Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown.Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug’s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes.Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early  Drosophila, Caenorhabditis elegans, zebrafish and mouse embryos have highlighted the substantial role that cytoplasmic post-transcriptional regulation plays in early embryos [Post-transcriptional regulatory mechanisms that function in the cytoplasm to control mRNA translation, stability and subcellular localization play essential roles in a wide variety of biological processes. While these types of controls function in a variety of cell types, they are particularly prevalent during early metazoan development where mRNAs synthesized from the mother’s genome direct the early stages of embryogenesis . Indeed, embryos -13.cis-acting elements that represent binding sites for microRNAs (miRNAs) or RNA-binding proteins. For example, miRNAs induce degradation of specific transcripts in both zebrafish and Drosophila[Drosophila embryo [During early embryogenesis, regulation of specific transcripts is achieved through rosophila,10. Simia embryo . Smaug ia embryo -18. SRE a embryo ,19-22.Drosophila Smaug can recruit the Cup protein to an mRNA and Cup in turn interacts with the cap-binding protein eIF4E [Upon binding to target mRNAs Smaug family members repress translation and/or induce transcript decay through their ability to recruit various factors to a transcript -18,23,24in eIF4E . The Cupin eIF4E . Typicalin eIF4E . In contin eIF4E .Smaug can also remove an mRNA’s poly(A) tail through its ability to recruit the CCR4/NOT deadenylase -31. In tnanos and Hsp83. Smaug represses nanos translation through two SREs in the nanos 3′ untranslated region (UTR) whereas loss of Smaug has only a modest effect on nanos mRNA stability [Hsp83 mRNA through eight SREs in the Hsp83 open reading frame, while having no detectable effect on Hsp83 translation [Smaug has two well-characterized target mRNAs, tability -16,28,33nslation . Thus, Snanos and Hsp83 mRNAs are localized to the posterior of the embryo and Smaug’s regulation of these two transcripts is intimately associated with their localization. nanos mRNA is inefficiently localized to the posterior and nanos mRNA that escapes the localization machinery is found distributed throughout the bulk of the embryo where it is translationally repressed by Smaug [nanos mRNA localized to the posterior is not repressed by Smaug and Nanos protein expression is thus restricted to the posterior of the embryo. Hsp83 mRNA is uniformly distributed in early embryos and, as embryogenesis proceeds, Smaug degrades Hsp83 mRNA in the bulk cytoplasm of the embryo while transcripts at the posterior of the embryo are protected [Hsp83 mRNA to the posterior of the embryo.by Smaug -16,34,35rotected ,31,36,37nanos and Hsp83 mRNA, Smaug is likely to regulate the expression of a large number of mRNAs in the early embryo through direct binding. For example, genome-wide experiments have shown that embryos collected from homozygous-mutant smaug females show stabilization of approximately 1,000 transcripts [smaug mutant embryos also show cell-cycle defects associated with a failure of DNA replication checkpoint activation and they also fail to undergo zygotic genome activation [nanos or Hsp83, this is consistent with a role for Smaug in regulation of the expression of additional mRNAs.In addition to nscripts . In additivation ,15. As nTo elucidate the global functions of Smaug in early embryos we employed two genome-wide approaches: 1) RNA co-immunoprecipitations followed by microarray analysis (RIP-Chip) to identify mRNAs that are bound by Smaug and 2) polysome gradients coupled to microarrays to identify targets of Smaug-mediated translational repression. Our data suggest that Smaug directly regulates the expression of a large number of mRNAs in the early embryo. Comparison of Smaug-bound mRNAs to those that are translationally repressed by Smaug (identified in this study), and those that are degraded in a Smaug-dependent manner  suggest nanos and Hsp83.To identify Smaug’s target mRNAs on a genome-wide scale we used RIP-Chip. Extracts, prepared from 0- to 3-hour-old wild-type embryos, were immunoprecipitated with an anti-Smaug antibody (hereafter denoted as Smaug RIPs) while immunoprecipitations using non-immune serum served as a negative control (hereafter denoted as control RIPs). Genes that were not expressed or were expressed at low levels in starting crude extracts were removed from further analysis and Significance Analysis of Microarrays (SAM)  was thennanos and Hsp83, were enriched in Smaug RIPs compared to control RIPs. In contrast, four mRNAs that, based on our RIP-Chip data, are not bound by Smaug showed little or no enrichment  to assay the enrichment of specific mRNAs in Smaug RIPs compared to control RIPs. Twelve selected mRNAs from the RIP-Chip target list with FDRs <5%, including t Figure .Smaug is a multifunctional regulator that is capable of both repressing translation and inducing the degradation of target mRNAs. To complement our identification of the targets of Smaug-mediated mRNA decay  and our 2-transformed version of this ratio. We removed genes from the polysome data that were not expressed or were expressed at only low levels. We also omitted the data from pool 2 in the TI calculation as it represents a mixed population of translated and translationally repressed mRNAs. We note that inclusion of pool 2 in the TI calculation has little effect on the calculated TI  as the logP < 3 × 10-16), indicating an excellent correlation between the two data sets.We then compared the TI for each gene in wild-type embryos to previously published polysome/microarray data from similarly staged wild-type embryos . In thatsmaug mothers (hereafter denoted as ‘smaug-mutant embryos’). We then compared the TI for each expressed gene in wild-type and smaug-mutant embryos  for the bulk of mRNAs present in smaug-mutant embryos , consistent with the fact that the majority of the mRNAs that are present in pools 3 and 4 of our gradients are indeed polysome-associated. Similarly, we also saw a significant decrease in the TI  for the 342 genes that are targets of Smaug translational repression (FDR <5%), consistent with the fact that, in smaug-mutant embryos, these mRNAs are highly associated with polysomes.To confirm that the increase in TI was indeed the result of the recruitment of mRNAs onto polysomes, smaug mutants. This suggests that a substantial subset of the genes with >5% FDR are potential targets of Smaug-mediated translational repression. Since SAM corrects for an average change in TI, if a large proportion of transcripts were in fact translationally repressed by Smaug, SAM would over-correct, thereby increasing the number of false negatives. To further evaluate the extent of Smaug-mediated translational repression we generated lists of genes that encode mRNAs that are unlikely to be bound by Smaug and are, therefore, unlikely to be targets of Smaug-mediated translational repression and then assessed their behavior in the polysome-gradient microarray experiments. We did this by identifying the 250, 500 and 1,000 genes whose mRNAs showed the lowest fold-enrichment in Smaug RIPs versus control RIPs. A comparison of the TI for each of these genes in wild-type and smaug-mutant embryos showed a distribution with little bias towards an increase in TI in the smaug mutant, confirming that few are likely to be targets of Smaug-mediated translational repression . This trend is highly significant .In addition to those genes that meet an FDR of <5%  for each of the top three sets of bound transcripts, respectively  as compared with the 250, 500 and 1,000 genes whose mRNAs were unlikely to be bound by Smaug . This analysis showed a peak change of TI in N and bottom N Smaug-binders , respectively. Based on this analysis, we estimated that 3,135, 3,094, or 2,728 are likely to be translationally repressed by Smaug using the distributions for N = 250, 500, or 1,000, respectively . We conclude that Smaug represses the translation of approximately 3,000 mRNAs in early embryos, representing about half of the 5,886 genes whose expression we detected in the polysome-microarray data set.To estimate the actual number of genes that are translationally repressed by Smaug, we deconvolved the distribution of TI changes for all genes Figure  to estim0-3 (where N is any base) [Smaug binds to and regulates its target mRNAs through SRE stem-loop structures and, as such, we would expect that mRNAs bound by Smaug as well as mRNAs translationally repressed by Smaug would be enriched for these stem-loops. The consensus sequence for the SRE loop is CNGGNny base) ,20. The ny base) . Thus, l0-4 . Our method assigned a probability for each potential SRE within a transcript based on the likelihood that it would fold into a stem-loop structure where the loop matches the CNGGN0-4 consensus. For each mRNA, an SRE score was then calculated as the sum of the probabilities for each SRE within that mRNA [smaug-mutant and wild type for translational repression) did not have higher SRE scores than the median for the bound or repressed mRNAs with FDR <5%.To ask whether SREs are predictive of Smaug binding and translational repression we searched all expressed genes in the RIP-Chip and polysome-microarray datasets for stem-loops with the loop sequence CNGGNhat mRNA . Strikin0-4 loops along with six altered stem-loops. The altered structures contained changes in the invariant nucleotides in the CNGGN0-4 loop that are predicted to lower their affinity for the Smaug RNA-binding domain. We found that the bona fide SRE was a significantly better predictor of both Smaug binding and Smaug-mediated translational repression than any of the altered stem-loops  and Smaug-mediated translational repression  than mRNAs whose decay is not regulated by Smaug. Using these two measures we also found a genome-wide correlation between Smaug binding and Smaug-mediated translational repression . Similar results were obtained for Smaug-mediated translational repression . Together these data suggest that a large fraction of the mRNAs that require Smaug for their degradation and/or translational repression that were scored as unbound in the RIP-Chip experiments are nonetheless directly bound by Smaug.The comparisons from Figure et