Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution.We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes.The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality. Spliceosomal introns are associated with a well-defined splicing machinery in the nuclear genomes of all characterized eukaryotes, but the origin of these introns remains a puzzle Protopterus aethiopicusFishes are the largest and the most diverse group of vertebrates that exhibit high level of biodiversity in their morphology, physiology, ecology and behaviour There are five different melanocortin receptors (MCR), melanocortin 1 receptor (MC1R), MC2R, MC3R, MC4R and MC5R, which have been reported from different tetrapod genomes. Taken together, they constitute the MC receptor protein family within the G-protein coupled receptor (GPCR) superfamily.The different MC receptor subtypes have very diverse physiological roles In the current study, we assembled MC receptor orthologs/paralogs from vertebrates and we have demonstrated that fishes have a variable number of MC receptors with two in lampreys to six in zebrafish. Interestingly, the exon-intron structure analysis demonstrates dramatic changes in structures of MC2R and MC5R genes from a group of ray-finned fishes that underwent genome compaction. Upon extending these analyses to other GPCRs, we traced similar patterns of intron insertions in P2Y receptors and CHRM3 gene from these fishes. Furthermore, ultrasmall introns are found in the MC2R gene from selected fishes, indicating such introns exist at least in fishes.table S1. The encoding protein sequences of these MCR genes are aligned as depicted in figure S1. There are five MC receptors namely MC1R–5R, which are present in mammals, birds, and other tetrapods. However, there are only four MC receptors– MC1R, MC2R, MC4R and MC5R in fishes such as Takifugu rubripes, Tetraodon nigroviridis, Oryzias latipes, and Gasterosteus aculeatus, with the exception of Danio rerio where there are six MC receptors with two copies of MC5Rs named as MC5aR and MC5bR and a copy of MC3R-like gene. When we traced for MC receptors in basal fishes, we detected three MC receptors, which are similar to MC1R , MC3R  and MC5R  from the elephant shark  genome. We further detected two MC receptors from draft assembly of lamprey, Petromyzon marinus. These were similar to MCA and MCB receptors, which have been described previously in another species of lamprey, Lampetra fluviatilisBranchistoma floridae (JGI accession id: e_gw.70.133.1).To understand the molecular evolution of any gene family or superfamily, defining the dataset is the critical initial step. In order to define our data set of MC receptors, we carried out similarity searches against different vertebrate genomes see . The detfigure S1). To understand the relationship among MC receptors, we reconstructed phylogenetic trees , O. latipes  and G. aculeatus . In the T. nigroviridis genome, MC2R and MC5R are located on two different chromosomes, chromosome Un_random and chromosome 8, respectively. Since this fragment is present in the closely related pufferfish T. rubripes on a single locus, it is highly unlikely that T. nigroviridis can have MC2R and MC5R on independent loci suggesting that it is a genomic assembly error rather than an evolutionary event. This architecture is maintained in D. rerio  with closely associated MC5bR and MC2R, whereas, MC5aR is localized on chromosome 19 flanked by dyad of marker genes, sperm-associated antigen 1 (SPAG1) and cleavage and polyadenylation specific factor 1 (CPSF1) on one side and vertebrate transmembrane prostate androgen-induced (TMEPAI) protein on the other side .To gain further insight into the evolutionary processes that generated the MC receptors, we compared the genomic locations and the gene orders surrounding the MC receptors. In the human genome, MC2R and MC5R genes are found to be closely linked on chromosome 18, flanked by the RNA (guanine-7-) methyltransferase (RNMT) gene on one side and a dyad of markers, zink finger protein 519 (ZNF519) and neurofibromin 1-like 5 (NF1L5), on the other side . This genomic architecture is maintained in several mammalian genomes such as mouse (chromosome 8), rat (chromosome 19) and opossum (chromosome 1). This synteny is also conserved in birds such as chicken (chromosome 19) and zebra finch (chromosome 11) with an additional marker gene, programmed dead cell 5 (PDCD5). However, we could not detect MC1R gene in turkey genome probably due to the fragmental nature of the present genomic sequence. In the anole lizard genome, MC1R is present on small scaffold_2608, where it is not possible to analyse the surrounding marker genes. However, X. tropicalis genome contains this conserved chromosomal fragment on scaffold_303. Upon searching this genomic fragment in different fish genomes, we found that it is conserved in T. rubripes (scaffold_14), T. nigroviridis (chromosome 5), O. latipes (chromosome 3), G. aculeatus (group II) and D. rerio (chromosome 18).We extended the micro-synteny analysis to other MC receptors in order to gain an insight into their origin. MC1R is located on chromosome 16 in the human genome flanked by Fanconi anemia, complementation group A (FANCA) and transcription factor 25 (TCF25) on one side, and differentially expressed in FDCP 8 (DEF8) and dysbindin (dystrobrevin binding protein 1) domain containing 1 (DBNDD1) on the other side in a 350 kb region . This chromosomal fragment is present in several mammalian genomes such as mouse (chromosome 2: 450 kb fragment), rat (chromosome 3: 470 kb fragment) and opossum (chromosome 1: 700 kb fragment). Surprisingly, even though this fragment is present in chicken genome on chromosome 20 in a region of ∼190 kb. However, the MC3R gene is missing at this locus. To understand this loss of MC3R gene, we searched this genomic fragment in two different bird genomes such as zebra finch and turkey using genome browsers. We found that this syntenic architecture is present in these two birds along with the MC3R gene on chromosome 20 in zebra finch and chromosome 22 in turkey, respectively with fragment size of ∼180 kb in both cases. We detected the MC3R protein sequence from chicken upon similarity searches against GenBank (Accession id: BAA32555.1), indicating the presence of a MC3R homolog in chicken genome, though this homolog is not yet located on the chromosomal map due to sequencing error in current genome assembly. In the anole lizard genome, MC3R is present on small scaffold_168, where it is not possible to analyse the flanking marker genes. However, X. tropicalis genome contains this conserved chromosomal fragment on scaffold_148 in a 280 kb region. On searching this genomic fragment in different fish genomes, we did not find MC3R to be conserved in T. rubripes, T. nigroviridis, O. latipes, G. aculeatus and D. rerio. However, the previously reported MC3R of D. rerio is localized on a separate location characterized by different sets of markers flanking both ends of this MC receptor. This fragment contains the MC receptor homolog flanked by a triad of PTP1B, CEBPB and SRXN1 on one side and a dyad of SLC13A3 and GRID2 on the other side . We investigated the existence of this loci in other ray-finned fishes and we found that this loci is maintained with same sets of markers in other ray finned fishes such as T. rubripes (scaffold_226), T. nigroviridis (chromosome 9), O. latipes (chromofsome 7), and G. aculeatus (group XII). However, this loci has another GPCR, thyrotropin-releasing hormone receptor 3 (TRHR3) as corroborated by homology searches (Ensembl id are listed in table S2). There are two duplicates of TRHR3 in T. rubripes and we named it as TRHR3a and TRHR3b, respectively. Conservation of this loci in different fishes, which is drastically different than the tetrapod MC3R loci, corroborates that it might be a consequence of fish-specific genome duplication event In the human genome, MC3R gene is surrounded by Cerebellin 4 Precursor (CBLN4) gene on one side and a tetrad of markers, AURKA, CSTF1, CASS4 and GCNT7, on the other side in a region of about 550 kb . Other mammals, which are known to carry this fragment, include mouse (chromosome 18), rat (chromosome 18) and opossum (chromosome 3). This fragment is maintained in bird genomes such as chicken (chromosome 2), turkey (chromosome 3) and zebra finch (chromosome 2). The anole lizard genome carries this fragment in scaffold_168; however, we could not localize the CDH20 marker in the current draft version of this genome. MC4R is also found at similar locus in X. tropicalis (scaffold_95). Five analysed fish genomes contain this architecture, T. rubripes (scaffold_96), T. nigroviridis (chromosome 5), O. latipes (chromosome 20), G. aculeatus (group XXI) and D. rerio (chromosome 2).In the human genome, MC4R is localized on chromosome 18 flanked by dyad of markers, CDH6 and RNASEN on one side, and CDH20 on the other side. This genomic organization is conserved from fishes to mammals  and lamprey (jawless vertebrates) remain too fragmentary to get more insights into the micro-synteny architecture of the MC receptor clusters (data not shown). However, when we summarize the orthology assessment of MC receptors, it is noteworthy to mention that there are only four MC receptor orthologs conserved in different fishes. Although Moreover, it is clear that from teleost fishes to human, MC receptors are maintained as such without much expansion or reduction over a period of 450 MYA. Overall, MC receptors are vertebrate-specific genes, since no homologs have been detected with requisite similarities to confirm its existence in invertebrate genomes, as we described above. A recent review also supported this view D. rerio, elephant shark and lampreys, with MC5R and MC2R from four ray-finned fishes being the only exceptions.To elaborate on the mechanism(s) of intron insertions, gene structures of MC receptors from different vertebrates are illustrated in Figure S5). There are three introns inserted at positions 41a, 77c and 140c (full-length human MC5R amino acid numbering with suffix a–c for intron phasing) in MC5Rs of four fishes, T. rubripes, T. nigroviridis, O. latipes, and G. aculeatus. However, these novel introns were not traced in MC5R like genes from D. rerio, elephant shark (C. milii) and tetrapods . We found only one intron inserted at the position 140c (full-length human MC5R amino acid numbering) in MC2Rs of the four fishes, T. rubripes, T. nigroviridis, O. latipes, and G. aculeatus . These novel introns are bordered by canonical GT-AG splice signals, the overall GC content extends from 37 to 55.9%. Two out of the three conserved introns interrupt the open reading frame between codons (phase c), and remaining