Acinus (apoptotic chromatin condensation inducer in the nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the exon junction complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using the cross-linking immunoprecipitation protocol (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC. There is an extensive coupling among different steps in eukaryotic gene expression, as shown by the intimate connection between transcription and RNA processing . In the Some of these RBPs associate with the mRNA in a splicing-dependent manner . In partDrosophila Sxl (Drosophila (Acinus (apoptotic chromatin condensation inducer in the nucleus) was initially identified as a protein required for apoptotic chromatin condensation . In addihila Sxl  and alsohila Sxl . It was hila Sxl  and was hila Sxl . This cohila Sxl , as wellhila Sxl  and has hila Sxl . Additiohila Sxl  and has hila Sxl . Interesosophila . Finallyosophila . It is tThe CLIP (cross-linking immunoprecipitation) approach with its many variations  has been successfully used to map in vivo RNA–protein interactions for several RBPs in different experimental systems , it was recently shown that it crosslinked to RNA as part of the mRNA–protein interactome of HeLa cells . We confACIN1 gene gives rise to three protein isoforms, termed Acinus-L, Acinus-S′, and Acinus-S, with all containing an RRM domain and a C-terminal RS-like domain . We isolated this complex and prepared libraries for each iCLIP replicate as well as a matching control library from cells transfected with an empty vector (pCG). We also performed two iCLIP experiments for the endogenous Acinus protein using an antibody that recognizes all three Acinus isoforms . We detected two specific signals corresponding to AcinusL/RNA complex and to AcinusS/S′/RNA complexes . As a control, we performed an immunoprecipitation using rabbit IgG. RNAs were extracted separately for Acinus L and S/S′ complexes and their respective controls. Sequencing data were processed as indicated in the Materials and Methods section and the number of reads for each experiment is shown in Supplemental Figure S1C. We analyzed the correlation between replicates for each different iCLIP experiment, using the read counts obtained per protein-coding gene. For the three iCLIP experiments, we obtained a Pearson correlation above 0.97 between the replicates . Therefore, we decided to pool the replicates for each iCLIP experiment  before identifying the binding sites. The annotation of the reads revealed that all Acinus isoforms bind mostly protein-coding genes ; however, we noticed that a large number of reads are also derived from repeats and long noncoding RNAs . For this study, we focused our analysis on the role of Acinus on mRNA biogenesis.In order to identify the in vivo RNA targets that are common to all Acinus isoforms, we carried out three independent iCLIP experiments in HeLa cells transiently expressing a T7-tagged Acinus construct, which we termed T7-Acinus S*, which contains the RRM domain and the RS-like domain A that arSupplemental Fig. S3). For further analysis, we thus defined a list of 36,271 peaks common to two out of three of the iCLIP experiments  that will hereafter be referred to as “common peaks.” These common peaks help us define the general binding properties for all Acinus isoforms. We also investigated separately each iCLIP experiment to eventually reveal specific characteristics of each isoform.The raw data revealed that Acinus, either the endogenous protein, or the transiently transfected T7-Acinus S*, does not seem to bind discrete positions of a few nucleotides but rather binds to larger regions. Therefore, we decided to identify crosslinked regions using the peak-finding algorithm Pyicoclip from the Pyicoteo tools  rather tSupplemental Fig. S4).The annotation of the 36,271 common peaks showed that most peak summits fall into exonic coding regions (70%) C. Peaks Supplemental Fig. S4B). Altogether, these data seem to exclude specificity of binding based on a particular isoform of Acinus.Using MEME , we searSupplemental Fig. S5A–C). The enrichment upstream of the exon junction is obvious for peaks associated with eIF4A3 and is decreased (or even lost completely for Acinus S and T7 Acinus S* iCLIP) when focusing on eIF4A3-negative peaks . Interestingly, a similar analysis for eIF4A3 peaks revealed that the observed eIF4A3 enrichment upstream of the exon–exon junction does not depend on its association with Acinus peaks . These observations fit a model whereby all EJCs at canonical sites are composed of the core components  but have a different composition of the peripheral factors, such as Acinus.Acinus was described as an auxiliary component of the EJC  and prefSupplemental Fig. S7). Importantly, the identified “T-rich” binding motif , suggesting that Acinus binding in introns does not occur in association with the core exon junction complex.Mapping of iCLIP peaks showed that Acinus also binds introns C. Interemotif 1, D is consmotif 1,  where AcSupplemental Table S1). We defined CLIP+ introns as those with a fivefold enrichment of read counts between Acinus iCLIP and control, or with a minimum of five reads in the Acinus iCLIP data. While the peak data analysis gave us 3792 introns with Acinus binding site, this read-count approach identified 22,813 introns defined as CLIP+ in two out of three iCLIP experiments. Moreover, we found 4838 common CLIP+ introns for the three iCLIP data sets   . To investigate whether Acinus-bound introns exhibit specific functional properties, we also defined a list of 23,654 CLIP- (CLIP negative) introns that contain a number of reads in all Acinus iCLIPs less or equal to the number of reads in the control iCLIPs. The comparison between CLIP− and CLIP+ introns revealed that introns bound by Acinus are significantly shorter, have a higher GC content, and display weaker 5′ and 3′ splice sites . Independent knockdowns and controls clustered together in our gene expression analysis . In Acinus-depleted cells, we found 408 up-regulated and 382 down-regulated genes with a twofold change and an adjusted P-value of 0.05 . We also quantified all alternative splicing events in each RNA-seq data in an unbiased way considering all hypothetically possible splice junctions from annotated and de novo splice sites, as previously described . Interestingly, those changes show enrichment toward one specific direction of the splicing change (2 test 0.006). This would indicate that Acinus is required for the inclusion of those alternative exons. As for IR events, the bias toward an increase in retention of introns in Acinus-depleted cells was striking (χ2 test 3.10–6). We validated several of these splicing changes using the same siRNA used for the RNA-seq data (knockdown [KD-1]) and also with another siRNA (KD-2), which gave a comparable depletion of Acinus protein . We validated the changes observed for the alternative cassette exons in PPHLN1 and ECT2 transcripts that showed a reciprocal response to Acinus depletion escribed . Using ag change A. For inepletion B. Periphepletion .In order to investigate whether the effect of Acinus on alternative exons was direct, we used the iCLIP data to draw an RNA map . From thP-value < 0.01). Indeed, 27 out of 106 introns with IR changes were defined as CLIP+ in the three Acinus iCLIP experiments, while 82 introns are CLIP+ in at least two iCLIP data sets , and MDM2, as well as for those encoding laminin β 2 (LAMB2), the CDK5 regulatory subunit-associated protein 3 (CDK5RAP3), and the splicing factor SRSF5 A. In eveata sets B.P = 3.4 × 10−7, Mann-Whitney U-test). This shows that CLIP+ introns have a higher proportion of introns presenting a change in their IR level (Δpir above and below five) and preferentially an IR retention level (Δpir above five). The link between Acinus binding in intron and intron splicing defect is perfectly illustrated for genes with more than one affected intron. For example, we observed a correlation between the number of reads in each intron and the level of intron retention change for the LAMB2 and ZMIZ2 transcripts . Altogether, these data indicate a direct role of Acinus in controlling the splicing of a subset of introns.Next, we investigated the relationship between direct binding of Acinus to an intron and the corresponding IR changes upon Acinus depletion. We compared the distribution of changes in IR level after Acinus depletion for all, CLIP+ or CLIP− introns C. For thDrosophila, the EJC as well as Acinus co-regulate the intron removal of a subset of introns . Interestingly, for the increased intron retention events, most of the events depend on both Acinus and eIF4A3 proteins and only a minimal number of events seem to be regulated by Acinus (or eIF4A3) only. We validated a few events experimentally by RT-PCR upon siRNA-mediated depletion of eIF4A3 . These data suggest that Acinus and the EJC co-regulate a subset of alternative splicing events.It was reported that EJC core proteins have a role in the regulation of alternative splicing in HeLa cells . In the  introns . We deci introns . To compAcinus was initially described to be required for apoptotic chromatin condensation . We searImportantly, the observed increase in intron retention upon Acinus depletion, together with binding to this intron, suggested a direct role for Acinus in this regulated AS event. The DFFA transcript containing the retained intron encodes a shorter DFFA protein isoform (DFFA-S/ICAD-S), which does not work as a chaperone for the caspase-activated DNase (DFFB or CAD) and consequently impairs the DNA fragmentation induced by apoptotic stimuli . The splSupplemental Table S3). For example, Acinus regulates an alternative cassette exon in the pre-mRNA encoding the mRNA export factor THOC2, as well as an AS cassette exon and an IR event in the pre-mRNA encoding the transcription termination factor TTF2 . We validated the impact of Acinus depletion on the retention of a specific intron for the splicing factor SRSF5  term enrichment analysis of the 4416 protein-coding genes bound by Acinus . We also analyzed separately genes that show Acinus binding in introns (2668 genes). We noticed enrichment for RNA processing, mRNA metabolic processes as well as pre-mRNA splicing pathways C,D. For or SRSF5 A. The inor SRSF5 .Surprisingly, Acinus also binds many RNAs encoding proteins involved in cell cycle regulation C,D. AmonHere, we used the iCLIP protocol to identify endogenous RNA targets for Acinus, an auxiliary component of the EJC, in human cells. We combined this with full transcriptome analysis of gene expression