al. . The founanos mRNA’s SREs are found in the 3′ UTR [Hsp83 mRNA’s SREs are found in the open reading frame [The e 3′ UTR -16 and tng frame ,31, raisnanos and Hsp83, whose mRNAs are localized to the posterior. Of these 44 genes, 38 are regulated by Smaug at the level of mRNA stability and/or translation 15), Replication Protein A 70 (RpA-70), Regulator of cyclin A1 (Rca1), Cyclin E (CycE), Minichromosome maintenance 3 (Mcm3), CDC45L, mutagen-sensitive 201 (mus201) and Msh6. Of these 32 genes, 29 are regulated by Smaug at the level of mRNA stability and/or translation , Nucleosome assembly protein 1 (Nap1), Nucleosome remodeling factor - 38kD (Nurf-38), anti-silencing factor 1 (asf1), Caf1-180, Caf1-105, and vig2. Of these 25 genes, 24 are regulated by Smaug at the level of mRNA stability and/or translation . These analyses suggest several previously unrecognized roles for Smaug in the early embryo  that are members of the Chaperonin Cpn60/TCP-1 family as defined by the Interpro database and are involved in protein folding. The last four of these proteins are subunits of the eukaryotic chaperonin TCP1-ring complex (TRiC), also known as the chaperonin containing TCP-1 (CCT), which consists of two rings composed of eight different subunits . ConsistRegulatory particle triple-A ATPase 3 (Rpt3), Regulatory particle triple-A ATPase 5 (Rpt5), Regulatory particle non-ATPase 1 (Rpn1), Regulatory particle non-ATPase 2 (Rpn2), Regulatory particle non-ATPase 7 (Rpn7), Regulatory particle non-ATPase 9 (Rpn9), Regulatory particle non-ATPase 10 (Rpn10), Regulatory particle non-ATPase 11 (Rpn11) and Regulatory particle non-ATPase 13 (Rpn13)). In contrast, Smaug does not interact with any of the mRNAs that encode the 20S core particle proteins. In addition, Smaug interacts with mRNAs that encode proteins involved in other aspects of the ubiquitin-proteasome system (Ubiquitin activating enzyme 1 (Uba1), Ubiquitin fusion-degradation 1-like (Ufd1-like), TER94 and CG9588). Consistent with a role for Smaug in control of the ubiquitin-proteasome system, 12 out of these 13 mRNAs  term ‘ubiquitin proteasome pathway’. The ubiquitin proteasome system plays a vital part in a variety of cellular processes through its role in the degradation of target proteins. This mechanism involves the post-translational addition of multiple ubiquitin moieties onto a protein, which, in turn, target the protein for proteasomal degradation . The 26SDrosophila fat-body tissue or from Drosophila embryos [Third, Smaug might play a role in regulating lipid storage and/or mobilization since the GO term ‘lipid droplet’ is enriched in the Smaug-bound mRNAs. Lipid droplets are ubiquitous organelles that are found in a wide range of organisms from bacteria to humans. They consist of a neutral-lipid core composed of triacylglycerols and sterol esters surrounded by a phospholipid monolayer, and they serve as storage sites for energy, sterols and membrane precursors . Several embryos ,57. CompHexokinase A (Hex-A), Phosphoglycerate kinase (Pgk), Phosphoglucose isomerase (Pgi) and both genes encoding Glyceraldehyde 3 phosphate dehydrogenase (GAPDH1 and GAPDH2)) and our data indicated that all nine are regulated by Smaug at the level of stability and/or translation repression  and Triose phosphate isomerase (Tpi) have FDRs in the RIP-Chip data of 5.15% and 6.08%, respectively, and both are targets of Smaug-mediated transcript degradation and translational repression  and Pyruvate kinase (Pyk) are regulated by Smaug at the level of stability and/or translation. In summary, our data suggest that 8 of the 10 glycolytic enzymes may be regulated by Smaug.Fourth, a direct role for Smaug in regulation of metabolism is suggested by the enrichment for terms such as the SwissProt keywords ‘oxidoreductase’ and ‘NAD’ and the GO terms ‘oxidation reduction’ and ‘cofactor binding’ within Smaug-bound mRNAs. Together these lists comprise a total of 37 metabolic enzymes that function in a wide variety of pathways, including fatty acid metabolism, pyruvate metabolism, amino acid metabolism, the citric acid cycle and oxidative phosphorylation. Our data suggested that 28 out of 37 of these genes are regulated by Smaug at the level of mRNA stability and/or translation 12, and Bicaudal C. Rpn7 is a proteasome regulatory particle subunit and was selected because of the observed enrichment for GO terms related to ‘proteasome regulatory particle’. Likewise, because of enrichment for the GO term ‘glucose metabolic process’ and the KEGG pathway ‘glycolysis/gluconeogenesis’, we assayed hexokinase, the first enzyme in glycolysis, and phosphofructokinase, which represents a critical point of regulation [smaug-mutant embryos [Drosophila oogenesis [smaug-mutant embryos could disrupt normal patterns of post-transcriptional regulation.To assess the role of Smaug in regulating the expression of the new target mRNAs, we selected five for further analysis: gulation ,59 and cgulation . Thus, S embryos . Bicaudaogenesis . Thus, Bsmaug-mutant embryos versus wild-type ones 12, Bicaudal C; Figure  Figures  and 10, Drosophila SRE consensus is more restricted than previously thought [Here we have used genome-wide approaches to identify mRNAs that are bound by Smaug and those that are translationally repressed by Smaug. Our results show that the presence of SREs is predictive of both binding and translational repression and, consistent with previous work on the yeast and human Smaug homologs ,45-48, i thought . Integra thought  has led nanos and Hsp83, are differentially regulated by Smaug [nanos and Hsp83, Smaug both translationally represses and degrades a large fraction of its target mRNAs. We hypothesize that the extent to which Smaug regulates the translational repression and/or destabilization of its targets is likely to be a consequence of additional cis-elements within target mRNAs. For example, the Hsp83 3′ UTR contains a translational enhancer that may mitigate Smaug-mediated translational repression [nanos mRNA observed in the absence of Smaug suggests that additional cis-elements within the nanos transcript function in its destabilization.Previous work has firmly established that Smaug can both repress translation and induce degradation of target mRNAs. However, Smaug’s two well-characterized target transcripts, by Smaug ,28,31,33pression . SimilarSmaug functions in the localization and regulation of its target mRNAs at the posterior of the embryo ,31,34-37nanos and Hsp83 mRNA is the fact that transcripts found at the posterior of the embryo escape Smaug regulation. The molecular mechanisms that underlie this spatial regulation of Smaug function are not understood, but Oskar protein has been implicated in blocking Smaug function at the posterior and has been shown to physically interact with Smaug [torso mRNA carrying the first 96 nucleotides of the nanos mRNA’s 3′ UTR, which includes one of the nanos SREs, is repressed at both the anterior and posterior of the embryo [torso mRNA carrying the first 185 nucleotides of the nanos 3′ UTR, which contains both nanos SREs, is repressed at the anterior but is expressed at the posterior [cis-elements mapping within nucleotides 97 to 185 of the nanos 3′ UTR that localize nanos transcripts to the germ plasm [nanos mRNA expression in the germ plasm. Our identification of several dozen posterior-localized, Smaug-bound transcripts should facilitate identification of any additional cis-elements.A critical aspect of Smaug’s role in the regulation of th Smaug ,15,36,63th Smaug ,64. Thise embryo . In addiosterior . Taken trm plasm  and/or aOur analysis of the mRNAs that are bound by Smaug has identified a number of mRNAs that encode proteins that are involved in cell-cycle control and transcriptional regulation. Mis-regulation of one or more of these mRNAs could underlie the cell-cycle and transcriptional defects that occur in the absence of Smaug. Our data also suggest that Smaug has several new and unanticipated biological functions, including control of protein folding and degradation, lipid droplet function and basic metabolism.smaug mutants.Our data suggest that Smaug downregulates the expression of 9 of the 19 subunits of the proteasome regulatory particle and 4 out of the 8 that encode the TRiC/CCT complex. In addition, three of the four remaining TRiC/CCT mRNAs and eight of the remaining ten proteasome regulatory particle mRNAs require Smaug for their degradation and/or translational repression .Lipid droplets are storage sites of triacylglycerols, hydrolysis of which yields fatty acids that can be metabolized for energy or serve as a source of membrane precursors. Thus, lipid droplets could function as the source of membrane precursors that are required during blastoderm cellularization, a process during which plasma membrane invaginates around the syncytial nuclei that are found at the embryo’s periphery. A role for Smaug in regulating lipid droplet function is intriguing as Drosophila embryos but are rapidly degraded [Drosophila, but also in frog and mammalian early embryos [Our data also suggest a widespread role for Smaug in regulating metabolism in the early embryo, including a role for Smaug in down-regulation of glycolysis. Previous work has suggested that maternal mRNAs encoding the glycolytic enzymes are present in early degraded -75. Glyc embryos ,77 but tOur data are consistent with Smaug directly regulating a large number of mRNAs in early embryos through translational repression and/or transcript degradation. This raises the question as to whether all of these repressive interactions are biologically important.trans-acting factors that control mRNA translation and/or stability will regulate a large number of transcripts and, as such, the same concepts should apply.In one model only a subset of Smaug’s targets are biologically relevant because the extent of downregulation by Smaug varies in a target-dependent manner. For the biologically relevant target transcripts, Smaug would effectively turn off their expression while, for the others, Smaug would reduce their expression insufficiently to have an effect on their biological function. A similar type of model has been suggested for repression mediated by individual miRNAs, which, as in the case of Smaug, regulate the expression of a large number of transcripts . Given tsmaug-mutant embryos.An alternative, but not mutually exclusive, model is that factors like Smaug, which