one after the first nucleotide (phase a), respectively. We did not detect any significant similarities to known complex repetitive elements in these novel introns from pufferfishes. However, we found some repetitive elements in O. latipes, and G. aculeatus. This explains the increase in intron size in these two fishes as compared to the shorter introns from the pufferfishes. All of the novel introns of MC5 receptors interrupt a highly conserved domain of a typical GPCR with introns at positions 41a, 77c and 140c, respectively, without any insertions or deletions . The GC content of novel introns from these MC5R genes is ranging from 37% to 55.9%. In contrast, MC2R has only one conserved novel intron at position 140c with GC content 37.6 to 44%. MC2R from T. rubripes contains an additional intron located at position 230c that in the loop region between helices TM5 and TM6, which is a predominant region of insertion and deletions . Similarly, MC2R from G. aculeatus also contains an additional intron at position 236a again in the loop region between helices TM5 and TM6, albeit closer to the beginning of TM6 region in this case. Both these non-conserved novel introns from T. rubripes and G. aculeatus are exceptionally smaller in size, 18 and 33 bp, and GC content 66.7% and 93.9%, respectively.G. aculeatus and O. latipes. Similar mutations are also found in non-conserved novel introns of MC2Rs from T. rubripes and G. aculeatus. Therefore, with a few mutations at some of the proto-splice sites, the intron insertion sites are largely maintained.Intron insertions have been proposed to occur at preferred locations (C/AAG↑) referred to as proto-splice sites ; and a single intron insertion at position 120c in the P2Y6 receptors . These introns are not found in the P2Y receptors from zebrafish, elephant shark and tetrapods.To find more cases as described above for the MC2Rs and the MC5Rs, we scanned various other GPCRs. Indeed, we found introns, which have been inserted in similar fashion as above in some other GPCRs. Purinergic P2Y receptors, such as P2Y2, P3Y3-like (P2Y3L) and P2Y6 receptors also have novel introns inserted in the genome compacted ray-finned fishes ; one speTakifugu, Tetraodon, stickleback and medaka (blue background), but not in CHRM3 genes from zebrafish, elephant shark and tetrapods , confined only in the large loop between TM5 and TM6.We also found that there are two introns insertions at positions 67a (close to the TM1), 166c  in CHRM3 receptor of four fishes - etrapods . Additiotable S3.We scanned for the proto-splice site is conservation in all these novel introns in P2Y and CHRM3 receptors as done for MC receptors. These receptors with their respective proto-splice sites are listed in Acanthopterygii) from D. rerio (or superorder Ostariophysi) just like the intron insertions described above for the MC2Rs and the MC5Rs, probably via a similar mechanism.Taken together, our data indicate that these are novel intron insertions that occurred after separation of genome compacted fishes  have DRY introns shared from fishes to mammals with evidences of gene duplication in fishes. We also identified a DRY intron in GPR161 gene that has two copies in D. rerio. In contrast, GPR176 has single copy in all of the species with the exception of medaka, which has no GPR176 gene. Two galanin receptors, GALR2 and GALR3, also share DRY intron, but only the GALR2 intron is conserved in all of the species whereas the DRY intron of GALR3 is limited only to mammals. The DRY intron in Gastrin-releasing peptide receptor (GRPR) gene is shared in all of the considered species with evidence of gene duplication in fish genomes. Melanin-concentrating hormone receptor 2 (MCHR2) gene has DRY intron in human and fishes, whereas rodents have no MCHR2 gene. Neuromedin B receptor (NMBR) gene has DRY intron in all of the species. Two Neuropeptide FF receptors (NFFR1–2) also possess this intron in all of the analysed genomes and NFFR2 has gene duplications in fishes. Two prokineticin receptors (PROKR1–2) share this intron in all of the genomes with a single copy of each gene. Prolactin releasing hormone receptor (PRLHR/GPR10) gene has DRY intron only in the fish genomes but not in the mammalian genomes that were taken into consideration. Two serotonin (5-hydroxytryptamine) receptors, HTR4 and HTR7 contain DRY intron in all of the analyzed vertebrates with two copies of HTR7 genes in D. rerio. Three tachykinin receptors (TACR1–3) have a DRY intron that is conserved from fishes to mammals with duplication of TACR2 and TACR3 in different fish genomes. We found that thyrotropin releasing hormone receptor 2 (TRHR2) has a DRY intron in the rodents and all of the fishes with two copies of this gene in D. rerio; however, the TRHR2 gene is missing from the human genome. Until now, the TRHR2 gene carrying DRY intron has not been reported in fishes To gain insight into the repertoire of DRY introns from non-compacted and compacted vertebrate genomes, we carried out further analyses of previously reported other GPCRs that possess DRY introns in vertebrates In summary, we conclude that out of 24 common GPCRs that possess DRY introns 23 are maintained from fishes to mammals with a few exceptions. We observed that duplications in these GPCR genes occur only in the fish genomes. Furthermore, we compared the variation in the codon usage of the DRY intron between non-compacted and compacted genomes of vertebrates and we noted that there was no much variation in the DRY codon usage Figure 6et al. (2007) have also reported that fishes have variable repertoire of these receptors and they further observed that the expression profile of MC1R was different in divergent fish lineages D. rerio does not share genomic organization with MC3Rs of tetrapods, inferring that it might not be an ortholog of tetrapod MC3R. Moreover, zebrafish MC3R branches out earlier than elephant shark MC3R in a highly accurate Bayesian phylogenetic tree  in orthologs of MC5R from a group of selected ray-finned fishes that are found after the split of the D. rerio lineage (or superorder Ostariophysi) from the superorder Acanthopterygii (other four ray-finned fishes belong to this superorder). Similarly, unusual gene structures of MC2R from the same group of ray-finned fishes were also found after diversification of D. rerio from these fishes. Some introns are ultrasmall in size as found in case of novel insertion at position 230c and 236a in MC2R from T. rubripes, and G. aculeatus, respectively. Such small introns are rare; however, it cannot be completely excluded as the T. rubripes genome has some smaller introns In different studies, unusual exon-intron boundaries of MC5R and MC2R have been reported previously in pufferfishes C. intestinalis, C. savignyi, C. elegans) and Fridmanis et al. (2007) suggested that the DRY intron may have been inserted several times independently in different GPCRs Acanthopterygii. Consequently, our data support that these introns are recently inserted in these selected GPCRs after separation of D. rerio from other ray-finned fishes. Fridmanis et al. (2007) also briefly suggested that the DRY intron of these two MC receptors are recent rather than ancient MC5R and MC2R from the four fishes share an intron at position 140c Figure 4We scanned updated genomes of vertebrates for differentiating DRY intron repertoire of non-compacted versus compacted genomes. We upgraded the existing catalogue of the DRY introns shared among different vertebrates We surveyed DRY motifs with/without an intron in different GPCRs and some examples are given in Acanthopterygii. The emergence of introns can be explained by several mechanisms such as involvement of transposons Previously reported cases of novel introns in vertebrates are also associated with the diversification of these fishes Daphnia pulex genome The origin of these newly acquired introns remains open for investigations. Largely, due to the fact that during our searches using different homology search suites, these novel introns do not have significant homology either to the flanking sequences within the locus or anywhere else in overall sequenced parts of these fish genomes. Every genome sequencing project faces some problems in sequencing process, which result in unavailability of small proportion of genomic sequences. Therefore, fractions of genomic sequences from these selected fish genomes, if missing, are likely to remain unsequenced. Thus, a remote possibility exists that the novel introns are derived from some unsequenced portion of these fish genomes. However, this possibility is highly unlikely as these hypothetical unsequenced portions escaped detection in multiple fish genomes. The probability that all the four genomes have similar sequencing errors is extremely low, unless all of them carry some sequence features that make sequencing difficult in these regions. Similarly, the source of introns is unknown for a documented case of strain specific novel intron creation in rab4 gene from There are different types of processes that may be accountable for intron insertions with/without events responsible for the primordial emergence of spliceosomal introns. Proto-splice site is the hotspot for these intron insertions D. rerio, which diverged at a time period closer to the fish-specific genome duplication event Gene level novelties are created either by whole genome duplication or by segmental duplications, which may conceivably lead to the gain of novel introns, as an unaffected gene copy remains within the genome. The appearance of novel introns in the MC5R and MC2R cannot be directly coupled to gene or genome duplication, as none of these introns are found in MC receptors from A recent study of multiple intron gains in serine protease inhibitor superfamily in the lineages of selected ray-finned fishes In summary, a group of ray-finned fishes exhibits multiple intron insertions in selected GPCRs and serpins T. rubripes, T. nigroviridis, O. latipes, and G. aculeatus) whose genomic content is reduced. Such insertion events are absent in genomes of zebrafish, lamprey, elephant shark and other tetrapods. Both these receptors share one intron inserted at common site in conserved DRY motif. We demonstrated that P2Y receptors and CHRM3 also had novel intron gains, suggesting that this mechanism is not restricted only to the MC receptors. Overall, we found that all novel intron insertions are found only in fish genomes, which had undergone reduction in genomic contents close to ∼320–190 MYA.We have compiled a comprehensive catalogue of MC receptors from evolutionary distant vertebrates in this study. With a complete repository of MC receptors at hand, we carried out a study of introns in these genes. We