Although stochastic and deterministic processes have been found to jointly shape structure of natural communities, the relative importance of both forces may vary across different environmental conditions and across levels of biological organization. We tested the effects of abiotic environmental conditions, altered trophic interactions and dispersal limitation on the structure of aquatic microfauna communities in Costa Rican tank bromeliads. Our approach combined natural gradients in environmental conditions with experimental manipulations of bottom-up interactions (resources), top-down interactions (predators) and dispersal at two spatial scales in the field. We found that resource addition strongly increased the abundance and reduced the richness of microfauna communities. Community composition shifted in a predictable way towards assemblages dominated by flagellates and ciliates but with lower abundance and richness of algae and amoebae. While all functional groups responded strongly and predictably to resource addition, similarity among communities at the species level decreased, suggesting a role of stochasticity in species-level assembly processes. Dispersal limitation did not affect the communities. Since our design excluded potential priority effects we can attribute the differences in community similarity to increased demographic stochasticity of resource-enriched communities related to erratic changes in population sizes of some species. In contrast to resources, predators and environmental conditions had negligible effects on community structure. Our results demonstrate that bromeliad microfauna communities are strongly controlled by bottom-up forces. They further suggest that the relative importance of stochasticity may change with productivity and with the organizational level at which communities are examined. Ecologists have long sought to understand which factors generate the variation within and among communities that is so ubiquitous in nature. Deterministic, niche-related processes such as environmental filtering , competiEcologists have yet to determine under which conditions, in which environments or for which organism groups particular structuring forces or their combinations prevail –13. Partin situ manipulations of community assembly. We focus on the microfauna communities in bromeliads, including all non-bacterial, non-viral microscopic organisms such as flagellates, ciliates, amoebae, algae and predatory microscopic metazoans such as rotifers and copepods. In nature, these communities are subject to varying environmental conditions within and among bromeliads such as water temperature and light regimes. Furthermore, resource input may vary strongly and the abundance of organisms at higher trophic levels may differ among bromeliads, resulting in differential strengths of bottom-up and top-down control on the microfauna. Dispersal is assumed to act as a neutral driver of community assembly since microfauna are mainly wind-, water- and vector-dispersed [In this study we use a natural system that can be relatively easily manipulated to conduct field tests of three deterministic structuring forces  and one stochastic force  identified as potentially important by more controlled but simplified systems . Tank brispersed  and diffHere, we examine the influence of environmental conditions, resources, predation and dispersal on bromeliad microfauna community structure. After homogenizing natural communities to exclude potential priority effects, we measured environmental variables and then explicitly manipulated resource input and predator presence in a field experiment. Because a spatial signal in community structure can result from effects of spatially correlated environmental variables as well as from dispersal limitation, we experimentally manipulated dispersal at local (among leaf axils) and regional spatial scales (among bromeliads). We then assessed effects of our experimental treatments by examining changes in the structure of microfauna communities consisting of five major functional groups .We hypothesized that:If deterministic processes dominate, then community structure will only respond to differences in environmental conditions and to the manipulation of species interactions such as resource competition and predation.If dispersal as a stochastic process dominates, then community structure will change as a response to the experimental manipulation of dispersal at both, local and regional scales.If deterministic processes and dispersal both contribute to community structure, then the experimental increase in regional dispersal rates (among bromeliads) should result in a much greater change in community composition than an experimental increase in local dispersal rates (within bromeliads). This is because distant sites will develop more different communities, through differences in environmental factors and species interactions, than communities within the same region, exposed to more similar conditions.http://www.acguanacaste.ac.cr), north-western Costa Rica under research permit N° ACG-PI-028-2010 . The station is situated at an altitude of 700m and is surrounded by primary and secondary tropical rainforest and horse pastures. Bromeliads occur in all of these habitats. We used three dominant tank bromeliad species: Werauhia sanguinolenta  J.R. Grant, W. gladioliflora (H. Wendland) J.R. Grant and W. kupperiana (Suessenguth) J.R. Grant and initiated the experiment after the first strong rainfall of the wet season.This study was carried out in April and May 2010 at the Estación Biológica Pitilla  in the Área de Conservación Guanacaste : 1.37±0.05m), in three habitats varying in canopy openness in order to include a large range in environmental conditions: secondary forest (relatively closed canopy with low light conditions), mixed habitat (mostly edge of the forest with mixed light conditions) and pasture (mostly open canopy with high light availability). We mixed water collected from 44 bromeliads growing in the same three habitats near our experimental bromeliads to include the same species