and splicing changes upon Acinus depletion and demonstrated a clear role for Acinus in splicing regulation. The iCLIP experiments identified RNA targets associated with all isoforms of Acinus, including the long isoform (Acinus-L) and the two short isoforms (Acinus S/S′). In addition, we also performed another iCLIP experiment with an overexpressed T7-tagged version of Acinus (T7-S*), which comprises a region that is common to all three isoforms, including the RRM and the RS-like domains A. Our anWe show that Acinus binds mostly protein-coding transcripts; however, we also detected reads coming from noncoding RNAs, preferentially repeats and long noncoding RNAs, suggesting a putative role for Acinus in their regulation. Importantly, we also detected Acinus peaks in introns downstream from the 5′ splice site as well as spanning the 3′ splice site. We obtained a more complete list of introns bound by Acinus by using a read count approach. This analysis revealed that Acinus preferentially binds to suboptimal introns .Drosophila following Acinus knockdown . There could be, nonetheless, physiological conditions upon which levels of Acinus could be altered. For instance, hypermethylation of this gene has been observed in early stage lung adenocarcinoma, which could lead to a decrease in its expression . This is in agreement with previous findings in Drosophila where the core EJC plus the accessory factors RnpS1 and Acinus promote efficient splicing of neighboring introns (The exon junction complex (EJC) is a dynamic complex and while most exon–exon junctions are apparently bound by the four core proteins, the composition of peripheral factors is supposed to be variable, depending on the different stage of the mRNA life and maybe depending on the exon junction position within the same transcript . The dyn pathway . In mamm pathway . A role  pathway . EJCs ha pathway . Surpris introns . Here, EThe genome-wide RNA-binding profile of Acinus is also suggestive of additional cellular functions. Supporting this idea, the GO-term enrichment for Acinus (this study) and eIF4A3 mRNA targets  revealedWe discovered that Acinus binds and regulates the splicing of the last intron of the DFFA/ICAD pre-mRNA, giving rise to the production of DFFA-L  that has a function as chaperone for the caspase-activated DNase (DFFB/CAD) and promotes DNA fragmentation upon apoptotic stimuli . As thisIn summary, we present here a genome-wide RNA-binding landscape for the peripheral EJC factor Acinus that reveals a crucial role of Acinus in the regulation of splicing events co-regulated by the EJC. The identified mRNA targets place Acinus within a cross-regulatory RNA processing network and support its role in cell cycle progression.2.HeLa cells were cultured in medium  at 37°C in an atmosphere containing 5% COHomo sapiens acinusS mRNA, complete cds, GenBank: AF124727.1) into the mammalian expression vector, pCGT7, which was previously described  or Dynabeads protein A (endogenous Acinus iCLIP). For the iCLIP of tagged T7-Acinus S*, a T7 Tag antibody Agarose from Novagen was used for the precipitation in all IPs. For the iCLIP of endogenous protein, the immunoprecipitation (IP) step was carried out using Acinus (Ab-2) PC552 Calbiochem antibody coupled to Dynabeads Protein A, whereas rabbit IgG was used for the control. Dephosphorylation of the RNA 3′ ends was performed on beads using Alkaline Phosphatase, Calf Intestinal (CIP) following the supplier's protocol. The RNA linker (5′Phosphate-UGAGAUCGGAAGAGCGGTTCAG-3′Puromycin) ligated to the 3′ end of the RNAs was described in HeLa cells or HeLa expressing T7-Acinus S* were cultured on a 15-cm dish and irradiated once with 400 mJ/cmprotocol , with mihttps://usegalaxy.org/. Taking advantage of the iCLIP specific barcodes, identical reads, corresponding to PCR duplicates, were removed. Sequencing artifacts were discarded. Adapter sequences at the 3′ end were removed from the reads using CLIP tool. Barcode splitter tool attributed reads to each replicates. We obtained around 22 million reads for the three T7-Acinus S* experiments, whereas the control experiments produced 4 million reads. All endogenous iCLIP libraries gave 17 million reads (15 million reads for the control). Reads were mapped to the hg19 annotation of the human genome (hg19 assembly) using Bowtie . A correlation plot was produced using the number of reads obtained for each ENSEMBL Id for the different replicates. The correlation was evaluated by calculating the Pearson correlation coefficient.https://bitbucket.org/regulatorygenomicsupf/pyicoteo)  . Mapped http://meme-suite.org/tools/meme) (The peak summit was used as the reference binding site and peaks were ranked based on the number of reads at the summit. We retrieved 20-nt sequences around the summit and searched for motifs on 1000 sequences (top or randomly selected). The search was done for a 6- to 10-nt-long motif using MEME (ls/meme)  allowingeIF4A3 CLIP-seq data described in https://bitbucket.org/regulatorygenomicsupf/pyicoteo. We obtained 29,741 peaks for T7 Acinus S* iCLIP, 23,970 peaks for Acinus L, 32,146 peaks for Acinus S and finally 121,145 peaks for eIF4A3 CLIP. We also defined a list of 13,231 Acinus “common transcriptomic” peaks.We generated a simplified and a representative transcriptome data set by keeping for each gene the transcript with the longest CDS. First, reads mapping to noncoding RNAs and repeats were discarded. Then, remaining reads were mapped to the sequences of the transcriptome using Bowtie, allowing at most one mismatch and selecting uniquely mapping reads. Peak calling on transcripts was done using Pyicoclip on these mapped reads. Coordinates of the peaks on the transcriptome were converted to genomic coordinates using software available at Transcriptomic peaks present in internal exons (first and last exons were removed) were analyzed. Distances between the peak summit and the 5′ and 3′ side of the exon were calculated. The frequency of each distance was evaluated and normalized by the number of analyzed peaks to compare between different data sets. We plotted the density of peaks depending on the distance from the 5′ and 3 exon ends. We also calculated the distance of intronic peak summits from the 5′ and 3′ side of the intron. Finally, histograms showing the number of peak summits depending on the distance were generated.Reads mapping to noncoding RNAs and repeats were discarded. We kept reads that overlap a data set containing all intron interval coordinates (minimum overlap of 5 bp). We calculated for each intron the amount of reads in each iCLIP experiments and the control. We took advantage of the RNA-seq data (described below) to remove introns from very low expressed genes  and we kept 129,270 introns. We defined CLIP+ introns as introns with fivefold enrichment in Acinus iCLIP versus equivalent control or if there is no read in control, we kept introns with a minimum of five reads. CLIP- introns were defined as introns having a number of reads in all Acinus iCLIPs less or equal to the number of reads in the control iCLIPs.http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html).The strength of the splice site was evaluated on line using the maximum entropy score method  (http://RNAs were extracted from Acinus-depleted or control HeLa cells in triplicates. RNA extractions were performed using RNeasy Mini Kit (QIAGEN) with DNase treatment on column following manufacturer's instructions. RNA samples were sent to Beijing Genomics Institute (BGI). The libraries have been obtained using Truseq Transcriptome kit [poly(A) transcript enrichment] and sequenced on Illumina HiSeq providing a minimum of 10 Gb of data per sample (90-bp paired end reads).Transcript quantification was done using Sailfish  on the EThe analysis of splicing changes  in the RNA-seq of Acinus-depleted cells was done as previously described , whereasr, the minimal probability of acceptance that is required to consider a comparison to be believable), difference cutoff  that there is a difference of at least the cutoff (10% by default), and coverage . This default is meant to provide a reasonably stringent baseline. The RNA-seq data for eIF4A3, described in In differential splicing analysis, stringency criterions for filtering of output include probability  following manufacturer's instructions in control cells or cells depleted of Acinus. RNAs were subsequently treated with DNase RQ1 (Promega) to remove any gDNA contamination. cDNA were obtained using Superscript III Reverse Transcriptase (Invitrogen). The PCRs to detect the splicing changes were done with GoTaq Hot Start Colorless Master Mix. The sequences of the used primers can be found in To investigate whether Acinus directly regulates alternative exons, we looked for Acinus “common” peaks in proximity of the events. We considered a region containing the cassette exon as well as the neighboring constitutive exons. In the case of different possible constitutive exons, we selected the shortest ones. We detected peaks for 31 skipped cassette exons and 30 included cassette exons after Acinus depletion. As a background, we selected 185 alternative cassettes from the list of analyzed exons (percent inclusion between 10 and 90) that are not affected by Acinus (|Δ_psi| < 5) but containing a peak in the event regions. The distribution in exons (50-nt downstream or upstream the splice site) was analyzed plotting a histogram of the normalized counts considering a bin interval of 5 nt. We analyzed the distribution of the relative distance in introns using a normalized count density plot.U-test .The intron retention changes upon Acinus depletion was assessed for all, CLIP+ and CLIP− introns. We first removed introns whose intron retention level could not be assessed in some of the RNA-seq samples. For the remaining introns, we calculated the Δpir that corresponds to the change in IR level between Acinus depletion and control and plotted its density distribution. The distribution of the intron retention changes between CLIP+ and CLIP− introns are significantly different as revealed by a Mann-Whitney https://david.ncifcrf.gov/home.jsp. As a background, we provided the list of expressed genes obtained from the RNA-seq .To find significant GO terms, we used The Database for Annotation, Visualization, and Integrated Discovery (DAVID tool), Raw sequencing data can be found in the Gene Expression Omnibus database with accession number GSE81460. Acinus iCLIP peak files as well as RNA-seq expression data can also be found under this accession.Supplemental material is available for this article.