found that introns are inserted in closely linked MC5R and MC2R from a selected set of actinopterygian fishes , G. gallusT. guttataM. gallopavoA. carolinensis, T. nigroviridisO. latipesG. aculeatus, D. rerio and M. domesticaC. milii genome from the Elephant Shark Genome Project Web site  and S. purpuratus genome from the Human Genome Sequencing Center (HGSC), Baylor College of Medicine (http://www.hgsc.bcm.tmc.edu/). All genomes used in this study are listed in table S5.We extracted the genomic DNA/cDNA/protein sequences from different eukaryotes via BLAST suite www.softberry.org). We used human MC5R as the standard sequence for projection of intron position and corresponding numbering of human MC5R is followed by suffixes a–c, indicating intron phasing according to their location after the first, second, or third base of the corresponding codon, respectively as described previously To ensure correct gene structures of all putative GPCR genes, we predicted gene structures using GENSCAN http://www.repeatmasker.org) and RepBase Censor (http://www.girinst.org/censor/index.php) We traced the repetitive elements in novel introns of MC5R and MC2R using Repeat Masker package version 3.2.6  genome. Lampreys L. fluviatilis and P. marinus have two copies of MC receptors named as MCAR and MCBR. There are total 69 protein sequences of MC receptors used in this alignment and all of these MCR have conserved DRY motif (marked as ###) at the end of transmembrane helix 3 (TM3). Seven transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 (PDF)Click here for additional data file.Figure S2Chromosomal mapping of MC1 receptors. This figure illustrates that MC1R ortholog is found conserved from teleost fishes to mammals.(TIFF)Click here for additional data file.Figure S3Micro-synteny analysis of MC3 receptors.A. Ortholog of MC3R is conserved in tetrapods. B. MC3R like gene is found in only zebrafish whereas other ray-finned fishes have another thyrotropin-releasing hormone receptor 3 (TRHR3) gene at this locus instead of MC3R. Takifugu has two copies of TRHR3, which are named as TRHR3a-b.(TIFF)Click here for additional data file.Figure S4Micro-synteny analysis of MC4R genes depicting presence of orthologs of MC4R gene from fish to mammals.(TIFF)Click here for additional data file.Figure S5Intron insertions in MC5 receptor during diversification of ray-finned fishes. There are three intron inserted at positions 41a, 77c and 140c  in MC5Rs of four fishes - Takifugu, Tetraodon, stickleback and medaka, but not in MC5R like genes from zebrafish, elephant shark and tetrapods. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 (PDF)Click here for additional data file.Figure S6Analysis of intron insertions in MC2R from selected ray-finned fishes. There is one intron inserted at position 140c  in MC2Rs of four fishes - Takifugu, Tetraodon, stickleback and medaka, but not in zebrafish and tetrapods. This intron is also conserved in MC5Rs of these selected ray-finned fishes. Takifugu and stickleback has one intron inserted in their MC2R at intron positions 230c  and 236a, respectively. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 (PDF)Click here for additional data file.Figure S7Intron insertions in P2Y2 receptor during diversification of ray-finned fishes. There are three introns inserted at positions 124a, 181c and 267a (human MC5R amino acid numbering with suffix a–c for intron phasing) in P2Y2 receptor of four fishes - Takifugu, Tetraodon, stickleback and medaka (blue background), but not in P2Y2 genes from zebrafish, elephant shark and tetrapods. Residues conserved above 70% are marked by white on black background. ### indicates location of highly conserved DRY motif. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 (PDF)Click here for additional data file.Figure S8Intron insertions in P2Y3-like (P2Y3L) receptor during diversification of ray-finned fishes. One novel intron is inserted at position 187c (human MC5R amino acid numbering with suffix a–c for intron phasing) in the P2Y3L receptor of four fishes - Takifugu, Tetraodon, stickleback and medaka (blue background), but not in P2Y3L genes from zebrafish, elephant shark and tetrapods. There is also species-specific introns in the P2Y3L gene such as S212a for stickleback, M234a in medaka and T252a for Takifugu , localized in the loop between TM5 and TM6. Residues conserved above 70% are marked by white on black background. ### indicates location of highly conserved DRY motif. Gallus P2Y3 receptor was used as it a typical representative of tetrapod P2Y3. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 (PDF)Click here for additional data file.Figure S9Intron insertions in P2Y6 receptor during diversification of ray-finned fishes. One novel intron is inserted at positions 120c (human MC5R amino acid numbering with suffix a–c for intron phasing) in the P2Y6 receptor of four fishes - Takifugu, Tetraodon, stickleback and medaka (blue background), but not in P2Y2 genes from zebrafish, and tetrapods. Residues conserved above 70% are marked by white on black background. ### indicates location of highly conserved DRY motif. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 (PDF)Click here for additional data file.Figure S10Intron insertions in CHRM3 receptor during diversification of ray-finned fishes. There are two introns inserted at positions 67a, 166c (DRY intron or 140c by human MC5R numbering) in CHRM3 receptor of four fishes - Takifugu, Tetraodon, stickleback and medaka (blue background), but not in P2Y2 genes from zebrafish, elephant shark and tetrapods. Various species-specific introns in the large loop between TM5 and TM6 are marked by red background. Residues conserved above 70% are marked by white on black background. ### indicates location of highly conserved DRY motif. In this case, human CHRM3 amino acid numbering was followed as human MC5R was not suitable due to variable size of these receptor proteins with suffix a–c for intron phasing. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 (PDF)Click here for additional data file.Table S1List of MC receptors from selected genomes, identified from Ensembl database release 59 (August 2010). At times data is gathered from additional databases as indicated.(XLS)Click here for additional data file.Table S2List of CHRM3 genes from fishes. Codon usage in DRY motif is shown and codons of the R residue is marked by red color. The absence of intron is marked by grey background in R residue.(DOC)Click here for additional data file.Table S3List of selected GPCRs used in this study, other than MC receptors. Codon usage in the DRY motif is shown and codon usage of the R residue is marked by red color. Proto-splice site forming codons are indicated by bold letters. The presence and the absence of intron is marked by blue background and grey background for R residue respectively.(DOC)Click here for additional data file.Table S4List of GPCRs sharing a common DRY intron from different vertebrates. Vertebrates with compacted genomes are marked in yellow background. Codon usage of DRY motif is shown with | as point of intron insertion. Only mammalian PRLHR gene has no intron, shown in orange background. # Only partial sequence available that renders detection of DRY motif. *Upon BLAT search in Tetraodon Genome browser (http://www.genoscope.cns.fr/externe/tetranew/) using Takifugu NMBR, we obtained putative full length NMBR from Tetraodon localized as fragements on chrUn_random in current assembly of tetraodon genome. @Accession id from Ensembl. $Gene is partial in databases starting from the middle of DRY motif.(XLSX)Click here for additional data file.Table S5List of genomes used in this study.(DOC)Click here for additional data file.File S1Sequences of novel introns inserted into MC receptors of selected ray-finned fishes. Only introns mapping to core MCR domain (TM1–7) are considered. The numbering of intron positions refers to the full length amino acid numbering of human MC5R.(PDF)Click here for additional data file.


Cardiac transcription factors are master regulators during heart development. Some were shown to transdifferentiate tail tip and cardiac fibroblasts into cardiomyocytes. However, recent studies have showed that controversies exist. Potential difference in tail tip and cardiac fibroblast isolation may possibly confound the observations. Moreover, due to the use of a cardiac reporter (Myh6) selection strategy for induced cardiomyocyte enrichment, and the lack of tracking signals for each transcription factors, individual roles of each transcription factors in activating cardiac gene expression in mammalian non-myoblastic cells have never been elucidated. Answers to these questions are an important step toward cardiomyocyte regeneration. Because mouse 10T1/2 fibroblasts are non-myoblastic in nature and can be induced to express genes of all three types of muscle cells, they are an ideal model for the analysis of cardiac and non-cardiac gene activation after induction. We constructed bi-cistronic lentiviral vectors, capable of expressing cardiac transcription factors along with different fluorescent tracking signals. By infecting 10T1/2 fibroblasts with Nkx2-5, Tbx5, Gata4 or Myocd cardiac transcription factor lentivirus alone or different combinations, we found that only Tbx5+Myocd and Tbx5+Gata4+Myocd combinations induced Myh6 and Tnnt2 cardiac marker protein expression. Microarray-based gene ontology analysis revealed that Tbx5 alone activated genes involved in the Wnt receptor signaling pathway and inhibited genes involved in a number of cardiac-related processes. Myocd alone activated genes involved in a number of cardiac-related processes and inhibited genes involved in the Wnt receptor signaling pathway and non-cardiac processes. Gata4 alone inhibited genes involved in non-cardiac processes. Tbx5+Gata4+Myocd was the most effective activator of genes associated with cardiac-related processes. Unlike Tbx5, Gata4, Myocd alone or Tbx5+Myocd, Tbx5+Gata4+Myocd activated the fewest genes associated with non-cardiac processes. Conclusively, Tbx5, Gata4 and Myocd play different roles in cardiac gene activation in mammalian non-myoblastic cells. Tbx5+Gata4+Myocd activates the most cardiac and the least non-cardiac gene expression. Cardiac transcription factors Nkx2.5, Tbx5, Gata4, and Myocd are master regulators during heart development. The deletion of any of these genes results in the failure of normal heart development and cardiomyocyte maintenance We therefore choose the mouse 10T1/2 fibroblast cell line to evaluate the ability of several important cardiac transcription factors to activate cardiac genes. 