repress the expression of a large number of mRNAs, do so in order to limit the total levels of available mRNA within a cell. This reduction could result from both Smaug-directed degradation of transcripts and/or Smaug-mediated translational repression, the former eliminating the mRNAs and the latter removing them from the pool of available mRNAs. In this model, Smaug would function to control the competition among transcripts for limiting cellular components, such as the translation machinery. We note, however, that our data do not support this model - at least in regard to the translation machinery - as we fail to see a decrease in the translation of mRNAs that are not bound by Smaug in Drosophila embryogenesis, nuclei are transcriptionally silent and development is driven by mRNAs synthesized by the mother and deposited into the egg during oogenesis. Subsequently, one- to two-thirds of these maternal mRNAs are degraded [A third model to explain the biological significance of the regulation of a large number of mRNAs by a single factor relates to a requirement for large-scale changes in a cell’s function. Under such a circumstance one might expect that the expression of a large number of mRNAs must be translationally repressed and/or degraded while a new group of genes is activated. For example, during the first 2 to 3 hours of degraded ,9,12 - tdegraded ,37 or ardegraded . Thus, SDrosophila embryo and a significant fraction of these mRNAs are both translationally repressed and destabilized. Smaug plays a major role in controlling the expression of mRNAs that are localized to the posterior of the embryo and regulates a diverse set of processes, including metabolism, lipid droplet function, protein folding and protein stability.Smaug directly regulates the expression of a large number of mRNAs in the early w1118 stock maintained in a large-scale Drosophila culture. smaug mutant alleles included smaug1[smaug47. The smaug47 allele was generated via imprecise excision of a P-element (GE21229) using standard methods [GE21229 is inserted 2,499 bp 5′ of the smaug start codon and 20 bp downsteam of the transcriptional start site of the smaug-RB isoform. All isoforms are defined as described at [smaug1 allele showed homozygous maternal effect lethality [smaug30 and smaug47, showed deletions removing large portions of the smaug gene, but not affecting the neighboring upstream and downstream genes - CG5087 and CG5280, respectively. Sequencing revealed that the smaug30 allele is a 4,514 bp deletion of the smaug gene beginning 2,480 bp 5′ of and ending 2,034 bp 3′ of the smaug start codon. Sequencing also showed that this allele retains 933 bp of the P-element. This deletion removes 2,020 of 2,997 bp of the open reading frame of smaug RA, RB, RC, and RE isoforms. The smaug47 allele is a 5,542 bp deletion beginning 2,483 bp 5′ of and ending 3,059 bp 3′ of the smaug start codon. This deletion leaves 39 bp of the open reading frame in the smaug RA, RB, RC, and RE isoforms.Wild-type flies consisted of the ed smaug1 and smau methods . GE21229ribed at . The oriethality  and we r2, 1 mM dithiothreitol (DTT), 1× protease inhibitor cocktail ). Extracts were centrifuged for 10 minutes at 4°C, and the supernatant was supplemented with 9 M urea to a final concentration of 2 M. Protein A beads were pre-incubated with either guinea pig anti-Smaug antibody [Embryos collected at 0 to 3 hours post-egglaying were dechorionated with 50% bleach and homogenized in a minimal volume of RIP lysis buffer  benzenesulfonyl fluoride hydrochloride (AEBSF), 2 μg/ml leupeptin, 2 mM benzamidine, 2 μg/ml pepstatin A). Lysed samples were diluted 1 in 12.5 in polysome lysis buffer and 30% triton was added to a final concentration of 1% and then spun at 6,000xg for 10 minutes and the resulting supernatant was diluted in polysome lysis buffer supplemented with 1% Triton to an A260 of 12.5.Embryos laid by wild-type or 2, 500 mM NaCl, 50 mM Tris pH 7.5 was created using a BioComp Model 117 Gradient Mate gradient maker  using a rotation angle of 80.5° and a rotation speed of 18 rpm for 1 minute and 58 seconds. After chilling the polysome gradient on ice, 400 μl of diluted embryo extract was loaded onto the top of the gradient, which was then spun at 36,000 rpm in a Beckman SW 41 Ti rotor for 2.5 hours. The gradients were then separated into four pools . A fixed amount of exogenous in vitro transcribed Arabidopsis spike-in RNAs was then added to each pool. Our microarrays contain probes that allow for the detection of these RNAs allowing for subsequent data normalization. We added 20% SDS, 0.5 M EDTA and 20 mg/ml proteinase K to each fraction to final concentrations of 0.8%, 0.01 M and 0.128 mg/ml, respectively, and then incubated them for 30 minutes at room temperature. Glycogen was then added to a final concentration of 80 μg/ml and samples were ethanol precipitated overnight and the resulting pellet was washed with 75% ethanol and resuspended in phenol-saturated water. Following two phenol-chloroform extractions, samples were precipitated by the addition of 7.5 M LiCl to a final concentration of 1.5 M and an overnight incubation at 4°C. The resulting pellet was washed with 75% ethanol, resuspended in water and ethanol precipitated in the presence of 80 μg/ml of glycogen and 0.3 M sodium acetate. The precipitate was then washed with 75% ethanol and resuspended in water. The integrity of RNA in each pool was confirmed via northern blots, which were probed for nanos mRNA . The lysed samples were split in half and cycloheximide was added to one sample to a final concentration of 0.5 mg/ml and puromycin was added to the other sample to a final concentration of 2 mM. Samples were left on ice for 20 minutes and then incubated at 30°C for 10 minutes. Both samples were then diluted 1 in 12.5 with polysome lysis buffer supplemented with either puromycin or cycloheximide and 30% triton was added to a final concentration of 1%. The samples were then spun at 6,000xg for 10 minutes and the supernatant was diluted with polysome lysis buffer supplemented with either puromycin or cycloheximide to give an A260 of 12.5 and these diluted samples were then fractionated as described above.For experiments that utilized puromycin embryos were lysed in puromycin lysis buffer  that contain probes for Arabidopsis spike-in RNAs (see below). Microarrays were scanned using Genepix Pro software on a Molecular Devices  GenePix 4000B or 4300A scanner and quantified using Nimblescan.RNA samples from RIP experiments were used to prepare single-stranded cDNA using anchored oligo(dT) primers and the Canadian iewed at . Anchore2 and the log2 using ‘two class unpaired analysis’ in SAM (FDR <5%).RIP microarrays were normalized using the Robust Multi-array Average (RMA) quantile method and transcripts that were expressed at levels significantly above background in total RNA collected 0 to 3 hours post-egglaying were determined using ‘one class unpaired analysis’ in SAM and transcripts with an FDR >5% were excluded from further analysis of the RIP data. mRNAs that were reproducibly enriched in Smaug RIPs versus control RIPs were then identified by comparing the logArabidopsis spike-in RNAs. The hybridization signals from the spike-in RNAs were utilized by applying a linear transformation to each sample with the parameters, a and b, determined by fitting the linear function Y = aX + b using the spike-in signal, where X is the expression level of the spike-in RNAs in a specific sample, and Y is the mean expression level of the spike-in RNAs across all the samples. The genes significantly expressed in wild-type or smaug-mutant embryos in each of pools 1, 2, 3 and 4 were separately determined using ‘one class unpaired analysis’ in SAM (FDR <5%). We defined the genes significantly expressed in the wild-type and smaug-mutant embryos as the union of the significantly expressed genes from the four fractions derived from that genotype. We then compared these two lists and defined their intersection as the list of genes significantly expressed in both wild-type and smaug-mutant embryos, and restricted all the following analysis to the genes on this list. To determine the list of genes with different polysome association in wild-type and smaug mutants, we compared the geometric mean of the expression level in pools 3 and 4  in wild-type and smaug-mutant embryos, using ‘two class unpaired analysis’ in SAM.Polysome microarrays were normalized using the RMA quantile method. We further normalized the data using . Levels of RpL32 mRNA in each immunoprecipitated sample were used to normalize the levels of the experimental mRNA in that sample.cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) and random primers according to the manufacturer’s instructions. Quantitative PCR reactions were carried out using the BioRad  Real-time PCR system as per the manufacturer’s instructionssmaug-mutant and wild-type embryo samples for the top N and bottom N Smaug-binders  was calculated using the R (version 2.14.1) algorithm sm.density in the sm package (version 2.2-4.1). The sm.density algorithm provided smoothed density estimates for 100 values of change in TI for the top and bottom N binders, with the 100 values calculated by the sm.density algorithm with each smoothed density estimate.The fraction of genes expected to have changed in TI in N and bottom N Smaug-binders . First, for each gene, the density of its change in TI under the positive and negative distributions as defined by N top and bottom binders, respectively, was set to be equal to that of the closest grid point higher than the change in TI. We then estimated the probability that a gene was a positive by taking the ratio of its density under the positive distribution and the sum of its densities under the positive and negative distributions. This procedure was repeated for each of our three sets of positive and negative distributions to give us three different sets of probabilities. For each of these three sets of probabilities, we estimated the expected number of Smaug targets for that set by summing the ‘positive probabilities’ for all genes.For every gene expressed in our polysome gradient experiments, the probability that it was a positive target  was estimated using the top 0-4 on a non-specific stem. First, we performed an initial scan using RNAplfold (version 2.0.7) [et al.