of inhabiting organisms but homogenize the initial composition of experimental communities. We used a suction device with a tube to extract the water from the bromeliads after careful mixing to make sure all organisms were suspended in the water. With a filter with a mesh size of 850μm we removed large detritus particles and macroscopic organisms such as insect larvae (we checked for insect larvae emerging from eggs throughout the experiment and removed them instantly). Then, we added 35ml of this water, containing a mix of algae, protozoans and microscopic metazoans (hereafter collectively referred to as microfauna), bacteria, fungi and small detritus particles, to 50-ml falcon tubes. We placed 243 tubes containing the experimental communities into 27 bromeliads: nine bromeliads in each of the three habitats, nine tubes per bromeliad . The experimental communities in the predator-addition treatment each received one large  and two small  Culex jenningsi Dyar&Knab mosquito larvae. Those numbers represent average natural densities in bromeliads at the field site. Control communities did not receive any additions. All tubes were then covered with fine mosquito mesh to prevent insects from ovipositing and to monitor emerging adult mosquitoes. To keep predation pressure relatively constant over the course of the study, mosquito larvae were replaced whenever they pupated and emerged as adults. We could not observe the number of larvae during the study without disturbing the community, so we could not replace larvae that had died. However, this number was relatively low and all tubes in the predator treatment still contained mosquito larvae at the end of our study. The tubes were placed into separate leaf axils in the bromeliad with no water exchange between the tube and the bromeliad.The 27 experimental bromeliads were divided into three “trophic” treatments: control, increase of bottom-up effects by resource addition, addition of top-down effects by predator addition. For the resource-addition treatment, we collected leaf litter from the forest floor, chopped the material into 1-cmWe nested a hierarchical dispersal manipulation within each trophic treatment in each bromeliad. In each bromeliad, three of the nine experimental communities served as manipulations of “regional dispersal” between bromeliads, i.e., the removal of any dispersal limitation at the regional scale. After gently mixing the contents of the tube, we extracted 1 ml of water from each of the three experimental communities in each of the three bromeliads of the same trophic treatment in the same habitat. We then mixed the resultant 9 ml in a common vial and redistributed 1 ml of this mixture into each regional dispersal community. Manipulations of “local dispersal” within bromeliads  were treated similarly but only the 3 ml from the three “local” communities within a bromeliad were mixed. In three no-dispersal “control” communities we pipetted 1ml out of each community and placed the same liquid back into the tube to control for disturbance from pipetting. In these control communities, natural dispersal was the only means of exchange between communities, so patterns of dispersal limitation were allowed to develop over the course of the experiment. We performed the dispersal manipulations every four days over the four-week experimental duration.At the start of the experiment, we measured the key structural variables that we expected to influence microfauna communities. These were bromeliad diameter, number of live leaves (as a measure of habitat complexity and of light availability in each leaf compartment), height above the ground, natural water content of the bromeliad  and natural water content of each experimental leaf axil (in ml). Canopy openness was estimated by taking a photograph of the canopy above each bromeliad plant using a 35-mm lens and calculating the proportion of sky by pixel count. At the end of our study after four weeks, we measured pH, water temperature, volume, dissolved oxygen and turbidity in each tube to further describe the environment that the microfauna communities were experiencing. However, since these parameters can change across time and even within a day, their measurement at the end of the study only represents a snapshot of the environmental conditions and only large effects will be picked up by the analysis. We then collected microfauna samples from all 243 experimental communities and fixed them with acid 5%-iodine Lugol’s solution. The fixed samples were transferred to the University of British Columbia  where all organisms were counted under an inverted microscope (in 50-μl subsamples and 200x magnification). We used and expanded a photographic identification key developed for the microfauna living in bromeliads at our field site  to identWe analyzed the effects of resource addition, predation and dispersal on the overall abundance and species richness  and on the abundance and richness of the main functional groups. All abundance data were natural-log-transformed to achieve normality and homogeneity of variance. This conservative approach removes samples with zero abundance from the analysis. We repeated all analyses after adding a value smaller than the smallest recorded density to the data before log transformation. These additional analyses showed the same results and are therefore not presented.nlme [vegan [To account for the hierarchical error structure we analyzed abundance and richness with mixed effects models using the function lme in package nlme  in R, venlme . These ae [vegan . We addie [vegan . SpatialWe used multivariate analyses to test for the effects of canopy openness, temperature, trophic treatment and dispersal on community composition . Our resTo explore effects of varying alpha diversity (richness) on differences in community composition ,25,26 weWe counted a total of 62 species of protozoa and micrometazoa in our experimental communities and classified them into five major functional groups: algae (11 species), flagellates (17 species), ciliates (11 species), amoebae (11 species), predatory microfauna .Overall, our trophic treatments had strong effects on microfauna abundance , but lar2,23 = 0.267, P = 0.768 and F2,22 = 1.520, P = 0.241, respectively), suggesting that differences in abundance were the main cause for the richness differences in these groups. Differences between trophic treatments in the other groups remained significant after rarefaction, indicating that changes in abundance did not completely explain effects of trophic treatments on the richness of algae, flagellates and predatory microfauna.Diverging patterns emerged for individual functional groups  and 3. AOnly two environmental variables had significant effects on the experimental communities: canopy openness and water temperature. The other environmental variables were removed from further analyses. Canopy openness and water temperature both showed a spatial signal . Canopy openness influenced overall microfauna abundance  with higThe dispersal manipulation had no effect on abundance or richness of the combined microfaunal community, or of any of the functional groups Tables  and 2. I2,234 = 1.8099, P = 0.005 for linear effects, F4,232 = 2.8058, P = 0.005 for the four selected PCNM vectors) but this did not qualitatively affect any results. In the following section we present only results from analyses in which we removed the spatial trends.The spatial arrangement of the communities had an influence on their composition . This means that communities receiving added resources were more dissimilar from each other (higher beta diversity) than control communities and communities with predators. Communities with different dispersal manipulations did not differ in their community composition . We detected a small effect  may define the relative importance of deterministic and stochastic processes.Bottom-up control was the strongest and most prevalent effect driving several community characteristics, irrespective of differences in environmental conditions or experimental dispersal levels. Adding resources in the form of leaf litter increased overall microfauna abundance, decreased rarefied richness and shifted species composition. Abundances of certain fast-growing species were boosted strongly by the additional resources while overall species incidences remained unchanged, resulting in communities with less species per number of individuals. The decrease in alpha diversity with increased resources agrees with studies from grasslands  where stIn our study, resource addition also shifted functional group composition of microfauna communities. Flagellates (the most abundant group) increased most strongly as a response to resource addition, driving overall abundance patterns. Flagellates have short generation times  and haveUnexpectedly, only flagellate abundance increased with predator presence while overall microfauna abundance and the abundance of other groups did not change. Flagellates might have benefited from predators through a reduction in competition or predation by other groups possibly arising from size-selective feeding by mosquitoes , but morMecistogaster modesta Selys [It is not clear why the other groups did not change in response to the predators. Although we mimicked average natural densities of mosquitoes in bromeliads in the area , ourta Selys ,37. Moreta Selys ,39. For Deterministic processes: environmental conditionsOnly canopy openness and temperature had effects on community structure, and those were entirely due to high-temperature or high-canopy openness outlier communities. In a parallel observational study a number of habitat variables also influenced the structure of natural microfauna communities , however, explained variance was relatively low .a, water volume and water colour [The microfauna communities in Brazilian coastal bromeliads also show significant, but weak (17% of the variance) correlations with environmental factors, notably temperature, chlorophyll-r colour . One potWhile we detected a spatial signal in community composition, its inclusion in the models did not change the strong influence of the trophic treatment or the lack of dispersal effects. Furthermore, we did not find an influence of spatial arrangement on abundance and richness, and dispersal treatments had no effect on community structure. Instead, communities appeared to maintain their initial composition over the duration of the experiment, with most species being present at low abundance and extinctions being rare. We considered dispersal limitation a stochastic factor. However, had it affected only certain species, so operating in a deterministic fashion, we would similarly have seen changes in community composition following our dispersal manipulations.Environmental differences between bromeliads in the field did not lead to strong species sorting in our experiment, although natural bromeliad communities differ from each other . One interpretation of our results is that by homogenizing microfauna communities we overcame any priority effects of dispersal limitation that underlay these natural community differences. Whereas dispersal limitation has been detected in pitcher plant microfauna , and lowOur study of species-rich natural communities strengthens the results from more controlled experimental mesocosms  by showiCertain species benefitted from resource addition more than other species but the identity of those dominant species varied among replicate communities. This increase in stochasticity with increased resource availability has been shown previously from grasslands, where beta diversity of plant communities was positively related to nitrogen enrichment  or site In contrast to the increased stochasticity at the species level with higher resource availability, our study shows that at the functional-group level, shifts in composition towards communities dominated by flagellates and ciliates were strong and predictable. The general pattern emerging from this and other studies is that not only are stochastic and deterministic forces jointly structuring communities  but we aS1 Supporting Information(DOCX)Click here for additional data file.