10T1/2 cells are derived from mouse embryonic cells with fibroblast morphology. Because 10T1/2 cells are non-myoblastic in nature de novo. Tbx5, Gata4, and Myocd contributed differently to cardiac gene activation and non-cardiac gene suppression. The combination of Tbx5+Gata4+Myocd was the most effective activator of genes associated with cardiac-related processes including muscle cell differentiation, sarcomere, striated muscle contraction, and regulation of heart contraction.After infecting 10T1/2 fibroblasts with human Nkx2-5 (N), Tbx5 (T), Gata4 (G), or Myocd (M) lentivirus, either alone or in different combinations, we examined cardiac and non-cardiac  gene activation in these cells. Here we show that only the combinations of T+M and T+G+M can induce Myh6 and Tnnt2 cardiac marker gene expression Tet-on lentiviral vector (pNLTREpitt-EGFP-ΔU3) and Tet transactivator vector (pNLrtTA2sM2) were provided by Dr. Jakob Reiser cis-element reporter (2xTBE-Luc) and its mutant (2xmuTBE-Luc) were provided by Dr. Eric Olson The Tbx5 luciferase GCAAGTGACAGAATGGGCTGCAAGTGACAGAATGGG-3′5′-CT,2xNKE: TATGGCAACCAGGCAGGCTTATGGCAACCAGGCAGG-3′5′-CT,2xmuNKE: GATGGGCAGATAGAGGAGAGACAGGA-3′5′-CAAAGGGCC,GATA site: TCAGGGCATGCAGAGGAGAGACAGGA-3′5′-CAAAGGGCC,muGATA site: CCAAATATGGCTACACCCAAATATGGCT-3′5′-ACAC,2xSRE: -GATCTGCCACTACAC-GATCTGCCACT-3′5′-ACAC.2xmuSRE: cis-element reporters were named 2xNKE-Luc, 2xmuNKE-Luc, GATA-Luc, muGATA-Luc, 2xSRE-Luc, and 2xmuSRE-Luc, respectively. Nkx2-5, Tbx5, Gata4, and Myocd lentiviruses were characterized in CV-1 fibroblasts  by using a dual-luciferase assay system (Promega). For the Myocd lentivirus, Srf expression vector pCGN-SRF or its control vector pCGN (provided by Dr. Robert Schwartz The The 10T1/2 Tet-transactivator cell line (10T1/2-tTA) was established by infecting 10T1/2 cells (American Type Culture Collection) with pNLrtTA2sM2-puro lentivirus, followed by puromycin selection. 10T1/2-tTA cells were then infected with different combinations of the tetracycline-dependent Nkx2-5, Tbx5, Gata4, and Myocd lentiviruses. To induce transgene expression, doxycycline was added at a final concentration of 1 µg/ml.+ or Tnnt2+ cells within a designated multitransgene-positive cell population.10T1/2-tTA cells that were infected with different combinations of Nkx2-5, Tbx5, Gata4, and Myocd lentiviruses were seeded on glass cover slips and grown in Basal Medium Eagle (BME) medium (Invitrogen) supplemented with 1 µg/ml doxycycline, 5% (v/v) horse serum (Invitrogen), and 2 mM L-glutamine. Ten days after doxycycline induction, cells were stained with monoclonal antibodies against Myh6  or Tnnt2 (Abcam) and Alexa Fluor 350-conjugated goat anti-mouse IgG (Invitrogen). Images were collected by using an Olympus FluoView FV1000 confocal laser scanning microscope. DAPI  nuclear staining was used when needed. Gene cotransfection efficiency was determined as the multitransgene-positive cell number divided by the total cell number. The induction rate of Myh6− or Tnnt2-expressing cells was determined as the percentage of Myh6+Myocd+ and Tbx5+Gata4+Myocd+ cell purities were determined under a confocal microscope after cell sorting. Sorted cells were then plated in 60 mm dishes and grown in BME medium supplemented with 1 µg/ml doxycycline, 5% (v/v) horse serum (Invitrogen), and 2 mM L-glutamine for an additional 12 days. Samples were from three independent experiments, and total RNA was isolated by using Trizol reagent (Invitrogen), followed by DNase I (Qiagen) treatment. Samples were compared by using Affymetrix MG 430 2.0 arrays. All data are Minimum Information About a Microarray Experiment (MIAME) compliant and the raw data have been deposited in the Gene Expression Omnibus (GEO) database (accession number GSE27329). Expression data were analyzed by using dChip2008 p value cut-off of 0.05 was used. Activated or inhibited gene lists were compared by using the GeneVenn web application p value was calculated by using a non-parametric bootstrapping approach p<0.05 and a nested gene change number ≥4 were considered as significantly activated or inhibited.10T1/2-tTA cells that were infected with different combinations of Tbx5, Gata4, and Myocd lentiviruses were grown in BME medium supplemented with 1 µg/ml doxycycline, 5% (v/v) horse serum (Invitrogen), and 2 mM L-glutamine. Forty-eight hours later, positively transfected cells were sorted out by using FACS Vantage SE DiVa system (BD Biosciences). Because peroxisomal EGFP (tracking for Gata4) and nuclear EGFP (tracking for Myocd) are of the same excitation/emission wavelength but with different subcellular localization ability, Gata4p<0.05 was considered statistically significant.All results were expressed as mean ± standard error of the mean. Statistical significance was evaluated by analysis of variance following one-way ANOVA procedure with SPSS11.0 software (SPSS Inc.). A value of cis-element luciferase reporters containing wild-type and mutated cognate sequences of Nkx2-5, Tbx5, Gata4, and Myocd binding sites in CV-1 fibroblasts. Compared with LacZ lentivirus, Nkx2-5 lentivirus significantly transactivated the 2xNKE wild-type reporter  but not the 2xmuNKE mutated reporter  but not the 2xmuTBE mutated reporter  but not the muGATA mutated reporter  but not the site-mutated 2xmSRE reporter .Additionally, because Nkx2-5, Tbx5, Gata4, and Myocd lentiviruses were designed to co-express peroxisomal DsRed-MST, nuclear DsRed-MST, peroxisomal EGFP, and nuclear EGFP tracking signals, respectively; multigene transfection within a single cell was able to be tracked under fluorescent microscope by the presence of different subcellular localized signals .Among 15 different combinations, only the T+M and T+G+M combinations induced ructures Figure 2+Myocd+ and Tbx5+Gata4+Myocd+ cells used was 78.1% and 57.4%, respectively. Generally, Tbx5, Gata4, Myocd alone, and different combinations of these factors extensively perturbed whole-genome gene expression . The number of inhibited genes was 833(T), 767(G), 616(M), 1041(T+G), 508(G+M), 524(T+M), and 713(T+G+M) . Enriched cardiac, smooth muscle and skeletal muscle gene expression profiles are shown in Supplemental Table S2, and their expression was further validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis . T+M induced Myh11 and Smtn smooth muscle gene expression , whereas T+G+M only induced relatively lower Myh11 expression . However, endogenous Nkx2-5, Tbx5, Gata4, and Myocd were neither activated nor inhibited by T+M or T+G+M .To determine the difference between T+M- and T+G+M-activated cardiac gene expression and to determine the individual roles of Tbx5, Gata4, and Myocd in activating cardiac gene expression in mammalian non-myoblastic cells, we conducted genome-wide gene expression analysis by using 10T1/2 fibroblasts transfected with Tbx5, Gata4, Myocd alone, or different combinations of these factors. The purity of Gata4 and Gene ontology analysis was summarized in The combination of T+G+M activated genes associated with the most cardiac-related processes, including muscle development, muscle cell differentiation, structural constituent of cytoskeleton, structural constituent of muscle, myofibril, sarcomere, contractile fiber, muscle contraction, striated muscle contraction, regulation of heart contraction, circulation, glucose metabolism and fatty acid oxidation  and activated more genes involved in non-cardiac processes . In the specifically activated gene list by T+G+M, cardiac-related structural genes were Ldb3, Hsbp7, Myl4, Tnni3, Tnnc1, Mybpc3, Des, Myom2, Myh7 and Dsp. And cardiac-related transcription factors were Mlf1, Rbl1 and Atf6. In the specifically inhibited gene list by T+G+M, Cited2, Foxp1, Elk3 and Fgfr1 are known to be related with multiple developmental processes which will be discussed later. In the specifically activated and inhibited gene lists , there are several genes with unknown functions.As the genes activated or inhibited by T+G+M are not a simple plus of the genes activated or inhibited by Tbx5, Gata4, or Myocd alone, we tried to find out the genes that were specifically activated or inhibited by T+G+M by excluding genes activated or inhibited by Tbx5, Gata4, Myocd, T+G, G+M, and T+M from the activation or inhibition gene list of T+G+M. We found that T+G+M specifically activated 213 genes and inhibited 119 genes , forced expression of exogenous human Tbx5+Myocd or human Tbx5+Gata4+Myocd in 10T1/2 fibroblasts did not activate endogenous mouse Tbx5, Gata4, or Myocd expression in 10T1/2 fibroblasts . This is somehow similar to the previously reported observation showing that after the addition of exogenous Tbx5, Gata4, and Mef2c, endogenous Tbx5 and Mef2c expression was not induced +Myocd+ or Tbx5+Gata4+Myocd+ 10T1/2 fibroblasts, it is difficult to give an exact answer. Unlike the uniform gene expression profile and biologic characteristics of individual ventricular cardiomyocytes, the expression levels of exogenous Tbx5, Gata4, and Myocd in Tbx5+Myocd or Tbx5+Gata4+Myocd transfected 10T1/2 fibroblasts may vary from cell to cell because of different copy numbers of the transgene integrated in the genome. Rare but appropriate expression levels and mutual expression ratios of exogenous Tbx5, Gata4, and Myocd may possibly account for the observation that only a small fraction (5.87–7.40%) of Tbx5+Gata4+Myocd+ 10T1/2 fibroblasts expressed cardiac marker proteins  in the future study. According to recent literature, it is controversial and confusing whether combination use of cardiac transcription factors Tbx5, Gata4, and Mef2c can reprogram mouse tail tip and cardiac fibroblasts into beating cardiomyocytes + cardiac progenitor cells .In our study, we found that Nkx2-5, Tbx5, Gata4, or Myocd alone did not induce the pression Table 1 Supplemental Table S2), both Myh6 and Nppa are not activated by Tbx5, Gata4, or Myocd alone. However, Myh6 and Nppa are activated by the combination of Tbx5 and Myocd, which is consistent with the observations of Wang and colleagues Although T+G+M and T+M up-regulated Smarcd3 and Mef2c expression by 1.69- and 2.73-fold, respectively, no beating cardiomyocytes were observed in our study during a 4-week observation period. In consideration of the recent report that Tbx5+Gata4+Mef2c was inefficient to reprogram fibroblasts into cardiomyocytes +Gata4+Myocd+ 10T1/2 fibroblasts (including Tbx5+Gata4+Myocd+/Myh6+ subpopulation) are non-beating cells, we assume that the whole-genome gene expression profile may be different between Tbx5+Myocd+ or Tbx5+Gata4+Myocd+ 10T1/2 fibroblasts and mouse adult cardiomyocytes, although cardiac structural genes are activated in Tbx5+Myocd+ or Tbx5+Gata4+Myocd+ 10T1/2 fibroblasts . Differentially expressed (activated or inhibited) genes  are related to cardiovascular system development. Region (a) genes are also related to striated