[We used a two-step procedure to computationally predict SRE stem/loops carrying the loop sequence CNGGNn 2.0.7)  with the) [et al.. Potenti) [et al. routine et al.[These analyses were carried out as described in Laver et al..Antibodies against Rpn7 , Su(z)12  and Bicasmaug-mutant embryos were collected from females homozygous for the smaug47 allele, while 'wild-type' embryos were collected from females homozygous for the smaug47 allele that were also homozygous for a genomic smaug rescue transgene that was inserted at the attP40 site on the second chromosome by Genetic Services  using PhiC31 integrase-mediated transgenesis [smaug transgene, which rescues the smaug mutant phenotype, is a modified version of a previously generated smaug rescue construct [For enzyme assays sgenesis . The smaonstruct  that exp2) and assayed in extraction buffer supplemented with 16.5 mM ATP, 20 mM beta-NADP and 0.67 M glucose. Hexokinase catalytic activity was measured by adding Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase  dissolved at a concentration of 300 IU/ml in extraction buffer. The production of beta-NADPH was monitored at 340 nm in a Thermo SPECTRONIC spectrophotometer. Experiments were conducted with an amount of embryo extract that was in the linear range of the assay and enzyme activity was normalized to protein concentrations in each homogenate measured using the Bradford assay (BioRad). Enzyme activity was calculated using the formula: Units/mg protein = ΔA340/minute ÷ 6.22 × mg enzyme/ml reaction mixture, as described by Worthington [For the hexokinase assay, embryos were homogenized in extraction buffer , which converts fructose-6-phosphate and ATP to fructose-diphosphate and ADP. The final product, NADH, reduces a colorless probe to a colored product with strong absorbance at 450 nm. The absorbance was measured with a TECAN INFINITE m200 microplate reader. Experiments were conducted with an amount of embryo extract that was in the linear range of the assay and enzyme activity was normalized to protein concentration.The data reported in this study have been deposited in NCBI’s GEO . The RIPAEBSF: 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; AGO: Argonaute; bp: Base pair; CCT: Chaperonin containing TCP-1; DTT: Dithiothreitol; FDR: False discovery rate; GEO: Gene Expression Omnibus; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; miRNA: MicroRNA; RIP: RNA co-immunoprecipitation; RIP-Chip: RNA co-immunoprecipitations followed by microarray analysis; RMA: Robust Multi-array Average; RT-qPCR: Reverse transcription followed by quantitative polymerase chain reaction; SAM: Significance Analysis of Microarrays; SRE: Smaug recognition element; TI: Translation index; TRiC: TCP1-ring complex; UTR: Untranslated region.The authors declare that they have no competing interests.smaug30 and smaug47 alleles by MHKC; mRNA localization enrichment analysis by JDL; enzyme assays by ZY and construction of the smaug rescue transgene by NUS. CAS wrote the first draft of the manuscript with input from LC and JGD and the manuscript was revised by CAS and HDL with input from all other authors. All authors read and approved the final manuscript.HDL and CAS conceived of the project and designed its overall goals. LC and JDL are co-supervised by HDL and CAS; JGD and MHKC by CAS; XL by QM and HDL; ZY and NUS by HDL. All experiments and analyses were carried out by the indicated trainees under the supervision of their respective supervisors. All microarray experiments were conducted with advice of JTW; RIP-Chip and RT-qPCR validation was carried out by LC; polysome gradient-microarrays by JGD; RIP-Chip data analysis by LC with the assistance of XL; polysome gradient-microarray data were normalized by XL and JGD; subsequent polysome gradient-microarray data analyses and SRE-enrichment analyses were carried out by JGD with suggestions from QM; comparisons of binding, translation and stability data sets were carried out by JGD and LC; GO term analysis by LC; generation of the A table listing replicate-to-replicate comparisons of transcript microarray signal intensities from RIP-Chip experiments.Click here for fileA table listing the genes that encode Smaug-bound mRNAs .Click here for fileA figure showing northern analysis of the distribution of 18S ribosomal RNA in polysome gradients run with or without EDTA.Click here for fileComparison of wild-type TIs calculated with and without data from pool 2.Click here for fileComparison of the change in TIs in smaug-mutant versus wild type with and without data from pool 2.Click here for fileTables listing the replicate-to-replicate comparisons of transcript microarray signal intensities from wild-type polysome gradients, smaug-mutant polysome gradients, and smaug-mutant polysome gradients with or without puromycin.Click here for fileA table listing the genes that encode mRNAs that are translationally repressed by Smaug .Click here for fileA figure comparing the TIs of the bottom 250, 500 and 1,000 Smaug binders in wild-type and smaug-mutant embryos.Click here for fileA figure showing the kernel density plots comparing the change in TI in smaug-mutant versus wild-type embryos of the top and bottom 250, 500 and 1,000 Smaug binders.Click here for fileA figure showing the FDR-based rank of genes from the polysome gradient-microarrays.Click here for fileThe simultaneous overlap between the Smaug-bound mRNAs and those mRNAs that are regulated by Smaug at the level of translational repression and degradation.Click here for fileCumulative density plots that show the SRE scores for the 5′ UTR, open reading frame and 3′ UTR of Smaug-target mRNAs that are translationally repressed, degraded and both repressed and degraded.Click here for fileA table listing the Fly-FISH degradation categories and localization patterns enriched among Smaug-bound mRNAs.Click here for fileA figure showing the Fly-FISH degradation categories and localization patterns enriched among Smaug-bound mRNAs.Click here for fileA table listing the genes that encode Smaug-bound mRNAs that are localized to the posterior of the embryo.Click here for fileA table listing the genes that encode Smaug-bound mRNAs that are annotated as having roles in cell cycle, checkpoint response and/or response to DNA damage.Click here for fileA table listing the genes that encode Smaug-bound mRNAs annotated as having roles in transcription and/or chromatin.Click here for fileA table listing the genes that encode Smaug-bound mRNAs encoding proteins in the Chaperonin Cpn60/TCP-1 family.Click here for fileA table listing the genes that encode Smaug-bound mRNAs encoding proteins found in the proteasome regulatory particle and the ubiquitin proteasome pathway.Click here for fileA table listing the genes that encode Smaug-bound mRNAs encoding proteins associated with lipid droplets.Click here for fileA table listing the genes that encode Smaug-bound mRNAs encoding metabolic enzymes.Click here for fileA table listing the genes that encode Smaug-bound mRNAs encoding enzymes involved in glycolysis and related pathways.Click here for fileA northern blot that assesses the integrity of nanos mRNA in polysome gradient pools.Click here for file


According to the Yerkes–Dodson's law, the relationship between arousal and performance resembles an inverted U-shape curve with a moderate level of arousal being associated with an optimal level of performance  and are better at distinguishing the target stimuli from the distractors (α), in comparison with the low arousal condition. In addition, we have found that as the level of participants' arousal increases the capacity of their visual short-term memory (K) tends to decrease  signal to the visual cortex via reciprocal neural connections, thus ensuring that a threatening object in the visual field receives a greater allocation of attentional processing resources (e.g., Sugase et al., If arousal is capable of increasing attentional selectivity independently of any emotional manipulation, then why do van Steenbergen and colleagues find a difference between the positive and negative conditions in their study? (Previous studies by, e.g., McNamara and Fisch, In principle, then, arousal and attentional selectivity can be separated, although arousal serves to increase the effects of attentional selectivity. We propose that a general increase in cortical arousal modulated via brain arousal systems such as the RAS is sufficient to cause a narrowing of the attentional focus in line with the Easterbrook hypothesis. On top of this, however, the appearance of threatening stimuli in an otherwise neutral environment (cf. McNamara and Fisch, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Solanum lycopersicum) plants subjected to water stress as a model system, we standardized a sensitive and accurate protocol for detecting and quantifying DMSP pool sizes, and potential DMS emissions, in cryoextracted leaves. The method relies on the determination of DMS free and from DMSP pools before and after the alkaline hydrolysis via Headspace-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS). We found a significant (2.5 time) increase of DMSP content in water-stressed leaves reflecting clear stress to the photosynthetic apparatus. We hypothesize that increased DMSP, and in turn DMS, in water-stressed leaves are produced by carbon sources other than direct photosynthesis, and function to protect plants either osmotically or as antioxidants. Finally, our results suggest that SPME is a powerful and suitable technique for the detection and quantification of biogenic gasses in trace amounts.Dimethylsulfoniopropionate (DMSP) and dimethyl sulphide (DMS) are compounds found mainly in marine phytoplankton and in some halophytic plants. DMS is a globally important biogenic volatile in regulating of global sulfur cycle and planetary albedo, whereas DMSP is involved in the maintenance of plant-environment homeostasis. Plants emit minute amounts of DMS compared to marine phytoplankton and there is a need for hypersensitive analytic techniques to enable its quantification in plants. Solid Phase Micro Extraction from Head Space (HS-SPME) is a simple, rapid, solvent-free and cost-effective extraction mode, which can be easily hyphenated with GC-MS for the analysis of volatile organic compounds. Using tomato ( Biogenic DMS (dimethyl sulfide), a compound mainly emitted by marine environment, is a significant natural source of tropospheric sulfur . DMS is Figure 