Unique electrodynamic response of graphene implies a manifestation of an unusual propagating and localised transverse-electric (TE) mode near the spectral onset of interband transitions. However, excitation and further detection of the TE mode supported by graphene is considered to be a challenge for it is extremely sensitive to excitation environment and phase matching condition adherence. Here for the first time, we experimentally prove an existence of the TE mode by its direct optical probing, demonstrating significant coupling to an incident wave in electrically doped multilayer graphene sheet at room temperature. We believe that proposed technique of careful phase matching and obtained access to graphene’s TE excitation would stimulate further studies of this unique phenomenon, and enable its potential employing in various fields of photonics as well as for characterization of graphene. The negλ0 = 1.55 μm.In this article, for the first time eight years after its prediction, we demonstrate direct optical probing of the TE mode supported by graphene, employing modified Otto configuration that allows very precise phase matching of the incident wave to the TE excitation, and electrostatically doped multi-layer graphene stacks at room temperature, performed at the telecommunication wavelength −7 RIU. It was previously suggested that small effective index of the TE mode  regime1415The physical nature of TE excitations in graphene can be understood as magnetic dipole waves, i.e. self-sustained oscillations of current  with no n2) and below (n3) the graphene layer. Here we propose a TE mode excitation scheme where graphene is situated between dielectrics with asymmetric indices, n2 > n3, and placed at distance d from the coupling prism, n1  makes it difficult to provide symmetry between indices of materials above . Considering this, we employ a five-layer graphene stack to derive significantly more effective coupling to incident waven3(1.55 μm) ≈ 1.4045) with applied silver electrodes (n2(1.55 μm) ≈ 1.4070), such that desired index asymmetry condition n2 > n3 is satisfied. Employing of an ionic liquid instead of an ion geld between graphene and the coupling prism  ≈ 1.5006), approaching desired values of d ~ λ0, in order to obtain precise phase matching. To measure an angular reflectance, we employ a setup schematically shown in In a stack of randomly oriented graphene layers  and TE (blue) polarized incident wave at λ0 = 1.55 μm, and room temperature T = 300 K. While TM curves show no response to doping, TE reflectance of doped graphene exhibits a distinct dip in the vicinity of the critical angle θc and can be observed for both samples, indicating an excitation of a guided TE mode in the structure. It is important to note that in the absence of the graphene, reflectance profile indicates lack of any specific TE mode in the structure under applied experimental conditions, which was confirmed by the control experiment . We conduct model parameters fitting based on both TE and TM reflectance responses, providing roughly estimated tolerances of ±0.1λ0 for the d, and ±5 meV for the EF , with assumed charge carriers’ mobility μ = 1 × 104 cm2(Vs)–1. In accordance with the RPA, fitted sheet conductivity is taken as σ1 = σu – i0.01σu for the undoped, and σ2 = 0.3σu – i0.4σu (model case 1) and σ2 = 0.7σu – i0.47σu (model case 2) for the doped graphene, corresponding to doping levels of EF ≈ 0.42 eV and 0.38 eV respectively; σu = e2/4ħ is the universal conductivity of grapheneσeff1,2 = 5 × σ1,2. Additional notes on multilayer graphene doping can be found in To confirm the experimental data, and to get an insight into the mode excitation dynamics, we use a numerical model of employed excitation scheme considering angular resolution Δr the EF . In the EF 142425 in the lσeff1,2, and d = 3.4λ0 (model case 1) and d = 2.5λ0 (model case 2) for both samples is shown in d and σ, along with the estimated tolerances, it can be noted that fitted model parameters are very close to actual experimental conditions, thus confirming the doping of multilayer graphene coming up to negative values of σ″. Together with the reflectance minima located beyond the critical angle in both experimental and model studies, this indicates an excitation of TE mode in multilayer graphene.Numerical reflectance obtained for the values of It must be noted that an approximately 1 nm thick Debye layer of ions at the graphene/ionic liquid interfaceE|2 profile of this mode. Leftmost profiles correspond to reconstructed experimental conditions  in doped graphene (solid lines) at shorter d, their magnitude becomes comparable with that of radiative losses (green), providing significantly enhanced coupling to incident light1314n2 = n3 (see inset in L = λP /(2πq″) ~ 1000 of plasmonic wavelengths.x-z axes, revealing mode expansion by several λ0 into the bulk on both sides. Obtained with the solver dispersion of the TE mode for model case 1 (black), case 2 (pink), and case 1 with n2 = n3 (dashed) is demonstrated in n3, following prediction for the symmetrical (n2 = n3) configurationd ≈ dcut for considered model cases. It is also clearly demonstrated , and cured at room temperature during 24 hours. 12.5 × 12.5 mm2 area five layer graphene was manufactured by chemical vapour depositionIn this study we use PVDF film with thickness of 0.254 mm purchased from CS Hyde; silver electrodes were deposited manually using Pelco−5 deg. All measurements were performed under normal ambient conditions at 300 K.Infrared reflectance measurements were performed with a tunable diode laser at 1.55 μm wavelength and a power meter head with removed cover glass to avoid interference effects under varying angle. Gold-covered spherical mirror of 1250 mm radius was mounted at fixed stage while angular scan was conducted by a flat mirror on a moving linear stage , providin1) and bottom  layers, where the plane x = 0 corresponds to the n1|n2 interface, and the multilayer graphene stack of effectively zero thickness occupies the x = d plane. TE-polarized plane wave is incident onto the n1|n2 interface at angle θ. The ansatz is made that the electric field in each medium m = 1,2,3 takes the form In the numerical model, the excitation scheme shown in ansatz into the Helmholtz equation k0 = ω/c) we obtain q = n1k0sin(θ) is the projection of the incident wavevector onto the z-axis. Then, assuming the harmonic time dependence exp(–iωt) of the magnetic field H, substituting it into the Maxwell curl equation n1|n2 and n2|n3 interfaces, noting that K(x = 0) = 0 and K(x = d) = σeffE(x = d), thus obtaining the system of linear equations in complex amplitudes Am and Bm. Taking B3 = 0  and A1 = 1 , solving the system, we obtain the reflection coefficient of the incident wave R = |r|2 (r = B1/A1). The same system of linear equations in complex amplitudes is used in a solver to find eigenmodes solutions supported by the structure, solving the determined system in the absence of the incident wave A1 = 0.Substituting the How to cite this article: Menabde, S. G. et al. Direct Optical Probing of Transverse Electric Mode in Graphene. Sci. Rep.6, 21523; doi: 10.1038/srep21523 (2016).


