, for measuring the thickness of the different films, were done using Gwyddion.The amount of exfoliated graphene was measured on a Cubis Ultramicro-Balance MSA2.7S-000DM using a membrane holder. The filters were weighed 5 times before and after the filtration process in order to average the mass of graphene. Dispersions were first transferred in 10 mm path length quartz cuvette and analyzed by means of UV-vis-IR absorption spectroscopy using a Jasco V670 spectrophotometer equipped with a Peltier thermostated cell holder at 20.00 ± 0.05 °C. Also the optical transmittance of graphene films was measured using the same spectrophotometer equipped with a transmittance holder. The number of layers of graphene flakes was determined by High-Resolution Transmission Electron Microscopy (HR-TEM) using a JEOL 2100 F microscope. TEM samples were prepared by drop-casting the graphene dispersions on standard Quantifoil® R 2/1 holey carbon copper grid, followed by solvent evaporation at 150 °C for 10 min. Micro Raman measurements were carried out with a Renishaw spectrometer in confocal mode and backscattering geometry with excitation energy of 488 nm, 514.5 nm and 633 nm. A 100X objective with a NA of 0.95 was used for the characterization. This ensured a small laser spot size of less than 400 nm. The spectral resolution of the spectrometer is 2–3 cmo-DCB or NMP were deposed on freshly cleaned quartz substrate using our spin-controlled drop-casting set-up. A custom high torque step motor, with 1.8°/step and 200 step/revolution was used. We explored different parameters, i.e. rotation angle (from 0 to 35 steps), speed (from 1 to 35 step/s) and temperature (from 20 to 80 °C). The best condition giving a homogeneous film was by setting the rotating angle and speed at 22 steps and 11 steps/s respectively while heating at 50 °C. One deposition corresponds to one cycle of drop-casting 70 μL of graphene dispersion, 30 minutes of rotation and 180 min of post annealing at 100 °C under vacuum. The numbers of deposition were varied between 1 and 9. Sheet resistances were measured with a four probes Jandel (RM3000) platform.Prior to use, quartz substrates were washed by applying 2 cycles of acetone and isopropanol and cleaned by UV ozone treatment. 70 μL of the as-prepared graphene dispersions in How to cite this article: Haar, S. et al. Enhancing the Liquid-Phase Exfoliation of Graphene in Organic Solvents upon Addition of n-Octylbenzene. Sci. Rep.5, 16684; doi: 10.1038/srep16684 (2015).


Experimental data are understood within density-functional theory approach and the observed effects are in good agreement with the theoretical data.Realization of a free-standing graphene is always a demanding task. Here we use scanning probe microscopy and spectroscopy to study the crystallographic structure and electronic properties of the uniform nearly free-standing graphene layers obtained by intercalation of oxygen monolayer in the “strongly” bonded graphene/Ru(0001) interface. Spectroscopic data show that such graphene layer is heavily  Analysis of the electronic structure of graphene, which can be accessed in ARPES experiments, is performed via unfolding of the graphene-related bands to the primitive (1 × 1) Brillouine zone of graphene.In the present work we demonstrate the possibility to form the graphene/O/Ru(0001) intercalation system at the relatively low partial oxygen gas pressure and the elevated temperature for the complete initial graphene coverage on Ru(0001). The formed system of very high quality demonstrate the weak moiré structure as clearly deduced in scanning tunnelling and atomic force microscopy (STM and AFM) experiments. These experiments combined with the respective density functional theory (DFT) results allow to discriminate between geometric and electronic contributions in the microscopy imaging of this system. Our spectroscopy (STS) and DFT results show that a graphene layer in the graphene/O/Ru(0001) system is electronically decoupled from the substrate and demonstrates the π and Ru Crystallographic and electronic structures of graphene/Ru(0001) and graphene/O/Ru(0001) were compared in local STM/STS and AFM experiments. 20on-the-fly in the middle of the scan that allows to carefully trace the imaging contrast between two modes and its relation to the geometric and electronic contributions. In our STM experiments for the bias voltages in the range of ±1 V, graphene/Ru(0001) is always imaged in the Relatively large real space corrugation of 1.27 Å of graphene on Ru(0001)  makes AFM measurements with atomic resolution quite demanding. The results of such experiments are compiled in C places .−4 mbar of oxygen measured by the ion-gauge placed in the UHV chamber. Oxygen was introduced via a stainless steel pipe, which end was placed 1 mm from the sample surface that allows to increase the local pressure drastically (approximately by two orders of magnitude). This preparation procedure was initially also verified by the independent XPS measurements confirming our STM/AFM findings for the oxygen-intercalation system, graphene/O/Ru(0001).The oxygen intercalation was performed for the full graphene monolayer on Ru(0001) at 200 °C and a partial pressure of 1.8 × 10As can be found from the large scale STM image of graphene/O/Ru(0001) , oxygen s ≈20 pm  comparedDecoupling of graphene from the substrate and its nearly free-standing behaviour can be also concluded from the STM imaging of graphene/O/Ru(0001) around the step edges . One canIntercalated oxygen layer underneath graphene forms an ordered (2 × 1) structure on Ru(0001) as