1). Because methionine synthesis requires nitrogen and sulfur, it was shown that nitrogen availability affected the concentration of DMSP , the crop plant Saccharum (sugarcane), the wild mesophyte W. biflora, a moderately salt-tolerant species, and seven tropical species are reported to emit DMS  and unsuitability of sulfur containing volatiles for thermal desorption techniques  have hinSolanum lycopersicum), using HS-SPME-GC-MS, showed that tomato leaves emitted DMS upon alkaline hydrolysis of its leaf extracts (data not shown). Tomato is an important model species for VOCs analysis  were germinated in 0.7 dm3 pots filled with a mixture of peat:sand (1:1). Plants were grown in a growth chamber under controlled conditions , regularly watered to full pot capacity and fertilized once a week in order to supply mineral nutrients at free-access rates until the day of the imposition of water stress (potweight). Then, part of the plants (n = 4) was water-stressed by withholding water, whereas the other part (n = 4) was daily watered to pot capacity. As indicator of soil water availability, the FTSW /, where Dailypotweight is the weight of the water-stressed plants recorded during the water stress cycle and Finalpotweight is the “pot weight” at which stomatal conductance approached zero. DMSP analysis was performed at FTSW of 30% (FTSW30), when stomatal conductance (gs) decreased on average about 50% of the value FTSW100 plants.Seeds of a dwarf variety of r stress . The expthe FTSW  was calca fluorescence measurements were performed using a Li-6400 IRGA . A portion of a leaf was enclosed in a 2 cm2 cuvette, and exposed to a photosynthetic photon flux density of 700 μmol m-2 s-1, at a temperature of 25°C and with the relative humidity of the air within the cuvette ranging between 45 and 55%. By infrared gas analysis, steady-state net photosynthesis (A) and gs were measured. By chlorophyll fluorescence [a sensitive indicator of PSII photochemistry (= ΔF/Fs/Fs)/ and by comparing retention times and mass spectrum with those of an injected authentic standard, DMS standard was purchased by Sigma-Aldrich (Milano) and its assay was ≥98%.An Agilent 7820 GC-chromatograph equipped with a 5977A MSD with EI ionization operating at 70 eV was used for analysis. A three-phase DVB/Carboxen/PDMS 75-μm 2 cm SPME fiber  was exposed in the headspace of the vials at 40°C for 10 min for VOCs sampling. DMS has a boiling temperature of 37.34°C, so we used 40°C as SPME incubation temperature to favor the partitioning of DMS on the headspace for fiber’s uptake. The best extraction time was established to be 10 min, which afforded DMS uptake without causing fiber’s saturation phenomena as established in preliminary experiments. A Gerstel MPS2 XL autosampler equipped with a magnetic transportation adapter and a temperature controlled agitator (250 rpm with on/cycles of 10 s) was used for ensuring consistent SPME extraction conditions. This device ensured homogeneous sample mixing and favored the partitioning of VOCs into the headspace during SPME extraction. Chromatographic conditions were: column HP-Innowax ; injection temperature 250°C, splitless mode, oven program 40° for 1 min, rate of 5°C minFigure 2). Control analyses on specimens before hydrolysis, indicated that the amounts of free DMS eventually present was negligible as compared to DMS after hydrolysis . On the other hand, a comparison of the “free DMS” between well-watered and water-stressed plants revealed that “free DMS” in water-stressed leaves was significantly higher compared to that of well-watered leaves, albeit the amounts were negligible as compared to the DMS recovered after alkaline hydrolysis . The limit of detection (LOD), the limit of quantification (LOQ) were calculated as reported by The DMS standard was also used to quantify DMSP with a calibration curve in the range of 50–2000 pg  .Physiological data and DMS variations were tested using student’s A, ϕPSII and qP showed a slight, non-significant decline in well-watered plants (FTSW100), but gs (the indicator used to assess stress level) remained stable at FTSW100 (data not shown). As expected . Stomatal conductance decreased from 0.20 ± 0.20 to 0.09 ± 0.01 mmol s-1  , whereas A dropped from 11 ± 0.86 to the value of 4.9 ± 0.64 μmol m-2s-1 when the FTSW of water-stressed S. lycopersicum pots reached 30% compared to FTSW100 . Similarly, at FTSW30, ϕPSII  and qP  had a reduction of approximately 54 and 60%, respectively, compared to FTSW100 plants.During the time course of the experiment expected , all the-2), was determined by using the calibration curve of DMS .We have developed a sensitive and reliable methodology to detect DMSP and DMS in higher plants. The DMSP amount, expressed on a leaf area basis , and statistical analysis performed showed significant differences between the two treatments with a P < 0.01 .The DMSP content retrieved in FTSWIn general, DMS emission by plants has been elusive, and only occasionally reported . EmissioSpartina alterniflora  and 1025.29 (±96.27) pg, respectively. Consequently, the calibration curve obtained by SPME technique allowed estimating DMSP content in S. lycopersicum leaves with an accuracy, calculated in the highest levels of calibration range, of 97.7% (Table 1). SPME DVB/Carboxen/PDMS fibers efficiently captured DMS from the headspace vials at the selected experimental conditions allowing good reproducibility of results. A possible limitation of this technical approach is its application for quantitative analysis, because some phenomena  may occur along with the experiments and, as a consequence, alter the linearity of calibration response. On the other hand, fiber saturation phenomena can be eventually corrected by using suitable internal standards . This may of course represent a problem, if the amounts of DMS in samples are close to the LOD (32.2 pg), which is not our case. As shown in Table 1, at the lowest calibration levels, the accuracy of the methodology was not as good (49.7%) as at highest calibration levels (97.7%), despite an high precision along the of calibration range.The LOD, the LOQ, the accuracy and the precision of the calibration curve were reported in tandards . As an aThe Headspace-Solid Phase Microextraction (HS-SPME) technique used for assessing both DMSP content and the potential emission of DMS in tomato resulted highly effective in the detection and quantification of these compounds. DMSP and DMS are mostly found in marine ecosystems and in halophytic plants, where they serve a protective function against different abiotic stresses. In our knowledge, this study is the first to report DMSP and DMS in higher plants such as tomato. Using this novel, powerful methodology, we found a strong positive effect of water stress on DMSP content in tomato leaves experiencing an inhibition of their photosynthetic apparatus. These results suggest a potential functional role for DMSP and for its volatile product DMS in terrestrial vascular plants.SC, LC, and MC designed the research. SC and LC performed the experiment; SC, LC, and SK analyzed data; All authors contributed to write the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The plasma growth enlarged, over one hour, the nuclei to graphene sheets larger than one hundred nm2 in area. Characterization by electron and X-ray diffraction, high-resolution transmission electron microscopy images provide evidence for the presence of monolayer graphene sheets.In this paper, we demonstrate the seeded growth of graphene under a plasma chemical vapor deposition condition. First, we fabricate graphene nanopowders (~5 nm) by ball-milling commercial multi-wall carbon nanotubes. The graphene nanoparticles were subsequently subject to a direct current plasma generated in a 100 Torr 10%CH Graphene layers, as a result of weak van der Waals attraction, cause the material to adopt a stacked multilayer configuration24527910111291011121316Recently we have shown that multi-wall carbon nanotubes (MWNTs) are composed of AA′ stacked graphene helices where width is of the order of a few nanometers18XRD analysis of the samples provides more specific information as to the structural changes of the MWNTs following milling. The data showed that 002, 020, 004, and 200 peaks of orthorhombic AA′ graphene stacked MWNTs2 in area when the nanographene seeds were subject to a direct current plasma, as shown in the HRTEM image in 582 exhibiting layer edges are evident in The GNPs grew to form graphene plates of hundred nm45The broad 002 peak with the d-value of 3.60 Å of the milled samples  indicateμm (see Methods). The intensity ratio of 2D peak to G peak, I2D/IG, is about 0.62, and this value is similar to those for the graphene samples grown on Co or Ni (see Table 1 of Ref. The intersections (black arrows) in We propose a mechanism for the plasma growth of graphene via the lateral growth of the zigzag lines of graphene which subtend angles of 120°  with twenty steel balls (each 2 g) and processed by a conventional (Spex) milling apparatus for 1 ~ 6 hours. For the novel CVD growth, the purified GNPs which were prepared on a molybdenum substrate were subjected to a direct current plasma generated at 100 Torr of a 10%CH4-90%H2 gas mixture for I hour. The temperature of the substrate was kept at 1,200°C.Commercial MWNTs , prepared by VCCVD technique, were used as a starting material for the fabrication of graphene nanopowders (GNPs) to be served as nuclei for the seeded plasma growth of graphene. Diameters and lengths of the MWNT samples were 10–20 nm and 3–20 μm.The milled samples, i.e., GNPs were confirmed by HRTEM observation as well as X-ray diffraction pattern analysis. The GNPs including metal particles originated from the steel balls were purified through a solution treatment where the samples are dispersed in ethyl alcohol with sonication and the metal impurities are removed by magnets. HRTEM observation for grown graphene sheets were performed by a JEM-2100F operating at 200 kV and a FEI Titan Cubed with aberration corrector and a monochrometer operating at 80 kV. Raman analysis was performed by a Renishaw In-Via Raman Microscope with laser excitation of 532 nm and spot size of 1–2 J.L. designed the experiments and performed data analysis. S.L., Y.P. and K.L. carried out the experiments. Y.K. worked on XRD analysis of the materials. J.K. and B.M. worked on HRTEM observation. J.L. and P.J. co-wrote the paper.