As a direct consequence, the absorptance of graphene depends on the refractive indices of the surrounding media. This explains the difficulty in determining separately the optical constants of graphene and their widely varying values found in literature so far. Finally, our results allow an accurate estimation of the potential optical losses or gains when graphene is used for various optoelectronic applications.The fundamental sheet conductance of graphene can be directly related to the product of its absorption coefficient, thickness and refractive index. The same can be done for graphene’s fundamental opacity if the so-called thin-film limit is considered. Here, we test mathematically and experimentally the validity of this limit on graphene, as well as on thin metal and semiconductor layers. Notably, within this limit,  A, reflectance R and transmittance T, is frequently discussed3411121316a, thickness d and refractive index n151618A, R and T do not depend on the parameters a, d or n individually, but only on their product adn. (ii) There is no dependency on the wavelength either, except through the dependencies of the parameters themselves. This explains why in the case of graphene—the thin film par excellence—considerable disagreement exists over the measured individual optical parameters20212223d and dielectric function ε lose their usual physical meaning and must rather be defined as tensors, relating to each other as N and The discovery of free-standing graphene2346789adn product has been related to the fundamental sheet conductance e being the electron charge and ħ the reduced Planck constant)ω the angular frequency, The graphene’s c is the speed of light in vacuum. For graphene we obtain:where ATFL, reflectance RTFL and transmittance TTFL of a layer between two media:To analyze the effect of the TFL on graphene we take equations recently derivedG and vacuum impedance Here, We first numerically investigate the range of validity of the TFL by comparison to rigorous Fresnel formulae. In a first case we analyze 30 monolayers (ML) on glass and investigate regions of validity within 10% accuracy. The validity regions are in general limited by high energy and high absorption coefficient thresholds stemming from the above mentioned assumptions: cannot distinguish between absorption coefficient, refractive index and thickness. This is valid in the near-infrared to visible range for any material with absorption coefficient below 105cm−1.In a second case we consider a 10× thinner sample (3 ML), 10× better accuracy (1%) and we obtain slightly broader regions of TFL validity than in the previous case. In this case, the reference data of a, d and n. For oblique incidence, additional simulations (not shown here) prove a similar difficulty to distinguish between a, d and n, for thin layers on a substrate. However, angles far from normal incidence, as in ellipsometry, always increase significantly the ability to distinguish between these parameters.In a third case, we remove the glass substrate, assuming thus a freestanding layer. The region of TFL validity for transmittance and absorptance changes slightly, but for reflectance, conversely, the validity of TFL shrinks to a negligible region around the adn product, thanks to one of the consequences of the TFL: The values ATFL, RTFL and TTFL are mutually dependent in such a way that by measuring only one of them we can calculate the remaining two. By combining Experimentally, the validity of the TFL can be verified independently from the actual value of the RTFL and TTFL, ATFL is calculated as 1−RTFL−TTFL. Noteworthy, this yields a ‘universal’ relation that applies to materials beyond graphene.Knowing adn product, we simulate in RTFL and TTFL: according to (6) for a freestanding layer, and according to (4) and (5), while accounting multiple reflections for the case of the layer on glass. The latter is also simulated for the case of immersion in carbon tetrachloride (CCl4). The advantage of the CCl4 is that at room temperature its refractive index is similar to the one of glass. As such, the situation of freestanding layers can be approached. Black symbols show the theoretical T vs. R relations, when graphene’s fundamental conductivity (2) is taken. To compare with experiments, the pairs of transmittance and reflectance values represent points in the graph, plotted by symbols. We see that the symbols for graphene fall well on the theoretical curves. In addition, the TFL was equally well fulfilled for an 11-nm-thick layer of evaporated aluminum over a broad spectral range, and also for a 110-nm-thick indium oxide layer, but only in the infrared region (<0.8 eV).For any value of the adn product from adn product is shown in n and k spectra of single-layer graphene, taken from literature42021242627n and k values of graphene samples, compared to their respective adn products. This is consistent with the fact that graphene on a substrate (measured in transmission and reflection) and freestanding graphene (when measured in transmission only) always fulfills the TFL over a broad wavelength range  and (5) that for an ultrathin layer on a substrate or a freestanding layer:Finally, we evaluate, based on the TFL, the losses or gains of using graphene as transparent functional layer. It follows from A/T ratio is also a good parameter for evaluating macroscopically-thin layers, because for a layer on a substrate the A/T ratio is virtually free from interference effects and free from direct wavelength dependenciesMoreover, the A/T ratio of a layer on a finite substrate by TFL and rigorously. In both cases the effect of multiple reflections in the substrate is accounted for by the Fresnel equations. We tested a set of thicknesses and absorption coefficients while keeping the value of adn product fixed to 0.0229. Three cases were considered: the embedded layer, the layer-on-substrate for layer-side incidence and the layer-on-substrate for substrate-side incidence. We see that for the embedded layer, as well as for the layer-on-substrate, the increase of refractive index of the surrounding medium or the substrate can indeed significantly reduce the ratio A/T. As long as the TFL describes well this phenomenon (well up to a film thickness of 10 nm) it is advantageous to embed graphene in, or place it on top of, a high-refractive-index medium. For macroscopically-thin layers (e.g. in our case 335 nm) this trend is weakened, and importantly, for lower values of refractive index of the surrounding the A/T ratio of macroscopically-thin layer is lower than that of ultrathin layer. This implies, that thinning down a layer while keeping the adn product constant is not favorable, unless a high-refractive-index surrounding medium, e.g. silicon, is used. Interestingly, for the substrate-side incidence the refractive index has no effect on A/T ratio. These effects are crucial when comparing optoelectronic applications of graphene with usual macroscopically-thin window layers.In a, d, and n. Graphene satisfies this limit over a broad spectral range and it makes the separate determination of its optical constants difficult, especially when graphene is on a substrate. The layer thickness, as a condition of the limit, should rather be compared to the vacuum wavelength; in the infrared and upon perpendicular incidence, the limit can be satisfied even by a 100-nm-thick layer on glass (e.g. of indium oxide below 0.8 eV). Within the thin-film limit, the plot of reflectance versus transmittance is, for a given surrounding medium, a universal curve, which was also used here for experimental verification. Another interesting quantity is the absorptance normalized to transmittance, which is perfectly suitable for comparing absorption losses in graphene and other window layers. It shows that if the thin-film limit is satisfied, the performance is strongly enhanced by the high refractive index of the underlying medium.Within a given spectral region and depending on the substrate and incidence angle, thin layers may satisfy the thin-film limit when their measurable optical properties are given only by the product of 4) in a custom-made setup. Our custom-made setup also allows photothermal deflection spectroscopy (PDS)−4 through heating of immersion liquid, e.g. CCl4. The refractive index of CCl4 is around 1.46 in our spectral range (0.6–3 eV)https://graphene-supermarket.com/Transparent-Conductive-Coatings/. We also used a layer of aluminum, thermally evaporated at pressure 5×10–5 mbar and a layer of indium oxide, sputtered in DC regime at 6 mbarThe transmittance-reflectance spectroscopy was done either in air by Perkin-Elmer Lambda 900 or in a carbon tetrachloride .


N-doped graphene with Curie temperature higher than room temperature is a good candidate for nanomagnetic applications. Here we report a kind of N-doped graphene that exhibits ferromagnetic property with high Curie temperature (>600 K). Four graphene samples were prepared through self-propagating high-temperature synthesis (SHS), and the doped nitrogen contents of in the samples were 0 at.%, 2.53 at.%, 9.21 at.% and 11.17 at.%. It has been found that the saturation magnetization and coercive field increase with the increasing of nitrogen contents in the samples. For the sample with the highest nitrogen content, the saturation magnetizations reach 0.282 emu/g at 10 K and 0.148 emu/g at 300 K; the coercive forces reach 544.2 Oe at 10 K and 168.8 Oe at 300 K. The drop of magnetic susceptibility at ~625 K for N-doped graphene is mainly caused by the decomposition of pyrrolic N and pydinic N. Our results suggest that SHS method is an effective and high-throughput method to produce N-doped graphene with high nitrogen concentration and that N-doped graphene produced by SHS method is promising to be a good candidate for nanomagnetic applications. Graphene has attracted tremendous attention since its first isolation by Novoselov and Geim in 20041356d and f elements. Unexpected magnetic properties have been found in some low dimensional materials. The reduction in one or more dimensions typically results in the reduction of the coordination number of atoms, which reduces the hopping tendency of electrons89Magnetism in nanomaterials is a scientific discipline at the forefront of fast emerging fields in nanoscience and nanotechnology. In current technological applications, magnetic materials are mainly based on et al. have prepared N-doped graphene by annealing reduced graphene oxide in ammonia, which can increase its magnetization at a relatively low temperature (≤600 °C)et al. pointed out that pyrrolic N could induce a net magnetic moment of 0.95 μB/N, compared with pyridinic N which has less influence on the spin polarization of the edge states2-coordinate carbon with graphene domains and the option to incorporate nitrogen especially on pyrrolic bonding sitesStudies on the magnetic properties of graphene have established the possibilities of developing magnetic materials with lightweight, high strength and high thermal conductivity. It has been recently shown that carrier-doped graphene has a very large diamagnetic susceptibility13The magnetic properties in graphene and related 2D carbon materials are often explained by the existence of different kinds of defects132425272829303132353638395 K/cm). SHS is widely applied in producing various materials, including carbon-free, this method has not yet been applied to synthesizing CNTs2 (dry ice)3 to produce few-layer graphene with a conventional calcination method50Self-propagating High-temperature Synthesis (SHS) has received considerable attention since it is a relatively simple, fast, low-cost, and efficient novel material production method4243d and 4f elements. N-doped graphene samples produced by SHS method exhibit both high Curie temperatures (higher than room temperature) and high coercive force. Thus, it is considered a good candidate for nanomagnetic applications.Here, we prepared four few-layer graphene samples with different nitrogen contents using a patented method based on self-propagating high-temperature synthesis (SHS). We are particularly interested in the N-doped graphene samples because of its unexpected magnetic properties without the addition of 33, 99.5%), carbamide, (CO(NH2)2, 99.5%) and carbon dioxide (99.9% purity) were used as starting materials. These materials were purchased from Sinopharm Chemical Reagent Co., Ltd.Magnesium , calcium carbonate  with different nitrogen contents were synthesized by the SHS patent methodThe phase composition of the as-prepared powders was analyzed by powder X-ray diffraction (XRD) analyses (Philips X’ Pert diffractometer) with CuKα radiation. Environmental scanning electron microscopy  and high-resolution transmission electron microscopy (HRTEM JEM-2100) were used to observe the morphology of the graphene sheets. The TEM specimens were prepared by dropping ethanol/water (38 v/v%) solution containing 1 wt % N-doped graphene onto a copper grid and drying at 100 °C. Raman spectra were obtained using a Raman Station  with a 532 nm wavelength laser corresponding to 2.34 eV, 30% of the laser power  was used on the samples. X-ray photoelectron spectroscopy  was utilized to determine the bonding characteristics of the samples. All XPS peaks are calibrated according to the C 1 s peak (284.6 eV). The composition was confirmed using X-ray fluorescence  in order to determine the presence of any metallic elements. 