The high ratio of pyridinic and pyridone N-doped graphene sheets have been synthesized by functionalizing graphene oxide (GO) with different oxygen groups on its surface. The typical N-doped graphene was determined to be ~3–5 layers by transmission electron microscopy (TEM) and atomic force microscopy (AFM), and the nitrogen content was measured as 6.8–8 at. % by X-ray photoelectron spectroscopy (XPS). The structure of the N-doped graphene with different surface functional groups was characterized by Raman spectroscopy. The research result indicates that the carboxylation of GO is the key factor to obtain pyridinic and pyridone N types during the N atom doping process. Compared to general N-doped graphene, the electrochemical test shows that specific capacitance of the GO-OOH-N sample reaches up to 217 F/g at a discharge current density 1 A/g and stable cycling performance (keep 88.8 % specific capacitance after 500 cycles at the same discharge current density) when applied to the supercapacitor electrode materials.The online version of this article (doi:10.1186/s11671-015-1031-z) contains supplementary material, which is available to authorized users. Graphene is an allotrope of a new-type carbon with two-dimensional carbon atoms tightly packed into honeycomb lattice . The speBy doping nitrogen into graphene, the bandgap can be opened, which makes it possible to adjust the conductivity type and the electronic structure of graphene; thus, the physical and surface performance of graphene can be modulated , 11, 12.sp2-hybridized carbons will hinder the N atoms doping into graphene lattice, the quaternary-type N is difficult to form, and often with lower N content or even the failure of doping [In general, the graphene and nitrogen atoms could be combined together in four ways –21. Consf doping . Oxidizef doping , fuel cef doping , 25, andf doping .Supercapacitors are new energy storage device, and they have many advantages attracting tremendous attentions, such as high power density, excellent cycling stability, and fast charge/discharge capability . GraphenThe effect of N doping types of graphene on supercapacitor performances has been previously investigated , 29, 30.In this paper, we obtained the N-doped graphene with different N types and content by adjusting the oxygenic functional groups on the graphene surface. As a result, the graphene with a high percentage of both pyridinic and pyridone N was obtained from carboxylic graphene oxide. The graphene with designed N types exhibits superior capacitance and cycling performance in the electrochemical tests, suggesting that special oxygen functionalization of graphene surface is a promising method to synthesize N-doped graphene with optimized N-doped types for high-performance supercapacitors.4 , N2H4·H2O , H2SO4 , H3PO4 , HCl , H2O2 , C2H5OH , ClCH2CO2H , NH3·H2O (30 %), and home-made secondary deionized water. All medicines were not treated with further purification.Materials used were as follows: natural flake graphite , KMnOλ = 690 nm), and Nanoscope IIIa controller atomic force microscope (AFM).All the graphene and N-doped graphene sheets were characterized with the JOL2010 transmission electron microscope , PHI-5400 X-ray photoelectron spectroscopy (XPS), Rxn1-785 Raman spectrometer (VH2SO4:VH3PO4 = 9:1). Then 18 g of KMnO4 was slowly added. The temperature was reached to 50 °C and kept at the temperature for 60 h accompanied by magnetic stirring. Finally, the solution was stewed and cooled down to room temperature. After that, deionized water of 300 ml was slowly added for dilution, and 10 ml of H2O2 and 100 ml of HCl were added to remove impurities. Then a large amount of distilled water was used to make washing and centrifugation, which were repeated for several times until the pH of the solution was neutral. The brown precipitate was frozen and dried for preservation.Three grams of natural flake graphite was added into 400 ml of ice-mixed acid (2CO2H (7.5 g) were added, and the solution was stirred for 3 h. After the reaction was complete, the solution was washed repeatedly to remove impurity ions until the neutral state was reached. Vacuum drying was conducted at 60 °C to harvest GO-OOH.The as-prepared GO (0.5 g) was dispersed in 250 ml of distilled water using ultrasonic equipment. Yellow transparent colloidal solution was regained. Then NaOH (10 g) and ClCH3·H2O. Then 20 ml of N2H4·H2O solution was added followed by stirring for 30 min. The solution was then transferred into the reaction vessel and kept for 5 h at 130 °C, and black product was obtained. Centrifugal washing was performed for several times with distilled water and ethanol. N-RGO was finally obtained by vacuum drying at 60 °C.Twenty-five milliliters of GO solution (6 mg/ml) was taken, and its pH was adjusted to 10 by NHThe process for preparation is the same as GO-N, except that the react temperature is 180 °C.The process for preparation of GO-OOH-N is the same as GO-N, except that the precursor solution GO is replaced by GO-OOH.N,N-dimethylformamide(DMF) until they formed a slurry with the proper viscosity, and then the slurry was uniformly laid on a piece of Ni foam about 1 cm2 that