We investigate the networked nature of the Japanese credit market. Our investigation is performed with tools of network science. In our investigation we perform community detection with an algorithm which is identifying communities composed of both banks and firms. We show that the communities obtained by directly working on the bipartite network carry information about the networked nature of the Japanese credit market. Our analysis is performed for each calendar year during the time period from 1980 to 2011. To investigate the time evolution of the networked structure of the credit market we introduce a new statistical method to track the time evolution of detected communities. We then characterize the time evolution of communities by detecting for each time evolving set of communities the over-expression of attributes of firms and banks. Specifically, we consider as attributes the economic sector and the geographical location of firms and the type of banks. In our 32-year-long analysis we detect a persistence of the over-expression of attributes of communities of banks and firms together with a slow dynamic of changes from some specific attributes to new ones. Our empirical observations show that the credit market in Japan is a networked market where the type of banks, geographical location of firms and banks, and economic sector of the firm play a role in shaping the credit relationships between banks and firms. One key economic questions in the description of markets is whether a market is characterized by an underlying networked structure . In factIn our approach we consider the Japanese credit market as a bipartite network of banks and firms. Bipartite networks are quite common in complex systems. Classic examples are networks of actors and movies, board members and companies, authors and scientific papers, etc. The customary investigation of bipartite networks is done by performing a one-mode projection for one or both of the two sets of vertices . This apComplex systems can be monitored over long periods of time. The analysis and modeling of these systems in terms of networks can be done by considering the network connections observed for the whole time period and/or by analyzing the network at successive time intervals as, for example, daily, weekly, monthly or yearly time intervals. Here we investigate the bipartite network of credit relationships yearly from 1980 to 2011 by obtaining 32 distinct credit networks. For each year we obtain the credit network and its community structure by using Barber’s BRIM (bipartite recursively induced modules) algorithm [To investigate the time evolution of the communities detected for different years, and, more generally, the time evolution of the underlying networked structure, we need a method that tracks the dynamics of the different communities over time. This method is needed because we are interested into the dynamics of the communities and due to an unavoidable level of uncertainty related to the statistical nature of the community detection process. In this paper, we propose a method which is able to track the time evolution of communities obtained at successive periods of time. The method is more general than just using transition probabilities and it is based on a statistical test which is robust with respect to the heterogeneity of the size of communities and therefore works for communities of different sizes. Our statistical validation procedure of the time evolution of communities allow us to track efficiently the evolution of the communities over time.The dynamics of the networked structure of the system is investigated by characterizing the communities obtained for different years in terms of the over-expression of attributes of banks and firms concerning (i) the regional location of firms, (ii) economic sectors of firms, and (iii) the types of banks. The statistical validation of the over-expressed attributes is done by using the method presented in . This meThe paper is organized as follows. In Subsection “Dataset” of Material and Methods we briefly discuss our dataset. Subsection “Community detection in bipartite networks” discusses community detection in the bipartite network of the Japanese credit market. Subsection of Results entitled “Time evolution of communities” introduces a method used to track the time evolution of communities detected in networks and obtained for successive time periods. Subsection “Over-expression of attributes” presents the empirical results obtained in the characterization of the over-expression of attributes of banks and firms in each community over the years and in Section “Discussion” we draw our conclusions.Our dataset is based on a survey of firms quoted in the Japanese stock exchange markets  and in Japanese over-the-counter (OTC) markets. The data were compiled from the firms’ financial statements and survey by Nikkei Media Marketing, Inc. in Tokyo, and are commercially available . The surThe number of banks of the database changes year by year by selecting the partition associated with the highest modularity among the 20 performed runs. It was 225 in 1980, remained approximately constant until 2001 and then decreased to 166 in 2011. The number of firms was first increasing from the value of 1414 in 1980 to the value of 3034 in 2006 and then decreasing to the value of 2706 in 2011. The number of firms increased from 1802 in 1995 to 2602 in 1996 in the presence of the first inclusion of the OTC firms in the database. During the same years the number of banks increased from 219 to 226. The density of links in the bipartite network defined as number of observed links over number of potential links was on average decreasing from the value of 0.0867 in 1980 to the value of 0.0398 in 2011. The variation of the density of links was not too large during the first inclusion of the OTC firm. In fact the density of links decreased from 0.0721 to 0.0601 from 1995 to 1996. In the supporting information we provide a summary statistics for the banks and firms of our database. Specifically, for each year we provide the occurrence of the different type of attributes characterizing banks and firms see  Tables. The Japanese credit market has been previously analyzed by considering one-mode projected networks , an eigeConcerning financial institutions, commercial banks are long-term, city, regional (primary and secondary), trust banks, insurance banks and government-related financial institutions including credit associations but excluding the Bank of Japan. We remark that failed banks are included until the year of failure, and that merger and acquisition of banks are processed consistently to identify surviving banks. For quoted firms, those who are active in each investigated calendar year are all included even if they failed later during the considered years.i and firm j when a credit relation (short and/or long) is present between i and j. Links are described by a binary variable (just indicating the presence or absence or a credit relationship), i.e., in the present investigation the bipartite network is an unweighted network.In our bipartite network a link is present between bank Community detection in large and dense bipartite networks has been considered in the past years by several authors , 9–12 anThe algorithm of Barber  is the oIn our analysis we have repeated the application of BRIM community detection algorithm a number of times for each year we investigate. Specifically, for each investigated year in each run we apply the algorithm 100 times and we perform 20 independent runs.To quantitatively evaluate the differences which are present among the partitions obtained in the 20 independent runs performed for each calendar year, we evaluate the adjusted Rand index (ARI)  among alTo provide an indication of the differences observed among the partitions obtained in independent runs, in t + 1 in which one detects an over-expressed amount of elements of a given community of year t. The community detection procedure has a certain degree of stochasticity and degeneracy with respect to small differences of the fitness measure and therefore the membership of an element into a given community might also just be due to chance. We therefore need a method detecting over-expression of the same composition in communities of two successive years that is based on a carefully devised statistical procedure which is robust to the size heterogeneity of the different communities.The communities detected by using the BRIM algorithm and discussed in the previous section are obtained year by year. It is therefore of interest to properly put communities detected on a given year in relation with communities detected in the following year. A time evolution of the communities can be detected by considering what are the communities of year t there are Nt communities i = 1, ⋯, Nt and in period t + 1 there are Nt+1 other communities j = 1, ⋯, Nt+1. For all the t we search amongst all Nt+1 communities of period t + 1 which communities t. Specifically, let us call Nt,t+1 the number of distinct vertices in the two consecutive periods t and t + 1. The probability that p-valuep-value threshold pt, the above methodology gives us a way to select the communities in year t + 1 that are linked to a given community in year t in a statistically robust way.We propose the following method. Suppose that in period p-value threshold must be corrected to take into account that we are performing a multiple hypothesis test comparison. Indeed, for each pair of consecutive periods we perform the test Nt ⋅ Nt+1 times against the null hypothesis of random distribution of elements among two partitions of communities of consecutive periods. Moreover we perform these tests for all pairs of consecutive years in our dataset, i.e., from 1980 to 2011. The most restrictive multiple hypothesis test correction is the Bonferroni correction, which prescribes that the modified p-value threshold pB is:pt = 0.01.To avoid the presence of false positive, the To better understand the key aspects of our method it is worth comparing the results of the statistical validation procedure with a simple indicator as the transition probability. Let us define the transition probability for community In t evolves into the largest community of year t + 1 for all the investigated years. In addition to this primary channel of community evolution we also detect that in some years other smaller communities merge part of them into the largest one . For the sake of clarity, among the communities merging into the largest community, only communities at one year distance from the largest community of each year are shown in the figure. In the following section we will investigate the over-expression of the attributes characterizing the elements of the largest community observed in each calendar year.In t with communities at year t + 1 but, differently than in t + 1 and communities at year t different from the one already shown in the figure. The additional incoming validated connections from other communities of the previous year are not shown to make the figure readable. As in In t to communities at step t + 1. In One way to visualize time evolving communities is the one using so-called alluvial diagrams . These dThe color pattern of The identification of bank-firm communities and the statistical validation of their time evolution provides the basis for detecting and understanding the networked structure of the credit system and its time evolution. A further step is to look for information characterizing the obtained communities and their time evolution. In other words it is important to characterize the communities in terms of attributes over-expressed by the elements belonging to the same communities with respect to an appropriate random null hypothesis. The method used here was introduce in . It shouRt where 0.01 is the univariate threshold and Rt = (NS + NP + NB) ⋅ Nt is the total number of tests done in the statistical validation of communities of the year t. More specifically, NS is the number of distinct economic sectors, NP is the number of distinct Japanese prefectures, NB is the number of types of banks, and Nt is the number of communities detected at year t. In this way, we minimize the number of false positive but unavoidably increase the number of false negative.The metadata available for the characterization of firms and banks allows us to identify the economic sector and the prefecture of the main office of firms and the type of bank. The summary statistics for each year and for each attribute is provided in the supporting information  Tables. The over-expressed prefectures are the prefectures of Kanagawa (14) and Tokyo (13), i.e. two prefectures of the so-called greater Tokyo area. The Table also shows the over-expression of the main economic sectors of the firms belonging to the community. The over-expressed economic sectors are Electric and electronic equipment (EEE) for the time period 1980–1993, and Services (S) and Wholesale trade (WT) for the time period 1996–2011.In summary the credit relationships preferentially detected in the largest community change type of over-expressed economic sector during the years 1995–1996. On the other hand, the over-expression of bank type and geographical location remains essentially the same before and after 1996.To rule out the possibility that the changes observed in the over-expression of economic sectors are due to the 1996 inclusion of the OTC firms we have repeated the community detection and the over-expression characterization by considering the listed firms and the OTC firms separately. Information about the number and attributes of listed and OTC firms is given in the supporting information  Tables.The results of the separate analyses are presented in In The second branch starts in 1980 and ends approximately in 2005 . The choice of a one-to-one correspondence between banks’ partitions and firms’ partitions also simplify our analysis of joint over-expression of attributes of banks and firms. With this approach we have been able to show the existence of layers of the credit market involving an over-expression of groups of firms characterized by specific (i) economic sectors, (ii) regional locations (prefectures), and (iii) specific types of banks. These empirical observations show that the credit market in Japan is a networked market.The robustness of our results is shown by the ability of our approach in detecting both the long term stability and the slow dynamics of the detected communities. The time evolving communities have been tracked from each year to the next one by using a newly introduced statistical method able to track the time evolution of communities detected in successive periods of time also in the presence of size heterogeneity of the communities. It is worth noting that our method presently used to track time evolution of communities can also be easily adapted to link communities detected in a multiplex network.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(TXT)Click here for additional data file.S11 Table(TXT)Click here for additional data file.S12 Table(TXT)Click here for additional data file.S13 Table(TXT)Click here for additional data file.S14 Table(TXT)Click here for additional data file.S15 Table(TXT)Click here for additional data file.