2 milligrams of N-doped graphene powder was spread out on the surface of boric acid powder , the effective test zone is a disk surface with a diameter of 20 mm. The magnetic properties were measured using a Quantum Design MPMS magnetometer based on a superconducting quantum interference device (SQUID).TEM observations were used to further investigate the high magnification morphology and crystalline structure of the pristine graphene and N-doped graphene sheets, and the typical TEM image of the pristine graphene sheet presents in The XRD patterns of N-doped graphene in 48−1 are corresponding to the G-band, D-band and 2D-band, respectively, according to previous study53552g mode of sp2 carbon domains. Both bands can be influenced by doping. D bands have been significantly enhanced in Sample 2 and Sample 3 with respect to pristine graphene and Sample 1, because pyrrolic and pyridinic N are usually accompanied by defects or edges in the graphene sheets. It is interesting to note that the G band of Sample 2 and Sample 3 moves to higher frequency which is sign of high density N doping; in the work of Zhao et al., a similar trend of the G band was also observedThe Raman spectrums of pristine and N-doped graphene sheets are shown in ID/IG) is a measure of defect quantity−1, which is a feature of graphene195960ID/IG in graphene increases with the increasing of carbamide in the reactants, which indicates that the defect concentrations in N-doped graphene increase with the increasing of the N-content in the samples. This is due to the fact that the N atoms break the sp2 C six-member ring structure in graphene to form pyrrolic or pyridinc nitrogenI2D/IG) can be used to estimate the layer number of graphene sheet, the values of I2D/IG for both Sample 2 and Sample 3 are higher than that of Sample 1, indicating thinner thicknesses for Sample 2 and Sample 3. In addition, pyrrolic N bonding configuration also attenuates the 2D mode, as the intensity of 2D mode is dependent on the electron-doping concentration, which results in the inversion relationship of the I2D/IG and the electron concentration. As a result, the reason for the value of I2D/IG for Sample 3 lower than that of Sample 2 may be caused by the higher concentration of pyrrolic N in Sample 3 .Both 2D-and G-band features are highly relevant to layer numbers and defects. The D-band and G-band intensity ratio (XPS is a common technique to measure the doping concentration and the N bonding configuration in N-doped graphene. As shown in 2-hybridized carbon (C-C)533 hybridized C atoms bonded with O, N or (C)3-N bonds, which could be originated from C-O, pyrrolic or graphitic N bonding configuration. Furthermore, different from the N-doped graphene directly grown by CVD system, a new weak peak located at 287.9 eV can be observed in our sample. This peak indicates sp2 hybridized C atoms bonded with N, C-N-C bonds, which is originated from either graphitic or pyridinic N bonding configuration2 hybridized aromatic N with two sp2 hybridized C neighbors in the form of C = N-C (pyridinic N) and the peak at 400.4 eV is assigned to the tertiary N in the form of N-(C)3 or H-N-(C)2 (pyrrolic N). These assignments are consistent with previous report2 hybridized N atoms with three sp2 hybridized C neighbors) in the N-doped graphene lattice would appear around 402.0 eV, it indicates graphitic N bonding configuration is very limited in our N-doped graphene. The pyridinic bonding configuration refers to the bond between N and two C atoms at the defects or edges of the N-graphene. The pyrrolic N bonding refers to the N atom bonding in a five-membered ring structure. Compared with the XPS spectrum captured for the pristine graphene (Sample 0), these changes observed for the C 1 s orbit in the N-doped graphene suggest that the N doping behavior indeed occurs in graphene lattice to a certain extent.The C 1 s peak of the samples in XPS can be split into three peaks, which locate at 284.3, 285.0 and 287.9 eV (Gaussian decomposition and Lorentz decomposition are employed in this fitting). The main peak at 284.3 eV corresponds to graphite-like spOn the basis of the XPS analyses, the N and O contents of N-doped graphene samples are shown at M-H) curves for pristine and N-doped graphene samples measured at room temperature (300 K) are shown in The magnetization versus magnetic field (Hc) and saturation magnetizations (Ms) can be obtained from the magnetic hysteresis loops in Ms at 10 K for Sample 0, 1, 2 and 3 are 0.072, 0.275 and 0.318 emu/g, respectively; and the values of Hc at room temperature for Sample 1, 2 and 3 are 63.1, 143.7 and 168.8 Oe, respectively. It is strange to see that the Hc and Ms and of Sample 0 (0.125 emu/g and 117.8 Os) are higher than those of Sample 1 and lower than those of Sample 2 and 3, which means that the pristine graphene has higher ferromagnetism than the sample with low content of N (2.53 at%). The result can be understood considering the result in D/IG for the pristine graphene is also higher than that of Sample and lower than those of Sample 2 and 3. Since it is well accepted that defects play the main role in the ferromagnetism and the ratio of ID/IG is positively corresponding to defect concentration, the higher ferromagnetism for Sample 0 can be understood from the point of view of defect concentration.The magnetic hysteresis loops shown in This result also suggest that since the introduction of carbamide can change the reaction temperature  and the reaction atmosphere, there may be differences in the mechanism for the two reactions with and without carbamide. As a result, Sample 0 only has limited reference value.The coercive forces and the saturation magnetizations at different temperature are shown in Ms of the four samples have similar trends with those of the coercive force. They decrease with the increasing of N content and temperature. The values of Ms reach 0.282 emu/g at 10 K and 0.148 emu/g at 300 K for Sample 3, which are comparable to graphene obtained in freestanding MoS2 nanosheet or other dopant-free diluted magnetic semiconductorsThe values of d or f element is not responsible for the observation of ferromagnetism in N-doped graphene. Therefore the presence of defects seems to be the main factor in the emergence of ferromagnetism in N-doped graphene.XRF measurement is utilized to check the content of the ferromagnetic impurities in our samples. The specific ferromagnetic impurities are 8.9 ppm Fe and 5.0 ppm Ni as shown in In addition, it was reported that Pyrrolic N-doped graphene can be produced based on a stoichiometric dehalogenation of perhalogenated arene and pyridine precursors by a transition metalM ~ T curves are shown in M ~ T curve with respect to temperature range 550–700 K are plotted as the insets in The magnetization behaviors of the four samples recorded at 3000 Oe are shown in To study the reason for the decreasing magnetism, take Sample 2 as an example, we have heated the Sample 2 in vacuum for 5 minutes at 600 K, 650 K and 700 K, respectively. XPS measurements were conducted for the samples heated at different temperature, the contents of C, O and N for Sample 2 were shown in To further support the XPS results, thermogravimetric (TG) analysis was conducted for Sample 0 and Sample 2. The TG curves are shown in Connected these results with the behaviors of magnetism of the samples, it is reasonable to conclude that the peaks at 625 K for the N-doped graphene samples are caused by the decomposition of N groups, with the loss of N groups which are the source of magnetic moment, the magnetization drops quickly. So the peak at 625 K is the result of thermal instability for N-doped graphene between 600 and 650 K.After 650 K, the relative contents of C, N and O are stable as shown in Hc and Ms increase with the increase of N content in N-doped graphene. However, it is interesting to find that the Curie temperature of Sample 3 is a little less than that of Sample 2. We would discuss the meaning of the phenomenon.As above mentioned, the N plays an important role in the magnetic properties of N-doped graphene, both the In the case of Curie temperature, although it is not clear why there is such strong interactions in these N-doped samples, which give rise to high Curie temperatures, we could speculate and discuss the influence factors of Curie temperature according to the well known physical principles, that is the magnetic response of the N-doped graphene could be determined by the competition between the RKKY interactions and the screening effect of electrons. We will discuss the issue from two aspects.B/N as proposed by Li et al.13Firstly, we should point out that pyrrolic N plays an important role in the formation of magnetic moments, because pyrrolic N can induce a net magnetic moment of 0.95 μSecondly, the ferromagnetism in N-doped graphene indicates that localized magnetic moments coming from pyrrolic N and defects can cause the ferromagnetism response by the magnetic coupling. This coupling effect could be realized through the RKKY interactions by delocalized electrons32It is worth emphasizing that the SHS method plays an important role in the formation of pyrrolic N and defects in the N-doped graphene. SHS method used in the present study is a process far from equilibrium technique, which utilizes the energy released by the exothermic combustion reactions of the starting materials; the combustion reactions can produce very high temperature instantly  and then cool quicklyIn summary, we have produced few layer pristine and N-doped graphene by SHS method. Room temperature ferromagnetism has been found and related to carbon defects and pyrrolic N arising from nitrogen doping in N-doped graphene. It has also been found that the saturation magnetization and coercive field increase with the increasing of nitrogen content in the samples. The Curie temperature is 673 K~678 K for the N-doped graphenes with higher N contents. The drop of magnetism between 600 and 650 K is caused by the thermal instability of N-doped graphene. N-doped graphene samples produced by SHS method exhibit both high Curie temperatures and high coercive force. The work proves that SHS method is a promising high-throughput method to produce N-doped graphene which may have potential applications in electromagnetism.How to cite this article: Miao, Q. et al. Magnetic properties of N-doped graphene with high Curie temperature. Sci. Rep. 6, 21832; doi: 10.1038/srep21832 (2016).