muscle proliferation. The different expression patterns of these genes in T+M or T+G+M transfected 10T1/2 fibroblasts indicate the extensive perturbation of the cardiogenic process by the combination of Tbx5, Gata4, and Myocd.Although T+G+M activated genes that are related with complement activation (e.g. Cd55) and regulation of ossification (e.g. Spp1 and Ptger4) processes, these genes are also closely related with cardiac functions. Cd55 is a normally expressed cardiomyocyte receptor for Coxsackie B viruses roblasts Figure 3Supplemental Table S4), Ldb3  and Dsp (desmoplakin) are important for cardiomyocyte sarcomere development. Defects in either of these genes are associated with severe cardiomyopathies Supplemental Table S5), Cited2 (Cbp/p300-interacting transactivator), Foxp1 (Forkhead box P1), and Elk3 (member of the ETS oncogene family) are transcription factors that contribute to multiple developmental processes such as blood vessel development (Cited2) In the list of genes specifically activated by T+G+M Click here for additional data file.Figure S2Molecular weights of the induced Myh6 and Tnnt2 proteins in 10T1/2 fibroblasts are same as those in mouse heart. Tbx5+Myocd and Tbx5+Gata4+Myocd transfected 10T1/2 fibroblasts are induced by doxycycline (1 µg/ml) for 48 hours and are subjected to RFP+ and EGFP+ double selection by fluorescence-activated cell sorting. Sorted cells are induced by doxycycline (1 µg/ml) for additional 12 days followed by western blot analysis. The purity of Tbx5+Gata4+Myocd+ cells is 46.2% as confirmed under fluorescent microscope. LacZ transfected 10T1/2 fibroblasts and mouse heart are used as negative and positive controls, respectively. TM, Tbx5+Myocd; TGM, Tbx5+Gata4+Myocd. 4–20% SDS-PAGE.(JPG)Click here for additional data file.Figure S3Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of cardiac marker gene expression in Tbx5, Gata4 and Myocd transfected 10T1/2 fibroblasts. Cardiac marker gene Myh6, Ryr2, Nppa and Tnnt2 expression were examined in microarray leftover samples. Each experiment was performed in triplicate. T, Tbx5; G, Gata4; M, Myocd. The copy number for each transcript is expressed relative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as a constitutive control.(JPG)Click here for additional data file.Figure S4Reverse transcription polymerase chain reaction (RT-PCR) analysis of cardiac transcription factor expression in adult mouse heart.(JPG)Click here for additional data file.Figure S5Difference exists in whole genome and cardiac cluster gene expression profiles between Tbx5+Gata4+Myocd transfected 10T1/2 fibroblasts and adult mouse myocardium. (A) Whole genome gene expression profile. (B) Cardiac cluster gene expression profile. Cardiac genes are selected by their annotation containing “heart” or “cardiac”. Genes in differential expression regions (a), (b), (c), (d) and (e) are listed. Mouse hearts include biological duplicates . TM, T+M; TGM, T+G+M.(JPG)Click here for additional data file.Table S1Activated and inhibited gene lists by Tbx5, Gata4, Myocd alone or different combinations. Activated or inhibited genes were defined as genes with an absolute fold change ≥2.0 relative to the LacZ group and genes with a signal intensity equals to 0 in the LacZ group but not in the transcription factor-treated groups or vice versa.(XLS)Click here for additional data file.Table S2Array signal values of several well-known cardiac marker genes in 10T1/2 fibroblasts.(DOC)Click here for additional data file.Table S3Array signal values of endogenous Nkx2-5, Tbx5, Gata4 and Myocd in 10T1/2 fibroblasts.(DOC)Click here for additional data file.Table S4Genes specifically activated by Tbx5+Gata4+Myocd in mouse 10T1/2 fibroblasts. Tbx5+Gata4+Myocd specifically activated gene list was generated by excluding genes activated by Tbx5, Gata4, Myocd, T+G, G+M, and T+M from the activated gene list of T+G+M. Gene lists were compared by using the GeneVenn web application. * Gene accession number was used if probe set does not have a gene symbol.(DOC)Click here for additional data file.Table S5Genes specifically inhibited by Tbx5+Gata4+Myocd in mouse 10T1/2 fibroblasts. Tbx5+Gata4+Myocd specifically inhibited gene list was generated by excluding genes inhibited by Tbx5, Gata4, Myocd, T+G, G+M, and T+M from the inhibited gene list of T+G+M. Gene lists were compared by using the GeneVenn web application. * Gene accession number was used if probe set does not have a gene symbol.(DOC)Click here for additional data file.


Graphene exhibits exciting properties which make it an appealing candidate for use in electronic devices. Reliable processes for device fabrication are crucial prerequisites for this. We developed a large area of CVD synthesis and transfer of graphene films. With patterning of these graphene layers using standard photoresist masks, we are able to produce arrays of gated graphene devices with four point contacts. The etching and lift off process poses problems because of delamination and contamination due to polymer residues when using standard resists. We introduce a metal etch mask which minimises these problems. The high quality of graphene is shown by Raman and XPS spectroscopy as well as electrical measurements. The process is of high value for applications, as it improves the processability of graphene using high-throughput lithography and etching techniques. Graphene has many potential applications including micro-nanoelectronics, sensors and transparent electronics. For applications in electronics, the reliability of processing of graphene is a major obstacle. The processing of graphene requires a transfer or growth on an insulating substrate, its patterning and subsequent contacting. With recent development of large scale synthesis of graphene layers -4, its uEach of these processing steps may affect the quality of the graphene as defects can be created, and contaminants can be introduced. While contaminants or solvent residues may be reduced by annealing and/or cleaning procedures, polymers residues are difficult to remove with these techniques. Harsh cleaning conditions may cause introduction of defects to the graphene layers or its delamination due to the absence of interfacial bonds to the substrate. Recently, the substrate effects and possible capture of contaminants under the graphene layers have been discussed .In this article, we show reliable processing of graphene on insulating substrates to produce high quality graphene field effect transistor (FET) devices. CVD graphene grown on copper was used, which was analysed by various methods after transfer. The patterning of graphene is of note, as this step was found to be unreliable using conventional methods, i.e. by the use of polymers as etch masks ,8. In ou2 were transferred onto SiO2 and characterised by XPS and AFM. The XPS spectrum is shown in Figure sp3 bonds, indicating that some contaminants were residing on graphene. An AFM scan  to G (approx. 1580 cm-1) peaks ratio shown in the inset supports this. The Raman spectrum also shows some defects as a small visible D peak (approx. 1350 cm-1). This could also indicate the presence of impurities.CVD-grown films on Cu of typically 1 × 1 cm2 atmosphere. The etch time ranged from 15 s to 2 min and plasma power between 100 and 500 W. The substrate holder was cooled by helium flow, and a heating of the substrate causing a possible crosslinking of the polymer resist cannot be ruled out. A 30 s treatment in a barrel asher (Diener) under O2 plasma was also tried. {AQ: Please supply missing word or phrase between "with" and "O2" in the sentence, "A barrel asher..." }A range of resist was investigated in our study which included AZ nLOF 2070 (~500 nm), maN-2403 (~ 300 nm) and S1813 (~ 1 μm). Bilayers photoresist masks using AZ nLOF 2070 as top layer and PMMA (450 k) or LOR-10B as bottom layers were also used. As delamination occurred during lift off of these resists, we created a processing procedure using a metal hard mask as shown in Figure D peak in Raman spectrum of etched graphene L/WVsd, we get μFE ~ 90 cm2/(V s). This value does not match the high values obtained from the exfoliated graphene in transistor measurements [The optical image of an FET device is shown in Figure urements , but to urements . HoweverCVD growth has been shown to give large area mono- or bilayer graphene. We produced FET devices arrays using optical lithography. Using metal hardmask, we were able to minimize delamination of underlying graphene films, which often occured when using standard resist. Further, the introduction of metal hardmask seems to have a minimal impact on the graphene and does prevent contamination by polymer residues. The large area processing shown here will open opportunities for graphene in industrial settings.3) to remove copper. The resulting film was cleaned with DI water. The graphene layer on the film was then pressed onto substrates while heated at the same time in a mechanical press. After 45 s, the support layer was dissolved in acetone and substrates were placed in chloroform for further cleaning. These substrates were then analysed with XPS, AFM and Raman spectroscopy. The XPS spectrometer used a monochromatised Al Kα X-ray source with a resolution of 0.7 eV. For AFM an Asylum MFP-3D with standard silicon tip was employed. The Raman spectra were taken with a laser excitation of 633 nm on a Horiba Jobin-Yvon Labram spectrometer using a 100 × magnification. Silicon substrate with 300 nm of oxide on top was used for characterising transferred graphene.Graphene was produced by CVD on copper foils at a temperature of 1000°C using methane as a carbon precursor . The foi2O3 on top as substrates. Liftoff pattern were applied on top of graphene using a resist bilayer. Nickel (30 nm) was evaporated on the substrates and followed by a liftoff. This produced an etchmask of Ni sitting on top of graphene. The samples were then etched in O2 plasma in a barrel etcher to transfer the pattern on Ni to graphene underlayer. Nickel coating was finally removed using dilute HCl . Another liftoff pattern was made on top for contacting graphene, using same process as above. Nickel contacts were deposited and a liftoff was performed again. The overall process is shown in Figure Electrical measurements were done on heavily doped silicon with 60 nm of AlCVD: chemical vapour deposition; FET: field effect transistor.The authors declare that they have no competing interests.SH carried out graphene synthesis and transfer, as well as SEM and Raman characterisations. NP did XPS studies. KHL and HYK conducted transport measurements and data analysis. GSD conceived of the study and participated in design and coordination. All authors read and approved the final manuscript.


Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF.AGO1 and AGO2  were lower compared with either slow or normal lung biopsies and fibroblasts.miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA  were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing. Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disorder characterized by excessive, irreversible scarring of the lungs Although a high degree of variability in IPF progression has been observed in patients Dicer1 is required for further processing There is growing evidence that the regulation of gene transcription in health and disease involves several non-redundant mechanisms. One such mechanism involves microRNA, which are 22 nucleotide non-coding RNA molecules that exert a biologically important effect on post-transcriptional gene expression MicroRNAs have been implicated in cancer, heart and neurodegenerative diseases, diabetes, and inflammation AGO1 and AGO2 (the core components of the RISC) were expressed at lower levels in rapidly progressive IPF biopsies and/or fibroblasts grown from the same biopsies compared with both normal and slowly progressive IPF biopsies and fibroblasts. Together, these data suggest that the development of IPF and/or the pace of its clinical progression might be a consequence of abnormal miRNA generation and processing.The aim of the present study was two fold: 1) to determine whether miRNA biosynthesis differed between IPF and normal lung tissues; and 2) to determine whether miRNA expression differed between IPF patients exhibiting varying speeds of disease progression. Herein, we report that the miRNA levels in diagnostic lung biopsies markedly differ in both rapidly progressive and slowly progressive IPF patients compared with normal individuals, albeit the large majority of the miRNA identified were significantly lower in the IPF biopsies compared with the normal biopsies. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT), a process of emerging importance in IPF CO) decrease of ≥15%. IPF patients that did not meet these criteria over the first year of follow-up were clinically diagnosed as slowly progressive . Samples taken from disease-free patient autopsies were used as normal controls . A University of British Columbia Institutional Review Board approved the procurement and analysis of these tissues.All surgical lung biopsies (SLBs) were obtained from the patient at the time of diagnosis of IPF. Patients were diagnosed with IPF using a multidisciplinary approach involving a clinicians, radiologists, and pathologists 2 qPCR-Grade miRNA Isolation Kit . Concentration and purity were again measured after miRNA isolation using the NanoDrop 1000 photospectrometer and samples were screened for an A260∶A280 ratio greater than 1.8. Conversion of miRNA to cDNA was carried out using 100 ng of miRNA according to manufacturer's instructions for the RT2 miRNA First Strand Kit .Left side upper and/or lower lobe surgical lung biopsies were stored at −80°C and thawed on ice immediately prior to RNA isolation. Approximately 1 ml of Trizol  was added to each biopsy. Each biopsy was homogenized using the Tissue-Tearor  and total RNA extraction was performed according to the manufacturer's instructions. Concentration and purity of each lung sample was determined using a NanoDrop 1000 UV-Vis photospectrometer . 1 µg of total RNA from each biopsy was converted into cDNA using Murine Moloney Leukemia Virus Reverse Transcriptase . MiRNA was isolated and purified from 10 µg of total RNA according to manufacturer's instruction for the RT2 miRNA PCR Array Human miFinder . Plates contained 88 of the most abundantly expressed and best-characterized miRNAs found in humans as well as relevant housekeeping miRNA. Samples were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) using the ABI 7500 Real Time PCR System  following the manufacturer's protocol. A dissociation curve was run to factor out wells with nonspecific amplification and primer dimers. Samples were grouped as normal, slowly progressive, and rapidly progressive and analyzed using the PCR Array Data Analysis Excel Template with the ΔΔCt method . Data were normalized using the small nuclear RNA housekeeping panel: SNORD 47, SNORD 44, and U6.MicroRNA expression values for each sample were measured using a RThttp://mirecords.biolead.org/) MiRecords  were analyzed using a preloaded custom TaqMan Array Microfluidic Card . Data were normalized to an internal standard, GAPDH in the 96 well format and to 18s in the card. Samples were grouped into either slowly progressive IPF or rapidly progressive IPF, and compared with normal using the ΔΔCt method.Circular DNA (cDNA) made from total RNA was analyzed using quantitative real time polymerase chain reaction (qRT-PCR) using the ViiA 7 Real-Time PCR System . Pre-mixed primers and probes were purchased from Applied Biosystems and used to detect human 2O2 solution to permeablize tissue sections and block endogenous peroxidase activity. Tissue sections were additionally blocked for with 5% BSA/ 1% rabbit serum, Avidin Block , and Biotin Block . The sections were incubated overnight at 4°C in anti-AGO1 rabbit polyclonal antibody  or anti-AGO2 rabbit polyclonal antibody . In-house non-specific rabbit polyclonal IgG was used as a concentration matched negative control. Sections were incubated with biotinylated anti-rabbit secondary antibody  for 1 hour then with HSS-HRP Streptavidin Peroxidase  for 30 minutes. Tissue was stained with DAB chromogen, counterstained with hematoxylin, and photographed with an Olympus BX40 microscope and IP Lab Spectrum software .Five-micron thick paraffin-embedded tissue sections were cut from formalin-fixed normal, slowly progressive, and rapidly progressive SLBs. Each tissue sections was de-parafinized and rehydrated by washing with xylene and a decreasing ethanol gradient. Sections were heated in 10 mM citric acid (pH 6.0) for 12 minutes for antigen retrieval and incubated for 30 minutes in a 10% methanol/ .4% H2 cell culture flasks with media. The media consisted of DMEM  with 15% fetal bovine serum , 2 mmol/L glutamine, and 1× Penicillin-Streptomycin-Amphotericin B . Cells lines were cultured in media at 37°C in a 7% CO2 incubator and were serially passaged 4 times to yield pure populations of adherent fibroblasts. All primary fibroblast cell lines from each patient group were used between passages 6 and 11. Prior to an experiment, fibroblasts from each group  were plated into a six-well tissue culture plate with 5×105 cells per well and activated for 4 hours with media alone. Trizol reagent was added to each well to terminate the experiment and mRNA was isolated and analyzed as described above.To culture primary human fibroblasts, histologically normal and IPF SLBs were finely minced and the dispersed tissue pieces were placed into 150 cmwww.R-project.org). The “Ward” method, which derives spherical clusters has been used as the agglomeration algorithm for forming clusters.Student's T-test or Mann-Whitney test for non-normally distributed data were used to determine statistical differences among the analyzed groups. Calculations were performed using PRISM 5.0 software for Macintosh . P≤0.05 was considered statistically significant. The statistical analysis performed on the EMT-associated genes used qRT-PCR generated dCT values following the (Ct of gene of interest (GOI) – Ct of housekeeping gene (HKG)) calculation. Data were clustered using the absolute value of correlation coefficients (distance measure) using hierarchical clustering to show the genes that are strongly related in each group  . These data revealed that many transcripts relevant to fibrosis are validated targets of the miRNA present in the normal and IPF biopsy samples including many EMT-related genes (Supporting Table S1).We next hypothesized that a decrease in miRNA expression might explain, in part, the increased expression of fibrosis-related mRNA transcripts observed in IPF biopsies compared with normal or non-fibrotic lung biopsies. Unfortunately, predicting miRNA gene targets is challenging because each target prediction method predicts thousands of mRNA transcripts per miRNA. Since each target prediction program's methodology differs, these programs also predict discordant mRNA transcripts for a given miRNA. To lessen the discordance in miRNA target prediction, we focused on compiling only experimentally validated miRNA targets after an analysis of interactions. A list of experimentally validated targets was compiled using the miRNA that differed between slowly progressive and rapidly progressive IPF biopsies compared to normal biopsies  mRNA levels were significantly decreased in slowly progressive biopsies and rapidly progressive biopsies in comparison to normal tissue  was apparent in rapid IPF versus slow IPF.We next determined whether EMT-related genes differed in the three groups of lung biopsies using TaqMan Array Microfluidic Cards, and these data are summarized in HMGA2Further statistical analysis of miRNA expression in biopsies from both groups of IPF patients revealed that 5 miRNAs were increased in rapid IPF versus slow IPF, and one miRNA was decreased in rapid IPF versus slow IPF. A summary of these miRNA and their validated targets is shown in AGO1, which is a core component of the RNA induced silencing complex (RISC) Supporting Table S1). Argonaute proteins function to bind the miRNA and correctly position it to recognize the appropriate mRNA targets AGO1 and AGO2 were analyzed at the transcript and protein level in IPF and normal biopsies. Compared to normal biopsies, AGO1 transcript levels in slow and rapid IPF biopsies were significantly lower compared with levels in normal biopsies  targeted AGO1 revealed very little regardless of the antibody dilution or biopsy group, perhaps due to the lack of a suitable antibody for formalin-fixed tissues. However, AGO2 was detected in both slowly progressive and rapidly progressive sections when this antibody was used at a dilution of 20 µg/ml  in IPF patients. Thus, the findings from the present study coincide with other published findings regarding the differential expression of miRNA in clinical and experimental fibrosis.Our findings are consistent with a number of recently published studies directed at the characterization of miRNA expression and function in pulmonary fibrosis. First, a recent study by Pandit DICER1 enzymatic activity is necessary for miRNA biogenesis and loss of DICER1 expression through gene silencing and knockout approaches has been shown to cause aberrant or decreased endothelial cell proliferation DICER1 is also associated with familial pleuropulmonary blastoma DICER1 caused overall reductions in mature miRNA levels, which contributes to the loss of transcript regulation. However, analysis of DICER1 expression revealed no differences in the amounts of this transcript amongst the biopsies and fibroblast lines analyzed, leading us to presently conclude that changes in the expression of DICER1 do not appear to explain the differential miRNA levels in IPF versus normal.ZEB1 and SIP1, which repress E-cadherin and promote mesenchymal marker expression. As expected CD44, COL1A2, VIM, and FOXC2 were significantly increased in rapidly progressive IPF over normal and/or slowly progressive IPF biopsies. CD44 is a membrane associated cellular adhesion receptor that plays a role in remodeling the lung COL1A2 has been previously described in IPF Vimentin is a definitive marker for the meshchymal cells derived from epithelium VIM were significantly increased only in rapidly progressive IPF. These data suggested that levels of a particular miRNA might be less important than the processing efficiency of a particular miRNA. Together, these data highlight that transcripts associated with EMT were significantly elevated in IPF, particularly the rapidly progressive form of this disease. The impact of differential miRNA expression on EMT during IPF progression is presently not clear and requires further investigation.EMT is the transition of differentiated epithelial cells into motile mesenchymal cells, and this process is prominent in both experimental and clinical pulmonary fibrosis AGO2 is the catalytically active “slicer” that cleaves mRNA transcripts Argonaute proteins bind miRNA and position it in a conformation that promotes mRNA target recognition In summary, our results demonstrate that miRNA profiling in diagnostic surgical lung biopsies differentiates normal from IPF, and rapidly progressive IPF from slowly progressive IPF. Further analysis of miRNA levels in circulating cells as a means of ascertaining IPF disease progression is certainly warranted given the present findings. Alterations in circulating miRNAs have been detected in other diseases such as cancer. Finally, our results indicate that aberrations in the miRNA processing pathway might be the cause of altered transcript expression leading to the fibrotic phenotype in IPF and the differing speed of progression of this disease.Table S1This table is a list of the experimentally validated gene targets compiled using the miRNA species that differed between slowly progressive and rapidly progressive IPF biopsies compared with normal lung biopsies.(DOCX)Click here for additional data file.