Boiling heat transfer (BHT) is a particularly efficient heat transport method because of the latent heat associated with the process. However, the efficiency of BHT decreases significantly with increasing wall temperature when the critical heat flux (CHF) is reached. Graphene has received much recent research attention for applications in thermal engineering due to its large thermal conductivity. In this study, graphene films of various thicknesses were deposited on a heated surface, and enhancements of BHT and CHF were investigated via pool-boiling experiments. In contrast to the well-known surface effects, including improved wettability and liquid spreading due to micron- and nanometer-scale structures, nanometer-scale folded edges of graphene films provided a clue of BHT improvement and only the thermal conductivity of the graphene layer could explain the dependence of the CHF on the thickness. The large thermal conductivity of the graphene films inhibited the formation of hot spots, thereby increasing the CHF. Finally, the provided empirical model could be suitable for prediction of CHF. Following the demonstration by Geim and Novoselov23810et al.−1m−1 (at room temperature). Although the thermal conductivity of these graphene films is significantly smaller than has been reported for single graphene flakes, it remains larger than that of many other substrates, and hence graphene remains attractive for heat transfer applications. However, despite the favorable heat transport properties, little information about applications of graphene on thermal system is available. The characteristics of a heated surface, including the surface wettability1314161821Since graphene typically exists in the form of ultra-thin microscale flakes, much effort has been invested to synthesize large-area films that retain the properties of graphene. Jang Here, we report that thin graphene films on a heated surface may result in more efficient dissipation of heat in the lateral direction, which inhibits the formation of dry/hot spots during boiling, leading to an increase and CHF. The role of graphene films on the heated surface was investigated using infrared high-speed visualization of the boiling process. We found that the graphene layer led to an increase in the CHF via lateral heat transport, which inhibited the formation of hot/dry spots. Pool-boiling experiments were carried out in the Pohang University of Science and Technology (POSTECH) pool-boiling experimental facility, which is illustrated in the schematic diagram shown in colloidV is the volume of the RGO flakes, A is the area of the film, δ is the thickness of the film, graphiteρ is the density of the graphite, and colloidρ is the density of the RGO colloid. The filtered graphene film was transferred to the surface of the silicon heater  high-speed visualization  for 2 minutes during each step of the experiment. At large heat fluxes, the power supply was shut down when a sudden increase in the wall temperature was observed, which corresponded to the CHF.Prior to each experiment, deionized (DI) water was boiled for 1 hour using a cartridge heater to degas the water. Two wires were soldered to each electrode to apply a voltage and measure the temperature using the correlation shown in All images of the infrared high-speed visualization were trimmed and processed using Matlab and Image J. Raw image data appeared too dark, and so nucleation could not be observed. Therefore, the brightness and contrast were adjusted for each experimental case; this adjustment did not result in a change in the recorded temperature. One- and two-dimensional data arrays of the temperature distribution were extracted for the selected domain to analyze the temperature distribution and the changes with time thereof. The overall temperature distribution, one-dimensional temperature profiles, and temperature plots were obtained from the extracted data.H.S.A. designed the experiments and all procedures, and was the main author of the paper. J.M.K. carried out the experiments, provided the figure artwork, performed the data analysis, and contributed some written material to the manuscript. T.J. K. assisted with the analysis of the graphene film. S.C.P., J.M.K. and D.I.Y. also carried out experiments, fabricated experimental apparatus, and acquired and interpreted the SEM and TEM data. Y.P. carried out the visualization experiments using the infrared camera and fabricated some of the experimental apparatus. K.W.H. analyzed the AFM data. H.J.J. assisted with the analysis of the surface characteristics and boiling performance. H.S.P. assisted in the analysis of the IR visualization. H.K. and M.H.K. equally directed the project. All authors discussed the results and contributed to the paper.Supplementary informationVideo clip of IR high-speed camera in real time


At the scan rate of 5 mV s−1, the specific capacitance of the polypyrrole-CNT-graphene (PCG) electrode film was 453 F g−1 with ultrahigh energy and power density of 62.96 W h kg−1 and 566.66 W kg−1 respectively, as shown in the Ragone plot. A nanofibrous membrane was electrospun and effectively used as a separator in the supercapacitor. Four supercapacitors were assembled in series to demonstrate the device performance by lighting a 2.2 V LED.Supercapacitors also known as electrochemical capacitors, that store energy via either Faradaic or non-Faradaic processes, have recently grown popularity mainly because they complement, and can even replace, conventional energy storage systems in variety of applications. Supercapacitor performance can be improved significantly by developing new nanocomposite electrodes which utilizes both the energy storage processes simultaneously. Here we report, fabrication of the freestanding hybrid electrodes, by incorporating graphene and carbon nanotubes (CNT) in pyrrole monomer via its  Given the current energy crisis and depletion of finite sources, high performance energy storage systems (ESSs), capable of storing energy from both renewable and non-renewable sources, are essential to meet the increasing energy demand. Electrochemical capacitors39Performance of the electrode can be further improved, without compromising the structural integrity and the operating life of supercapacitors, by fabricating hybrid nanocomposites11121315161719202223242526273132Although nanocomposite electrodes exhibit high specific capacitance, they are limited by their low energy/power densities and high discharge rate. One way to improve the performance of these films lies in the fabrication technique. Several methods have been proposed to fabricate hybrid thin films such as layer-by-layer (LBL) electrostatic self-assembly and spin-coating homogeneously mixed solutions on a substrate3435in-situ polymerization of PPy. Supercapacitor performance depends also on electrochemical window provided by different types of electrolytes, such as aqueous, organic, solvent free ionic liquids or solid state gel electrolytes. It is important to compare the performance of nanocomposite electrodes in various electrolytes. In this study, we analyzed the nanocomposite in three different aqueous electrolytes, namely, ascorbic acid (Vitamin C), sodium sulfate, and sulfuric acid. The highest specific capacitance was observed in the electrode containing graphene and CNT in presence of sulfuric acid electrolyte. Furthermore, while fabricating the supercapacitor device, a novel nanostructured electrospun fiber membrane, made from polycaprolactone (PCL), was used as a separator. Lastly, the performance of this electrode based supercapacitor was measured using a 2.2 V light emitting diode (LED), revealing a slow discharge rate when compared to conventional commercial capacitor. After charging it for 30 s, the LED sustained a light for 3 minutes using a stack of 4 devices in series. The hybrid nanocomposite electrode polypyrrole-CNT-graphene (PCG) will be termed as PCG-n, where n is the % concentration of the graphene and CNT combined in PPy, henceforth.In this study, we report a hybridized electrode utilizing EDLC materials such as graphene and CNT within polypyrrole, a pseudocapacitive material, forming a stable and high performance freestanding electrode. This was achieved by electropolymerization of the monomer Py in the presence of graphene and CNT, yielding a nanocomposite polypyrrole electrode. Several nanocomposite electrodes were prepared with increasing concentration of graphene and CNT, such as 0.01 wt%, 0.05 wt % and 0.1 wt% in the A hybrid PPy-graphene-CNT freestanding electrode was fabricated using potentiostatic electrochemical polymerization, as shown in As evident from the SEM images , the cono, which is a characteristic peak of amorphous PPy, and arises mainly due to the scattering from PPy chains at the interplanar spacing. In the case of the PCG 0.01 nanocomposite a sharp peak is observed at approximately 2θ = 25.75o suggesting the incorporation of graphene and CNT in PPy matrix, improving the crystallinity of the structure. Using Bragg’s law, the distance between