was used as a current collector and then dried at 80 °C for 2 h. The Ni foam coated with the composite was pressed for 1 min under 1.0 MPa and dried at 120 °C for another 12 h. A Pt electrode and Ag/AgCl electrode filled with saturated KCl aqueous solution were used as the counter electrode and reference electrode, respectively. Cyclic voltammetry (CV), galvanostatic charge/discharge, and electrochemical impedance spectroscopy (EIS) were measured on a CHI760E electrochemical workstation in a three-electrode system. The specific capacitance is calculated according to equation C = It/V, where I is the charge/discharge current density (A/g), t is the discharge time (t), and V is the voltage (1 V). The reported specific capacitance and energy density are all normalized to the weight of sample.A mixture containing 80 wt % active materials (3 mg), 10 wt % acetylene black, and 10 wt % poly-tetrafluoroethylene (PTFE) was well mixed in Figure −1 and D band near 1350 cm−1, representing the degree of crystallization and the degree of disorder or defect in the samples, respectively. The former is caused by in-plane stretching vibration under the Eg2 mode, and the latter is caused by the scattering of phonons at the boundary of the disordered hexagonal Brillouin zone [ID/IG = 0.14). After oxidization treatment, the crystallization degree of GO decreased obviously due to the oxygen-containing groups induced. For the carboxylic graphene, broadening of D band indicates more disorder of the structure, which is related to the increase of oxygen content and the size reduction caused by bond breaking in a transition from C-O-C to -COOH. By nitrogen doping and reduction treatment, N-doped graphene samples were obtained, with a small reduction in ID/IG value (from 1.08 to 1.01). This is the result of the combined action of several factors: (1) reduction of oxygen-containing groups led to decrease of ID/IG value; and (2) more structural defects of graphene caused by N doping would increase the value of ID/IG; the ID/IG values of GO-N, GO-N-180, and GO-OOH-N were similar, indicating close defect density of the N-doped samples but different defect type and distribution, as confirmed by XPS results.Figure uin zone . It can sp2 C-C peak was enhanced. The possible reason is that the carboxyl groups with high binding energy were introduced to the edge of graphene, while the epoxyl groups were distributed inside in-plane [As an effective tool to characterize elemental contents and bonding states in the samples, XPS technique can be used to understand the changes in oxygen and nitrogen content and their existing forms in the samples before and after reaction. Figure in-plane . The redAfter nitrogen doping, C1s peak of all NG samples shows only one main peak at 284.6 eV and can be divided into three sub-peaks centered at approximately 284.6, 286, and 289 eV , constant current (A), discharge time (s), potential window (V), and mass of the active material (g), respectively. As shown in Fig. + and N atoms, giving rise to abundant electrolyte ions occupied on the electrode surface [2/g, respectively. The pore size and distribution are close for both samples , indicating that this markedly enhanced capacitance ability of GO-OOH-N ought to be attributed to the high N content and favored N structures benefiting from an artificial carboxylation on graphene oxide precursor instead of pore structure difference. Unexpectedly, the Csp of GO-OOH-N undergoes a visible drop from 1 to 2 A/g and reaches to a lower capacitance level compared to that of GO-N; this observation may be elucidated by the instability of the pseudocapacitive redox pairs at high current density [The specific capacitance of the electrode is obtained from the equation: Csp =  surface . Clearly density , which c density , 28.The impedance analyses of all products are measured in the frequency range of 100 kHz–0.01 Hz at open circuit potential to evaluate the kinetic and fundamental behavior of electrode materials and are shown in Fig. Figure In this article, the changes of N types in graphene were investigated by using different functionalized graphene oxide precursors under varied experimental conditions. The research result shows that low temperature was favorable for the generation of pyridone nitrogen, while high temperature was beneficial to the generation of pyridinic nitrogen. When GO-OOH was used as the precursor, the presence of carboxyl groups was favorable for the both generations of pyridinic nitrogen and pyridone nitrogen. The GO-OOH-N shows the highest capacitance, and an inferior of GO-OOH-N to GO-N in rate capability at high current density convinced the instability of pseudocapacitance in the fast charge/discharge process. Moreover, GO-OOH-N also showed an impressive specific capacitance and cyclic stability after 500 cycles at a discharge current density 1 A/g. These observations demonstrate that the regulation of nitrogen structures in graphene could be achieved by the well-controlled oxygen groups on graphene surface. We believe that the present way is beneficial for the supercapacitor applications or other fields.


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