can be deduced from the Fast-Fourier-Transformation (FFT) image of the STM data shown in on-the-fly in the middle of the scan. Both parts of this panel demonstrate the atomic resolution. Panel (b) of this figure shows the AFM scan of gr/O/Ru(0001) and the respective height profiles extracted from the similar places of STM and AFM are shown in panel (c). The extracted atomic and mean moiré lattice corrugations extracted from the STM data are ≈30 pm and ≈10 pm, respectively. The same values extracted from AFM images are ≈6 pm and ≈4 pm, respectively.Similar to gr/Ru(0001) we also employ the combined STM/AFM measurements to the gr/O/Ru(0001) system. This allows to trace the true crystallographic structure of the system via discrimination between electronic and structural contributions in STM and AFM imaging. T = 150 °C in UHV conditions After STM/AFM studies of gr/O/Ru(0001) the same sample was transferred through air (maximum exposure time was 1 min) in the other experimental station for low temperature STM/STS studies. Due to the protection properties of graphene and its extreme inertness242526T = 1 K) STM/STS studies of the graphene/O/Ru(0001) system. Panel (a) presents combined topographic K points of the Brillouine zone (BZ) of graphene2829K and E relative to K point of the graphene BZ. [The possible deviation of the shape of the later features from the circle (see lower inset (FFT-ii) in the panel (b) might be due to the trigonal warping of the scattering vector map at higher binding energies with respect to p-doped.Series of the topographic EF)n-doping of graphene in gr/O/Ru(0001) with In order to verify this result the single ) system . Our valweak and strong interaction between graphene and Ru substrate with the minimal distance between C and Ru of 2.24 Å  of 2.10 Å) [covalent-like bonds at the graphene-Ru interface for the strongly interacting places of the structure. These results are in very good agreement with previous works323334In order to justify our experimental findings we perform analysis of these data within the framework of DFT .  2.10 Å) . The ori 2.10 Å) . This cap-doped systems.According to the previous13p-doping of graphene and formation of the localized dipole moment at the graphene-O-Ru interface.The side view of gr/0.5 ML-O-(2 × 1)/Ru(0001) is overlaid with the difference charge density , which sinterface states are formed in the energy range n-doped.The electronic structure of graphene (DOS) on Ru(0001) and on 0.5 ML-O-(2 × 1)/Ru(0001) is shown in   eV see . The dopσ and π states at the Γ and M points of BZ, respectively, and therefore their energy positions and intensities might be affected, for such big system , by the slight variation of the k-points sampling in BZ during integration.Oxygen intercalation (0.5 ML) in graphene/Ru(0001) leads to the almost complete recovering of the free-standing character of the graphene-derived electronic states. Comparing DOS for this system with the one for free-standing graphene, the position of Dirac point is strongly interacting system with the formation of several interface states at the K-point of BZ as a result of hybridisation between graphene and valence band states leading to the complete destruction of the Dirac cone. Thus, as it was already postulated in ref. weakly and strongly interacting regions within the graphene/Ru(0001) system, due to the metallic character of graphene in this system, the electronic structure of graphene in its original BZ is defined by the bonding strength and the electronic structure at the most perturbed graphene places. Upon the oxygen intercalation hybridisation between graphene and metal valence band states is absent (see π (as well as σ) branches are clearly recognisable in the electronic structure of graphene/0.5 ML-O-(2 × 1)/Ru(0001) [Similar conclusion regarding effective electronic decoupling of graphene from metal via oxygen intercalation can be made when comparing the results of band structure calculations performed for the studied systems . For grasent see . The maiRu(0001)  and theyIn order to prove our results we also perform simulations of STM images in the framework of the Tersoff-Hamann formalismstrongly interacting graphene layer from the Ru(0001) substrate via intercalation of an oxygen layer. The modifications of the crystallographic structure before and after intercalation are traced by the combined STM/AFM measurements. Strong variation of the imaging contrast is found in STM, reflecting the electronic structure of the system, whereas the corrugation obtained in AFM experiments is very close to the one deduced from DFT calculations. Decoupling of graphene via oxygen intercalation leads to the strong p-doping of a graphene layer with the Dirac point located at In our work we successfully decouple a Graphene/Ru(0001) system was prepared in ultrahigh vacuum system via cracking of ethylene: in situ via Ar+ -sputtering. In presented STM images the tunnelling bias voltage, The STM and AFM measurements were performed in constant current or constant frequency shift modes, respectively. In this case the topography of sample, et al.k-meshes for sampling the supercell Brillouin zone are chosen to be as dense as 6 × 6 and 3 × 3, respectively, and centred at the Γ-point. The STM images are calculated using the Tersoff-Hamann formalismThe DFT calculations were carried out using the projector augmented wave (PAW) methodHow to cite this article: Voloshina, E. et al. Restoring a nearly free-standing character of graphene on Ru(0001) by oxygen intercalation. Sci. Rep.6, 20285; doi: 10.1038/srep20285 (2016).