Programmed Death-1 (PD-1) and its ligand, PD-L1, are regulators of immune/ inflammatory mechanisms. We explored the potential involvement of PD-1/PD-L1 pathway in the inflammatory response and tissue damage in cardiac injury models.Ischemic-reperfused and cryoinjured hearts were processed for flow cytometry and immunohistochemical studies for determination of cardiac PD-1 and PD-L1 in the context of assessment of the growth arrest- and DNA damage-inducible protein 153 (GADD153) which regulates both inflammation and cell death. Further, we explored the potential ability of injured cardiac cells to influence proliferation of T lymphocytes.m) as well as apoptotic and necrotic cell death. Importantly, while isotype matching treatment did not affect the aforementioned changes, treatment with the PD-L1 blocking antibody reversed those effects in association with marked cardioprotection. Further, ischemic-reperfused cardiac cells reduced proliferation of T lymphocytes, an effect partially reversed by PD-L1 antibody. Subsequent studies using the cryoinjury model of myocardial infarction revealed significant increases in PD-1, PD-L1, GADD153 and IL-17 positive cells in association with significant apoptosis/necrosis.The isolated ischemic-reperfused hearts displayed marked increases in expression of PD-1 and PD-L1 in cardiomyocytes; however, immunofluorescent studies indicate that PD-1 and PD-L1 are not primarily co-expressed on the same cardiomyocytes. Upregulation of PD-1/PD-L1 was associated with a) marked increases in GADD153 and interleukin (IL)-17 but a mild increase in IL-10 and b) disruption of mitochondrial membrane potential (ψThe data suggest that upregulation of PD-1/PD-L1 pathway in cardiac injury models mediates tissue damage likely through a paracrine mechanism. Importantly, inhibition of T cell proliferation by ischemic-reperfused cardiac cells is consistent with the negative immunoregulatory role of PD-1/PD-L1 pathway, likely reflecting an endogenous cardiac mechanism to curtail the deleterious impact of infiltrating immune cells to the damaged myocardium. The balance of these countervailing effects determines the extent of cardiac injury. The immune system has evolved to distinguish and defend against foreign antigens while simultaneously avoiding self-reactivity. Protection against self-reactivity is primarily relegated to central tolerance mechanisms which cause depletion of most self-reactive T lymphocytes. Yet, some T lymphocytes that are specific for self-antigens escape into the periphery, which likely underlies the evolution of peripheral tolerance mechanisms in order to protect against autoimmunity. An important mechanism regulating peripheral tolerance and autoimmunity is the expression of the programmed death-1 (PD-1) receptor .The PD-1 receptor is a coinhibitory member of the B7/CD28 superfamily of molecules which is expressed on T and B lymphocytes. Binding of PD-1 with ligand partners, namely PD-L1 and PD-L2, results in the upregulation of the suppressive arm of immunity, thereby protecting against self and microbial antigens –6. Indeein vivo relevance of PD-1/PD-L1 pathway in cardiac injury, additional studies utilized the cryoinjury model of myocardial infarction  while control ischemic-reperfused hearts received isotype matching antibody  via a side arm placed above the heart. Thereafter, cardiac tissue was filtered through a cell strainer  and centrifuged  to obtain single cell suspension for flow-cytometry based assays or cytospin-preparations for immunofluorescent studies. Also, myocardial tissue was fixed in buffered formalin for subsequent immunohistochemical studies  overnight at 4°C, preparations were then washed three times with Tris-buffered saline (TBS) for 5 min each time. All slides were then incubated with the secondary fluorescence-labeled antibody  for 1 hour in the dark at room temperature, washed twice in TBS for 5 min each time in the dark and then counterstained using DAPI . Further, cytospin preparations of about 20, 000 sorted cells per sample chamber were centrifuged , air-dried, fixed in 10% formalin and washed twice in PBS. All cytospin preparations were then treated as described above. Microscopic analysis and imaging was done by an Olympus BX40 using Q-Capture software.PBS.Tissue sections were prepared as described above. Following de-paraffinization, sections were washed for 10 min in distilled water. All subsequent procedures with tissue preparations were carried out at room temperature ,22. Endom as a surrogate marker of mitochondrial permeability transition (MPT) pore opening, which is a critical event in cell death.As described previously –18, flowAssessment of necrosis and apoptosis utilized the flow-cytometry-based annexin V/7-Amino-Actinomycin D (7-AAD) protocol as described previously –18. AnneT cells (CD3+) enrichment was performed using CD3 MicroBeads  as described previously . Cell se5 cells/well. Cardiac cells or APCs (as stimulators) were used at 5 x105/well. Following 72–96 hours of incubation at 37°C in a humidified 5% CO2 environment, all cells were harvested into flow cytometry tubes. After one wash with PBS, all samples were then incubated with anti-rat CD71-PE-conjugated antibody (a marker for activated and dividing T cells) for 20 minutes in dark on ice. Samples were then washed with PBS and T cell proliferation was measured by using flow cytometry analysis for CD71 expression. The average of the triplicate samples was recorded for each heart (n = 3 hearts/group/condition).The MLR is a commonly used functional assay to determine the proliferative capacity of T lymphocytes in response to antigen presentation ,22. ThisData were analyzed using Student’s t-test (for comparison of cryoinjury and sham groups) or the analysis of variance followed by Newman-Keuls post hoc test to establish significance (p<0.05) among groups for all other data. Data are reported as means ± SEM.GADD153 is believed to regulate inflammation and cell death –18, 20. Given marked upregulation of PD-1/PD-L1 pathway in ischemic-reperfused cardiac cells, we next explored the influence of these cells on the proliferative capacity of T lymphocytes. Fig –7E showsin vivo relevance of PD-1/PD-L1 pathway in cardiac injury, subsequent studies utilized the cryoinjury model of myocardial infarction. Cryoinjury was associated with significant increase in GADD153 positive cells . Further, as shown in As an initial step in establishing the m and marked necrotic/apoptotic cell death. Importantly, treatment with the PD-L1 antibody, but not isotype matching antibody, largely reversed the aforementioned changes in the ischemic-reperfused hearts. These observations indicate that upregulation of cardiac PD-1/PD-L1 pathway, in response to an IR insult, is a major determinant of tissue injury in the isolated buffer-perfused heart. Importantly, ischemic-reperfused cardiac cells inhibit proliferative capacity of T lymphocytes, an effect partially reversed by PD-L1 blocking antibody, thereby substantiating the inhibitory immune regulatory role of PD-1/PD-L1 pathway. Thus, while corroborating the antigen presenting role of cardiomyocytes, these novel observations establish the remarkable ability of an IR insult to influence proliferative capacity of T lymphocytes. Importantly, upregulation of PD-1/PD-L1 pathway is also a feature of the cryoinjury model of myocardial infarction thereby establishing its in vivo relevance for the damaged heart.The present study indicates that an IR insult upregulates cardiomyocyte expression of PD-1 and PD-L1 in association with increased GADD153 expression and promotion of a proinflammatory environment culminating in disruption of ψThe role of programmed death pathway in regulation of tolerance and autoimmunity is well-established –3. This Myocardial injury is associated with upregulation of endogenous inflammatory responses –18. RelyUpregulation of cardiac PD-1/PD-L1 pathway was accompanied with a robust increase in expression of GADD153 and promotion of a pro-inflammatory environment as revealed by a marked increase in IL-17 positive cells but a mild increase in IL-10 positive cells; we recently showed that the cardiomyocyte is a major source of inflammatory cytokine generation in isolated ischemic-reperfused heart , 19. Sinm as well as necrotic and apoptotic cell death in the ischemic-reperfused heart. One likely mechanism for this association could relate to the marked increase in GADD153 expression since knockdown of GADD153 confers cardioprotective effect [m and markedly reduces cell death. Nonetheless, it is noteworthy that while the anti-PD-L1 treatment abrogated GADD153 expression and cytokine expression, significant differences in JC-1 aggregates/monomers ratio and cell death  persisted between the untreated and treated ischemic-reperfused heart. The reason for this observation likely relates to multiple and divergent mechanisms that regulate the MPT pore and cell death in the ischemic-reperfused heart [Upregulation of PD-1/PD-L1 pathway was associated with disruption of the ψe effect . On the e effect . Thus, ied heart .Given the marked upregulation of PD-1/PD-L1 pathway in the isolated ischemic-reperfused cardiac cells, we conjectured that co-culturing of these cells with T lymphocytes would be of consequence for their survival/proliferative capacity. This notion is based on the well-established interaction of the PD-L1, expressed on APCs, with PD-1, expressed on T cells, culminating in their apoptosis of the latter . Since tThe isolated heart and MLR studies are suggestive of an important role for cardiac PD-1/PD-L1 in the ischemic-reperfused rat heart. As an initial attempt to further establish the relevance of this pathway in cardiac tissue injury, we utilized the murine cryoinjury model of myocardial infarction. This model produces standardized infarcts and is suggested to more closely resemble infarcts in clinical conditions than the coronary ligation model . Interesm as well as apoptotic and necrotic cell death; these effects are blocked by disruption of the PD-1/PD-L1 pathway, resulting in improved cardiomyocyte survival. We also demonstrate that cardiomyocytes are capable of inducing T cell stimulation, a process that is diminished by IR injury, which may reflect the upregulated PD-1/PD-L1 signaling in these cells. Collectively, our observations suggest that upregulation of PD-1/PD-L1 in response to cardiac tissue injury may have countervailing effects on cardiomyocytes and the immune system, the balance of which determines the extent of cardiac injury. Thus, the challenge remains how to harness the beneficial effects of cardiac PD-1/PD-L1 activation while preventing/reducing its deleterious effects on cardiac cells . Importantly, determination of the relevance of cardiac PD-1/PD-L1 pathway as a potential immunotherapeutic target to curtail T cell-mediated cardiac graft rejection and associated complications represent fertile grounds for future investigation.In conclusion, the present study shows an important role of PD-1/PD-L1 pathway in cardiac injury. The expression of PD-1 and PD-L1 by different populations of cardiac cells  is suggestive of a paracrine mode of action in the ischemic-reperfused heart. Upregulation of PD-1/PD-L1 is associated with GADD153 expression, proinflammatory changes accompanied with disruption of ψ