In this article, a multiscale investigation of few graphene layers grown on 6H-SiC(000-1) under ultrahigh vacuum (UHV) conditions is presented. At 100-μm scale, the authors show that the UHV growth yields few layer graphene (FLG) with an average thickness given by Auger spectroscopy between 1 and 2 graphene planes. At the same scale, electron diffraction reveals a significant rotational disorder between the first graphene layer and the SiC surface, although well-defined preferred orientations exist. This is confirmed at the nanometer scale by scanning tunneling microscopy (STM). Finally, STM  and Raman spectroscopy  show that the FLG stacking is turbostratic, and that the domain size of the crystallites ranges from 10 to 100 nm. The most striking result is that the FLGs experience a strong compressive stress that is seldom observed for graphene grown on the C face of SiC substrates. The truction -15. EpitC and (3 × 3) SiC reconstructions, and the graphene layers have several orientations on top of each surface reconstruction [2 V-1 s-1) [On the C face, the situation is completely different. There is no need for a buffer layer anymore but two different pristine surface reconstructions exist below the graphene layers: (2 × 2)truction -18. The V-1 s-1) . It alsoV-1 s-1) . BesidesV-1 s-1) . It is tV-1 s-1) ,18, SiC in situ under UHV conditions according to the procedure of ref. [The sample graphitization and first characterization  were performed  of ref. . The sur of ref.  was obtaSTM measurements were done at room temperature using mechanically cut PtIr tips. Typical STM images are gathered in Figure XYZ piezoelectric stage. It is then possible to measure at the same time, during the acquisition of Raman spectra, using the same laser beam as a probe, the power transmitted through the sample. Raman spectra were collected at room temperature using a Jobin Yvon-Horiba T64000 spectrometer in the confocal mode, with a ×100 microscope objective. The 514-nm line of an Ar ion-laser was used for excitation. The spot size was 1 μm, with 1-mW incident power under the objective. Using this original combination of techniques, a 16 × 16 μm2 mapping of the FLG area located at the center of the sample was performed. The step size was 0.25 μm along both X and Y directions. Since no bare SiC surface could be found at the probe size, a SiC reference spectrum was collected by focusing the laser beam in the SiC substrate deeper than the confocal field depth. FLG's Raman spectra were obtained by subtracting the SiC reference spectrum from the experimental spectra. Typical spectra, collected on the thinnest and thickest FLG parts, are compared to the one of a highly oriented pyrolytic graphite (HOPG) sample in Figure -1, respectively. These three bands are blue shifted compared with standard FLG and HOPG Raman spectra. As discussed later, this blueshift can only be explained by a high compressive strain of the graphene lattice. The 2D band has a single Lorentzian shape meaning that the FLG stacking is not Bernal but, rather, turbostratic. This first observation is in perfect agreement with the previous STM results. The D band around 1380 cm-1 comes from the breakdown of the wavevector selection rule and reveals the presence of crystalline defects inside or at the edges of FLG flakes. The in-plane size of the crystallites can be deduced from the ratio between the G and D band integrated intensities (ID/IG). Using the expression given by Pimenta et al. [To investigate the quality and thickness uniformity of the FLG, micro-Raman spectroscopy and microtransmission measurements were simultaneously performed. As has already been shown , these ta et al.  the domaLa ranges from 20 to 60 nm, which are in excellent agreement with previous STM observations.The in-plane sizes of the crystallites To estimate the average graphene thickness, one should use the relative extinction deduced from our microtransmission measurements . UnfortuLaser power fluctuations are corrected thanks to an additional low-noise photodiode that measures the laser power during the acquisition of the Raman map, and the estimated thickness found is shown in Figure -1, the G band around 1610 cm-1, and the 2D band around 2750 cm-1 whereas neutral relaxed graphene has a D band around 1350 cm-1, a G band centered at 1582 cm-1, and a 2D band at 2690 cm-1. The average up shift is then of 28 cm-1 for the G band, and of 60 (30) cm-1 for the 2D (D) band, respectively. The G and 2D band Raman shift maps are shown in Figure -1 and the 2D band from 2736 to 2764 cm-1. Such high blueshift cannot be explained by a doping of the graphene layers. Although high p-type doping (3 × 1013 cm-2) and n-type doping (4 × 1013 cm-2) induce a blueshift of the G band up to 1610 cm-1, the G band is then narrowed with a FWHM around 8 cm-1 [-1. This G band broadening refutes the high doping hypothesis. The doping hypothesis is also refuted by the 2D band position that cannot be explained by the n-type doping that shifts the 2D band to lower energies (down to 2660 cm-1) and by the p-type doping that shifts the 2D band up to 2700 cm-1, which is much smaller than the observed shifts. These blueshifts can actually be explained by a high compressive strain of the graphene lattice. This strain must originate from the strong difference in the in-plane thermal expansion coefficients of the SiC and the graphene. It is created during the cooling down of the sample after the growth. It can be assumed that it is biaxial. The strain and stress can then be deduced from the relationships of Table Finally, the most striking result is the strong blueshift observed on this FLG. The D band lies around 1380 cmd 8 cm-1 . In thisUsing these relationships, it can be estimated from the G band that the strain ranges from -0.2 to -0.7% with an average of -0.5%. The corresponding stress values range from -3 to -8.7 GPa with an average of -6 GPa. From the 2D band, a strain comprised between -0.3 and -0.5% with an average of -0.4% is found. The corresponding stress ranges from -3.7 up to -6 GPa with an average of -5 GPa. The strain/stress derived from the 2D band is slightly smaller than the one deduced from the G band. It can arise from the uncertainty on the graphene/graphite Grüneisen parameters . In FiguID/IG ratio) ranges from 20 to 60 nm, which is in excellent agreement with the graphene crystallites size probed by STM. Finally, the most striking result is that the D, G, and 2D bands are highly blue shifted . This means that the graphene lattice is highly compressively strained (around -0.4/-0.5%). Usually, FLG grown on the C face of SiC are fully relaxed by forming wrinkles to release the thermal stress during the cooling down of the sample after the growth. For this particular case, the strain might arise because graphene crystallite edges are bound to the SiC surface.The FLG has been grown on 6H-SiC (000-1) in UHV conditions. An average thickness of one to two graphene planes was found from AES, whereas Raman spectroscopy results indicate an average thickness of two to three graphene planes. This small discrepancy might occur because of the uncertainty of one to two layers for thickness determined from Raman spectroscopy and from the difference of the spot size between these two techniques. LEED and STM experiments show (i) a (3 × 3) SiC surface reconstruction, (ii) a wide distribution of disorientation between the first graphene sheet and the SiC surface, and (iii) rotational stacking fault between the graphene layers corresponding to a turbostratic stacking for the multilayers. The single Lorentzian shape of the 2D Raman band measured on these FLG confirms this turbostratic stacking. The FLG domain size (deduced from the AES: Auger electron spectroscopy; FLG: few layer graphene; HOPG: highly oriented pyrolytic graphite; LEED: low energy electron diffraction; STM: scanning tunneling microscopy; UHV: ultrahigh vacuum.The authors declare that they have no competing interests.FH and AM prepared the samples and carried out the LEED, Auger and STM measurements. FH and PM performed the analysis of the LEED, Auger and STM data. AT and JRH carried out the micro Raman spectroscopy and microtransmission experiments. AT, JRH and JC performed the analysis of the Raman and transmission data. AT drafted the manuscript. JC, JRH, CN and JYV participated in the writing of the manuscript. All authors read and approved the final manuscript.