A first-order model for graphene CVD on transition metal catalysts that combines kinetic and thermodynamic considerations is developed and experimentally verified. The chemical vapour deposition (CVD) of graphene on three polycrystalline transition metal catalysts, Co, Ni and Cu, is systematically compared and a first-order growth model is proposed which can serve as a reference to optimize graphene growth on any elemental or alloy catalyst system. Simple thermodynamic considerations of carbon solubility are insufficient to capture even basic growth behaviour on these most commonly used catalyst materials, and it is shown that kinetic aspects such as carbon permeation have to be taken into account. Key CVD process parameters are discussed in this context and the results are anticipated to be highly useful for the design of future strategies for integrated graphene manufacture. The question of which catalysts can be used is not only of fundamental importance but also a key issue for integrated graphene manufacture. Roll-to-roll approaches demand reusability of the catalyst, low cost and ease of graphene removal,8 whereas approaches without graphene transfer where the catalyst is part of the device structure or removed while the graphene stays on the target substrate demand graphene growth on a wide range of different catalyst materials at suitably benign CVD conditions.10 In spite of numerous surface science studies on the atomic structure of graphene layers on different single-crystal metal surfaces,12 the conditions and challenges for scalable graphene CVD are notably distinct and with a lack of basic understanding of graphene formation, catalyst selection criteria are yet to be well established.Chemical vapour deposition (CVD) has emerged as the dominant method to synthesise large single-crystalline domains and continuous films of “electronic-grade” graphene and other two-dimensional (2D) materials.14 to avoid graphene layer formation by carbon precipitation on cooling.18 Uniform growth of single layer graphene (SLG) on polycrystalline catalysts with significantly higher carbon solubility, including Ni19 and Pt,20 can, however, be routinely demonstrated,22 and it has been shown that single and few-layer graphene growth in particular for lower process temperatures occurs predominantly during hydrocarbon exposure at temperature and not during cooling.24 While simple thermodynamic considerations of carbon solubility are insufficient to adequately describe even basic growth behaviour on these most commonly used catalyst materials, we show that kinetic aspects such as carbon permeation have to be taken into account. Our simple model allows us to highlight key CVD parameters including the catalyst thickness alongside the growth temperature, carbon precursor pressure, exposure time and cooling rate. This captures well the behaviour apparent in our graphene CVD calibrations on Co, Ni and Cu and we expect our results to be highly useful for the design of future strategies for integrated graphene manufacture.Here, we systematically compare graphene CVD on three polycrystalline transition metal catalysts, namely Co, Ni and Cu, and propose a first-order growth model that can serve as a reference to optimize growth on any elemental or alloy catalyst system. A rationale widely adopted in current literature is that a catalyst material with low carbon solubility, such as Cu, is necessary for single layer growthvia continued hydrocarbon exposure and dissociation at the catalyst surface at constant temperature, which we refer to as isothermal growth, or it can also be crossed vertically at a given carbon concentration via catalyst cooling and the reduction in carbon solubility, which we refer to as precipitation on cooling. For a basic CVD process, consisting of heating up and pre-treatment of a catalyst , exposure to a hydrocarbon at constant temperature and cooling down in an inert atmosphere, graphene formation typically proceeds via isothermal growth but additional growth may also occur during cooling. Hence it is often argued that for the CVD of SLG, a catalyst with low carbon solubility is essential, and that for high carbon solubility metals, additional layers grow by precipitation upon cooling leading to multilayer formation.18JI, and that related to carbon diffusion into the catalyst, JD, with the difference in fluxes, JG, feeding the growing graphene layer  pressures lead to the direct nucleation of multilayer graphene due to a high supersaturation developing at the catalyst surface. Low precursor  pressures relative to the permeability  of carbon in the catalyst will lead to an effective filling of the catalyst bulk with carbon, which in turn can precipitate depending on cooling rate (see below). The thickness of the catalyst dictates the size of the potential carbon reservoir and hence plays an important role in this kinetic model. A thick catalyst will allow continued carbon diffusion into the bulk, JD, which acts as mediating sink for carbon via the flux balance between JI, JD and JG at the catalyst surface and hence provides robust conditions for isothermal SLG growth. The bulk of thinner catalysts will quickly saturate throughout with carbon, rapidly lowering JD and leading to FLG formation and inhomogeneous growth as JI ≫ JD. Exposure time to the carbon precursor is another important parameter. For SLG CVD conditions, too short exposures lead to isolated graphene domains, whereas too long exposure leads to the isothermal formation of additional layers even after complete SLG coverage has been achieved. In this context, catalytic graphene CVD is often described as inherently being self-limited, since coverage of the catalyst with SLG will lower JI. However, this only refers to conditions of low chemical potential i.e. low precursor pressures where a window of exposure time exists over which SLG can be uniformly stabilised. For typical catalyst metals it can be shown (see below), that at higher precursor pressures and/or longer growth times additional graphene layers nucleate at the interface between the catalyst and the initial graphene layer fed through intrinsic defects (including grain boundaries) in the initial SLG. This presents a possible pathway for the controlled CVD of bi- and tri-layer graphene films. The effect of catalyst cooling will depend on the amount of carbon in the catalyst bulk. For slow cooling rates, the direction of JD will reverse and assuming sufficient carbon in the catalyst bulk additional graphene layers can form. For fast cooling rates, however, JD will be suppressed and the carbon will remain in the catalyst bulk, i.e. a high cooling rate helps prevent the precipitation of additional layers upon cooling.The insets of 25 and Ni (∼0.19 atom% at 600 °C),26 to a common low carbon solubility catalyst, Cu (0.0007–0.0280 atom% at 1000 °C).28 For the latter it should be noted that while the carbon solubility is relatively low , there is little data available on the diffusivity of carbon in Cu,27 meaning that significant carbon permeation into thicker Cu may be possible, and thus the role of the catalyst bulk still needs to be considered. This is also relevant to growth strategies that target graphene growth at the back interface of the catalyst, i.e. growth at the catalyst-substrate interface or growth based on catalyst foil pockets.32The growth model outlined in 2-supported sputter-deposited films of Co, Cu, and Ni as catalysts, and adopt a relatively simple CVD process24 in which these are heated to growth temperature in a H2-containing atmosphere, exposed to a hydrocarbon precursor , and then cooled to room temperature directly following removal of the precursor . JD ≈ 0, JG ≈ JI) and thus the supersaturation necessary to nucleate the new layer is rapidly reached. In contrast, the red line shows graphene growth evolution for unsaturated catalysts. In this case, the catalyst bulk provides a sink into which carbon arriving at the catalyst surface can diffuse, mediating the SLG formation at the surface (JG = JI – JD). The unsaturated catalyst thus provides a broader processing window, under which SLG can be stabilized before the nucleation of the second layer.We investigate here the growth of graphene on commercially available polycrystalline foils and SiO2(300 nm)/Si show contrast indicative of uniform monolayer graphene coverage,34 and the uniformity confirms SLG formation across large areas on each of the catalysts. This is further confirmed by Raman spectra of the graphene grown on the Ni, Co and Cu foils (–1 and I2D/IG ratio >2), with 2D peaks well fitted with single Lorentzians all with fwhm of ∼35 cm–1, and with I2D/IG ratios of 2.8, 3.7 and 3.0 respectively. The ID/IG ratios, which relate to defects within the graphene lattice, are all low with values of Cu (7%), Co (8%) and Ni (10%) and thus indicative of high graphitic quality. We note that the growth conditions under which this uniform SLG is achieved vary greatly for the different catalysts in terms of the temperature, precursor pressure, and exposure time which will be discussed in more detail below.To demonstrate these two scenarios, we grew graphene on polycrystalline films and foils of Co, Cu and Ni following the first-order framework we have developed. We first focus on graphene growth on foils. Cu foils  which sh2(300 nm)/Si, optical micrographs of the graphene grown on Co  or an increase in precursor partial pressure (step 3) leads to the nucleation and growth of additional layers beneath the existing SLG37 resulting in FLG as shown in 38Despite the demonstration here that SLG can be stabilized across different transition metal catalysts with a broad range of carbon solubilities, we note that FLG can also be produced on these same catalysts under different conditions, even on Cu for which a self-limited growth behaviour is often suggested in the literature.