Graphene is a leading contender for the next-generation electronic devices. We report a method to produce graphene membranes in the solution phase using polymeric imidazolium salts as a transferring medium. Graphene membranes were reduced from graphene oxides by hydrazine in the presence of the polyelectrolyte which is found to be a stable and homogeneous dispersion for the resulting graphene in the aqueous solution. A simple device with gold contacts on both sides was fabricated in order to observe the electronic properties. The unique physical, electronic, and optical properties of graphene have been reported many times -4 and prTwo steps are involved in making water dispersible graphene: (1) first chemical oxidation of graphite to hydrophilic graphite oxide and (2) exfoliating it into graphene oxide (GO) sheets in aqueous solution. GO sheets are graphene sheets having oxygen functional groups. These GO sheets are prevented from agglomeration by electrostatic repulsion alone . The insOrganic salts based on the imidazolium moiety are an interesting class of ions. Low molecular weight imidazolium salts can have a low melting point and are then termed ionic liquids (ILs). Thus, ILs are molten salts at the room temperature and consist of bulky organic cations paired with organic or inorganic anions. Imidazolium ionic liquids have many advantageous properties, such as no flammability, a wide electrochemical window, high thermal stability, wide liquid range, and very small vapor pressure . They ar2SO4  and kept at 80°C for 5 h. The resulting solution was cooled down to room temperature. Mild sonication was performed in a water bath for 2 h to further delaminate graphite into a few micron flakes. Sonication time and power are very critical as they define the size of the resulting graphene oxide sheets. Excessive sonication leads to extremely small flakes. Then, the solution was diluted with 0.5 L deionized (DI) water and left overnight. The solution was filtered by Nylon Millipore™ filters . The resulting powder was mixed with KMnO4 and H2SO4 and put in a cooling bath under constant stirring for 1.5 h. The solution was diluted with DI water, and 20 mL H2O2 (30%) was added to it.Graphene oxide was prepared by the modified Hummer's method ,13. The -1 and strongly shook the solution for a few minutes. The imidazolium salt used by us was polyquaternium 16 (PQ-16) sold under the trade name Luviquat Excellence by BASF , a copolymer with 95% molar of imidazolium chloride and 5% molar of vinylimidazole. Use of this polymeric salt for graphene dispersion is not found in literature. Then, the solution was reduced by hydrazine monohydrate at 90°C for 1 h to obtain a stable dispersion of graphene in aqueous solution.The supernatant was collected after 12 h and dispersed in dilute HCl in order to remove the metal ion residue and then recovered by centrifugation . Clean GThis aqueous PQ-graphene dispersion was found to be stable even after 2 months, whereas the reduced GO without the addition of PQ-16 formed agglomerates soon after reduction with hydrazine. Thus, PQ-16 is the main cause of a stable dispersion of graphene membranes in aqueous solution. The underlying mechanism has been affiliated with adsorption of some of the polycations on the surface of the graphene membranes by non-covalent π-π interactions between the imidazolium rings of the salt and graphene, soon after reduction with hydrazine monohydrate [2 (SiO2 thickness approximately 300 nm) substrates by dip-coating. Schematic of the whole process is shown in Figure ohydrate . The gra2 to further reduce the graphene oxide and also to sublimate the solution residue. The optical microscope images were taken in order to identify graphene [The sample was rinsed with DI water and dried with nitrogen. The dried samples were further treated at 400°C for 2 h in Ar/Hgraphene . Atomic graphene . Graphengraphene  over rel2/Si substrate. The average thickness of a GO sheet was approximately 1 nm  after drop casting them on a relatively flat SiO2/Si substrate by drop casting, is shown in Figure AFM image of our chemically reduced GO sheet after addition of PQ-16, deposited on SiOTransmission electron microscopy (TEM) is also a very important tool for investigating the quality of exfoliated graphene. We dropped a small quantity of the dispersion on the holey carbon grid by pipette and dried the samples. Figure Id) vs. gate voltage (Vg) curve of FET prepared with this reduced monolayer graphene membrane. The FET gate operation exhibits hole conduction behavior. Pure two-dimensional graphene has a zero bandgap that limits its effective application in electronic devices. We believe that this reduced GO from PQ dispersion has a kind of doping effect that makes it more favorable for applications due to its improved electronic properties. There were theoretical simulations [2) substrate has also led to the energy gap opening [We fabricated a bottom-gated graphene field-effect transistor (FET) by putting a monolayer of reduced GO membrane in between thermally evaporated gold electrodes. The channel length between source and drain electrodes was 5 μm. The schematic and the scanning electron microscope (SEM) image of the device are shown in Figure ulations ,21, whiculations  that the opening . Here, w opening . Thus, c opening , althoug2/Si substrate by simple drop casting and were further reduced by annealing in H2/Ar. A simple device with gold contacts on both the sides was fabricated in order to observe the electronic properties. We conclude that chemical functionalization is a possible route to modify and improve the electronic properties of graphene.In summary, we report a method to produce and functionalize graphene membranes in the solution phase using polymeric imidazolium molten salts as a transferring medium. Graphene membranes were reduced from graphene oxide by hydrazine in the presence of a polyelectrolyte which was found to be a very stable dispersion for the graphene membranes in the aqueous solution. The reduced GO membranes were transferred to a SiOThe authors declare that they have no competing interests.All authors contributed equally, read and approved the final manuscript.


The creation of magnetic storage devices by decoration of a graphene sheet by magnetic transition-metal adatoms, utilizing the high in-plane versus out-of-plane magnetic anisotropy energy (MAE), has recently been proposed. This concept is extended in our density-functional-based modeling study by incorporating the influence of the graphene edge on the MAE. We consider triangular graphene flakes with both armchair and zigzag edges in which a single ruthenium adatom is placed at symmetrically inequivalent positions. Depending on the edge-type, the graphene edge was found to influence the MAE in opposite ways: for the armchair flake the MAE increases close to the edge, while the opposite is true for the zigzag edge. Additionally, in-plane pinning of the magnetization direction perpendicular to the edge itself is observed for the first time. Since 2004, graphene , a one-aAs the system of choice, triangular hydrogen-saturated graphene flakes (or graphene quantum dots) were investigated, comprising 90 and 97 carbon atoms for two different edge types, armchair and zigzag (AGQD and ZGQD), respectively. The triangular shape was chosen as the simplest geometry, providing the same edge type on all sides. On a chosen hollow site (above a carbon ring center) a ruthenium adatom was placed, and the distance was optimized by minimization of the total energy using density functional theory. The stability of the Ru adatom on the hollow site, surrounded by less preferable positions over the C–C bridge and atop a C-atom, was reported for infinite graphene . The graWithin our work we used density functional theory (DFT)  with the) direction, pointing perpendicular to the flake plane; (2) the in-plane-minimum  direction, i.e., the direction parallel to the flake plane with the lowest total energy; and (3) the in-plane-maximum  direction, for the highest total energy in-plane. Note, direction 2 may depend on the chosen site, and directions 2 and 3 are not necessary perpendicular to each other. Using the defined directions, the following two kinds of MAE are defined. The in- versus out-of-plane (EIO) MAE is defined asTo determine the magnetic anisotropy of the system at hand, the magnetization direction was varied, and the resulting total energies were compared. Three magnetization directions and corresponding energies were used: (1) the out-of-plane  is defined asand is negative when the easy axis points out of plane. If EIP is per definition always positive and would be equal to zero for an adatom on an infinite graphene sheet, due to the underlying symmetry.The B. This spin polarisation of the ZGQD arises from a highly localized pz-type edge state [We first consider the electronic structure of the armchair-graphene (AGQD) and zigzag-graphene (ZGQD) quantum dots in the pristine state, i.e., without decoration by a Ru atom. , as well as the large binding energy of the Ru adatom which we find to be of the order of eV  we find that (i) EIO > 0 for all absorption positions on both flakes, i.e., the easy axis is in-plane and (ii) EIO is significantly larger on the AGQD as compared to the ZGQD, see EIO is plotted for all 9 (8) symmetrically inequivalent sites of the AGQD (ZGQD).We now turn to the question of the detailed magnetic structure of the graphene flake with the Ru adatom. Considering first the in-plane versus out-of-plane anisotropy ) for Ru on the AGQD and ZGQD are shown. Clearly while in the former case the Ru acts to weakly polarise the flake with ferromagentic coupling, in the latter case the coupling is strongly antiferromagnetic (note that blue and red indicate negative and positive m isosurfaces). The net result of this antiferromagnetic coupling is to strongly reduce the moment of the Ru adatom on the ZGQD: while moments of 1.7–1.8 μB are found for the AGQD, for the ZGQD these values fall to 0.8–1.4 μB. To bring out the relation to the in-plane versus out-of-plane anistropy, EIO, we plot this quantity against the Ru adatom moment in As we shall now demonstrate that the origin of this difference in the magnitude of EIO. This is, in fact, a rather natural result as the physics of anisotropy is, upon expansion of the Dirac equation in powers of v/c, governed by the spin–orbit coupling term which is proportional to  with  the spin moment.In short, the electronic structure of the graphene substrate determines the polarisation of the absorbed Ru atom and this in turn governs the value of EIP for all the symmetry-inequivalent absorbate positions, as shown in EIP is generally an order of magnitude smaller than EIO and attains its maximum value, as one would expect, at the edges of the graphene flakes. This follows from the fact that it is the lowering of the symmetry of the local environment that is crucial for the anisotropy , and hence, in the centre of even the rather small flakes presented here the anisotropy is substantially lower than at the edges. Interestingly, we find that for the edge positions the spin always points perpendicular to the boundary of the flake.Having thoroughly probed the physics of the in-plane versus out-of-plane anisotropy we now consider the question of which in-plane direction the Ru moment assumes, i.e., the question of what is the in-plane easy axis of the spin. To this end we calculated Using first-principles DFT methods we have investigated the magnetic properties of Ru adatoms on two types of graphene flakes: the armchair (AGQD) and zigzag (ZGQD) edged triangular graphene quantum dots. The geometry of these flakes is such that each has only one specific type high-symmetry edge (armchair or zigzag), allowing the clear separation of the physics of these two common edge types. We find that for all flakes and adatom positions investigated, the Ru magnetic moment prefers to lie in the plane of the island, and that the difference in energy between the most favourable and least favourable in-plane positions for the moment is of the order of 0.1 eV. For adatoms at the edge positions we find that the moment points perpendicular to the edge of the island.EIO as compared to values at the centre of the flake, with the opposite trend seen for the armchair-edge flakes. The origin of this lies in the antiferromagnetic coupling of the adatom to the spin-polarised pz edge state in the ZGQD.Interestingly, the in-plane versus out-of-plane anisotropy dramatically depends on the edge type, with the zigzag edge showing a marked reduction in both the Ru moment and the corresponding