–6 mbar for 15 min followed by an increase in pressure to ∼10–5 mbar for 5 min. On varying the exposure temperatures from 400 °C to 800 °C , we observe a general improvement in graphitic quality with increasing exposure temperature. At higher temperature however, the improvement in graphene quality is accompanied by the formation of additional graphene layers with inhomogeneous FLG coverage observed , as well as by diffusion into or out of the catalyst bulk.41 Typical graphene CVD processes adopt a pre-treatment phase to reduce carbon contamination within the catalyst and thus provide a more defined starting point prior to growth. The gas-phase supply/removal of carbon is complex, affected by parameters such as temperature, catalyst activity, precursor/etchant chemistry, pressure and the boundary layer that can be present for growth conditions with higher total pressures.49 Nevertheless, the growth atmosphere is typically adjusted to deliver a net flux of carbon to the catalyst surface, with graphene growth at the catalyst surface (JG) fed by the balance between this gas-phase supply (JI) and diffusion into the catalyst bulk (JD). Although different hydrocarbon precursors are selected here for each catalyst , this is to ensure sufficient catalytic dissociation at the chosen growth temperature whilst avoiding undesired pyrolytic dissociation, and is not observed to significantly alter the growth evolution otherwise. For graphene grown on Cu, CH4 is mixed with Ar and H2 during the exposure period. An inert buffer gas (e.g. Ar) is used in conjunction with CH4 because it allows the dilution of carbon required without the need for stringent pumping . H2 is expected to participate more actively in reactions occurring at the catalyst–gas interface and it has been suggested to play a key role in limiting the formation of copper oxide at the catalyst surface,50 helping to activate surface bound carbon needed for growth51 and promoting the desorption/etching of small active carbon species.51 The detailed role of the CH4/H2 balance in the growth reactions has been discussed in previous reports, on the basis of adsorption modelling52 and thermodynamic analysis.53On the basis of our results and existing literature, we now rationalize the observed growth behaviour and develop a first-order model for graphene CVD on catalyst surfaces that can be applied generally to transition metal catalysts. We focus on isothermal growth, as despite the non-negligible carbon solubility of Ni and Co, our extensive JI = JD, and the local concentration of carbon at the surface increases until it reaches the catalyst's solubility limit and a local supersaturation (Δc) develops. Graphene islands subsequently nucleate and grow with continuing exposure (JG = JI – JD) until they eventually impinge on one another to form a complete graphene layer with grain boundaries and defects where the graphene grains merge.36 Despite the graphene coverage, carbon continues to be supplied to the catalyst surface through leakage pathways such as defects and grain boundaries, causing the carbon concentration at the catalyst surface to again increase until additional graphene layers nucleate , the catalyst thickness (l) and its permeability (P). The relation between these parameters is explained in detail by Weatherup et al.19 and it provides a lower bound for the thickness required to avoid catalyst saturation prior to complete SLG coverage given by:Whether or not a catalyst becomes saturated with carbon throughout its thickness prior to complete SLG coverage depends on the rate at which carbon is delivered to the catalyst  can also account for the different behaviour of Cu in comparison to the Ni and Co, when the catalyst thickness is reduced (12 atoms per m per s and Co at 700 °C ∼2 × 1013 atoms per m per s),54 based on the values of diffusivity available in literature,57 and indeed both catalysts show a similar transition from uniform SLG formation to inhomogeneous FLG formation as the catalyst thickness is reduced. There is little data available on the diffusivity of carbon in Cu,27 and reported values of carbon solubility at 1000 °C vary between 0.0007 atom% and 0.0280 atom%,28 however the lack of the transition from uniform SLG formation to inhomogeneous FLG formation indicates Cu has a significantly lower carbon permeability under these conditions. We therefore suggest that the broad processing window for SLG formation on Cu is related to this low permeability , and is not solely the result of the low carbon solubility of Cu. We note that catalyst alloying provides new opportunities for rational catalyst design by allowing properties such as permeability to be tuned to obtain a desired outcome by matching the catalyst with the growth process.This inequality indicates that a low value of  islands . For thi reduced . Whilst 60 The increase in graphene domain sizes at higher growth temperatures, and improved graphitic quality, is thus attributed to the increase in surface diffusivity with temperature.41 We further note that increased catalytic dissociation and increased probability of defect healing may also contribute to this improvement in quality observed for higher growth temperatures.61 This might suggest that higher growth temperatures are in general preferable, however this must be balanced against the increased likelihood of saturating the catalyst throughout with carbon due to the increase in permeability with temperature, as revealed by the inhomogeneous FLG growth observed at 800 °C on Co  without compromising quality.19 Further reductions in growth temperature to those compatible with back-end CMOS integration (≤450 °C) can be achieved with Au–Ni alloy catalysts, whilst still maintaining reasonable graphene quality.62The improvement in graphene quality observed with increasing growth temperature Fig. S1 can also Fig. S1i. The pot31 as schematically indicated in 63The catalysts considered herein , all present simple bulk phase diagrams for the growth temperatures used, consisting of only graphite and a metal-carbon solid-solution, without the involvement of other bulk intermediate phasesin situ observations of isothermal growth, nor by the extent of FLG formation we observe. Instead, our results indicate a distinction in growth behaviour based on whether a catalyst becomes saturated with carbon throughout its thickness during the growth process. For the conditions at which we obtain high-quality and uniform SLG on Co, Ni, and Cu, we remain in a regime in which the catalyst is not filled with carbon throughout its thickness. The catalyst bulk thus provides a sink into which carbon arriving at the catalyst surface can diffuse, mediating the SLG formation at the surface. For conditions at which the Cu, Ni and Co catalysts become saturated with carbon throughout, the undesired formation of inhomogeneous FLG is instead observed, although the low permeability of Cu leads to a particularly broad window of processing conditions over which SLG can be stabilized.In summary, we have developed a first-order model for graphene growth on transition metal catalysts with which we rationalise our systematic CVD calibrations for high-quality uniform SLG on Co, Ni, and Cu. We thereby identify key CVD process parameters  that must be adjusted to achieve the desired outcome based on consideration of the catalyst properties  and the kinetics of growth. SLG and FLG formation on all of these catalysts occurs predominantly at temperature during precursor exposure  for the process conditions adopted herein, rather than by precipitation on cooling. The simple distinction previously made in literature between catalysts with low carbon solubility, where surface segregation/adsorption of SLG is proposed to dominate, and higher carbon solubility catalysts, where FLG formation by precipitation on cooling is assumed, is therefore not supported either by our 66 and to provide a framework for the rational design of catalysts and processes for achieving graphene with properties tailored to specific applications. We expect this to be important in the design of growth strategies to obtain a desired growth outcome on a specific catalyst material, and/or where constraints are placed on the process conditions that can be used, e.g. direct integration into device structures.10The understanding developed herein provides important insights into the CVD of graphene on transition metal catalysts. Given the general nature of these insights, we expect them to be relevant to a range of different catalyst materials including alloys,2 and commercially available polycrystalline 25 μm Ni(99.99% purity), Cu(99.999% purity) and Co(99.95% purity) foils are studied.Graphene is synthesized by chemical vapour deposition in a custom-built cold-wall reactor for Co and Ni catalysts whilst a commercially available Aixtron BM Pro (4 inch) machine is used for graphene growth on Cu. Polycrystalline sputter-deposited films  on SiO2 (1 mbar) heating at ∼300 °C min–1 to the growth temperature. The chamber is then quickly pumped down and once the base pressure ∼10–6 mbar is reached, samples are exposed to a hydrocarbon precursor [C2H2 with pressures in the range 10–6–10–3 mbar for 5 s to 180 min] and subsequently cooled down to room temperature in vacuum at ∼100 °C min–1.For graphene grown on Co and Ni, samples are annealed for 15 min in H2/Ar (50 sccm/200 sccm) heating at ∼100 °C min–1 to the growth temperature. CH4 diluted 0.1% in Ar is then introduced to the chamber for 180 min promoting growth under a CH4/H2/Ar (12 sccm/26 sccm/250 sccm) atmosphere and finally cooled down to room temperature in Ar.For graphene grown on Cu, samples are annealed for 30 min at 250 mbar in a mixture of Hex situ using scanning electron microscopy . When imaging graphene with secondary electrons (SE), it generally appears darker than the catalyst surface due to the low generation of SE in graphene.67 The greater the number of graphene layers the darker they appear. Electron channelling contrast  can also be seen from the variations in contrast within the graphene regions in SEM images. Optical microscopy and Raman spectroscopy  are performed after graphene is transferred to SiO2(300 nm)/Si substrates and corroborate the SEM results.As grown graphene is characterised 3 or 0.5 M (NH4)2S2O8 aqueous solutions are used as etchants and for graphene grown on Co 10 M HCl acid is used. Graphene synthesized on Ni catalysts is transferred using an electrolysis-based bubbling technique in an aqueous NaOH (1 M) solution.68 The graphene/polymer film is rinsed in DI water and then transferred to SiO2(300 nm)/Si substrate. The polymer support is subsequently removed by immersion in a suitable solvent [acetone for PMMA and ethyl acetate for PS] followed by a bath in IPA and drying with an N2 flow.Graphene transfer is performed by depositing polymer supports [polymethylmethacrylate (PMMA) or polystyrene (PS)] onto the graphene/metal-catalyst sample. Wet etching techniques are employed to remove the metal. For graphene grown on Cu, 0.5 M FeCl