crystals can be calculated and it is found that the average d_spacing of PPy is 3.024 Å and that of PCG is 3.456 Å. The characteristic peak of the PCG nanocomposite has shifted due to the intercalation of the graphene and CNT in the PPy matrix, resulting in an increase in d_spacing.The effect of crystal formation with and without the presence of graphene and CNT can be observed with the XRD patterns. Cyclic voltammetry (CV) tests were performed to identify the charge storage mechanism of the control PPy and hybrid PCG electrodes in three different electrolytes, namely sulfuric acid (SA), sodium sulfate (SS) and ascorbic acid (AA). As shown in −1 at 5 mV s−1, in comparison to PPy electrodes in SS and AA electrolytes. Along with the highest specific capacitance, the SA electrolyte also exhibited a low decrease in the specific capacitance with a corresponding increase in the scan rate as shown in −1 the specific capacitance saturated to ~100 F g−1 even with a corresponding increase in the scan rate. Furthermore, −1 with the PCG 0.1 electrode at scan rate of 5 mV/s.Based on the CV tests, the specific capacitance was calculated with respect to various scan rates as shown in A supercapacitor device was fabricated as shown in To further demonstrate the performance of the supercapacitor device, a circuit was implemented to charge and discharge the supercapacitor to an external load, in this case a 2.2 V light emitting diode (LED) was used. In order to light up the LED, a stack of 4 supercapacitor devices were assembled in series and they were enclosed tightly within a CR-2032 button cell holder. The circuit was implemented as shown in in-situ synthesis of polypyrrole-CNT-graphene nanocomposite resulting in a controlled layered structure with high porosity. The resulting electrode has high ion accessibility, due to the fact that no organic binders were used to fabricate the thin film, which would otherwise reduce the ionic access to the active materials in the supercapacitor electrodes. This fabrication technique is also useful to develop micro-supercapacitors and biosensors because of its ability to precisely deposit the conductive nanocomposite material on the existing circuits.The novelty of the electrode fabrication technique lies in the ease of the −1 and a power density of 566.66 W kg−1 at the scan rate of 5 mV/s. For the PCG 0.1, an ultrahigh energy density was obtained in the range of 9.62 W h kg−1–62.96 W h kg−1 and a power density in the range of 1416.66 W kg−1–8665 W kg−1 at various scan rates. These electrodes exhibit high power density from EDLC materials and simultaneously exhibit high energy density from the pseudocapacitive type of materials which could results in many high power applications. The power density  and the energy density  of the electrode material was calculated by using the following expressionsThe electrochemical performance of the nanocomposite electrode was improved by increasing the concentration of graphene and CNT when incorporated in PPy as shown in the Ragone plot in Here, V is the initial voltage during discharge; m is the mass of electrodesv is the scan rate (Vs−1)Here The use of electrospun nanofibers as a separating membrane has also improved the performance of the supercapacitor device. Owing to small fiber diameter (~600 nm) and high surface area, the fibrous nanostructures act not only as an efficient permeable membrane across the hybrid electrodes but also as an electrolytic reservoir, which helps to slow the overall discharge in device. The supercapacitors can be integrated into many applications such as microelectrode arrays38402SO4 as supporting electrolyte in 25 mL of DH2O for 1 h. To make PCG nanocomposite with concentrations 0.01% w/v (PCG0.01), 0.05% w/v (PCG0.05) and 0.1% w/v (PCG0.1), an equal amount of graphene and CNT  were dispersed in 25 ml Py solution. This solution was stirred magnetically for 1 h, followed by sonication for 2 h to ensure that graphene and CNT were dispersed thoroughly. PPy and PCG films were synthesized potentiostatically at the scan rate of 20 mV s−1 and 10 mV s−1 respectively. The potential window for both samples was from [800–900 mV] and they were scanned for 100 cycles to obtain free standing films.The polypyrrole (PPy) film was prepared using pyrrole (Py)  monomer solution. The Py solution was prepared by stirring 0.87 ml of Py along with 0.355 g of NaThe performance of electrodes was studied in the presence of different electrolytes such as ascorbic acid (AA), sodium sulfate (SS) and sulfuric acid (SA). AA electrolyte was prepared using 10 mM Ascorbic Acid in 50 mM Phosphate Buffer Saline (PBS). 0.5 M of SS electrolyte was prepared by adding weighted amount of sodium sulfate salt to deionized water and stirring until a clear solution was achieved. As SS is a neutral salt, its pH is near 7. Lastly, sulfuric acid, which was the strongest electrolyte used, had a pH close to 0 in a 1 M solution.The nanocomposite electrodes were characterized electrochemically  using cyclic voltammetry. A three-electrode setup, consisting of a Pt counter electrode, an Ag/AgCl reference electrode and a PPy or PCG-n as working electrode was used for the electrochemical analysis. The capacitance values were calculated from the cyclic voltammogram using the following equationC is the specific capacitance (F g−1), m is the mass of the active electrode material (g), [V = V+−V−] is the potential difference and v is the scan rate (m V s−1). The total charge transferred during the cycle can be calculated by integrating the area under the curve of CV. The potential difference for each electrolyte was, VAA [−300 mV–800 mV], VSS , and VSA [−200–800 mV]. The electrodes were weighed before and after the deposition on graphite electrode in order to calculate the mass of the active nanocomposite material. A 3 mm cylindrical graphite rod was used for all experiments and it was covered using an insulating tape on its surface leaving only the base uncovered for deposition. This ensured that same surface area was maintained during each experiment.Where o–80o using CuKα radiation (λ = 1.5406 Å) operated at 40 kV and 40 mA. Using Bragg’s law, the distance between crystals were calculated as followsThe structural morphology of the nanocomposite films were characterized using scanning electron microscope . X-ray diffraction study was performed using Bruker Advanced diffractometer. The XRD patterns were recorded in the 2θ range of 10How to cite this article: Aphale, A. et al. Hybrid Electrodes by In-Situ Integration of Graphene and Carbon-Nanotubes in Polypyrrole for Supercapacitors. Sci. Rep. 5, 14445; doi: 10.1038/srep14445 (2015).


Highly confined surface plasmons on graphene attract substantial interest as potential information carriers for highly integrated photonic data processing circuits. However, plasmon losses remain the main obstacle for implementation of such devices. In near-field microscopic experiments performed at the wavelength of 10 μm we show that a substantial reduction of plasmon damping can be achieved by placing a nanometric polymer nano-dots spacer between the graphene layer and the supporting silicon oxide slab making graphene quasi-suspended. We argue that reduction of plasmon losses is attributed to weaker coupling with substrate phonons in the quasi-suspended graphene. Oph ≈ 0.2 eV) and sufficiently high doping level (ħω < ħωinter)16171819211213212526Mid-infrared graphene plasmons (GPs) have attracted tremendous interest in recent years owing to an unprecedented spatial confinement and tunability by electrostatic gating1234567810111214GPs damping due to the coupling with the substrate optical phonons can be reduced by shifting an excitation wavelength out of the corresponding phonon line, as has been demonstrated2228292 and SiCAdditionally, as it follows from theoretical analysis313233In this work we experimentally study the effect of quasi-suspended graphene on mid-infrared GPs. The monolayer graphene is placed above a chemically engineered nanostructured spacer (NS) on the substrate, that leads to a “quasi-free-standing”-like graphene38401 = 10 μm or λ2 = 11.2 μm correspondingly. A p-polarized beam is focused on the tip. This generates, in each point during the scanning, cylindrical GPs travelling in all directions out of the tip along the graphene flake. Launched GPs are reflected back from the graphene edges, collected by the tip and scattered out in the far-field. Pseudo-heterodyne interferometric detection of the scattered light allows us to record the amplitude of tip-scattered field, Es, as well as topography in each point of the scan (see Methods section for more details).To experimentally image GPs, we use scanning plasmon interferometry technique2411 = 10 μm due to the strong coupling between GPs and SiO2 optical phonons222 wafer, taken at λ1 = 10 μm and λ2 = 11.2 μm. Corresponding cross-sections of Es along yellow dotted lines are plotted in 2 = 11.2 μm, GPs appear as half GP-wavelength spaced fringes, at λ1 = 10 μm plasmon field is significantly damped and nearly invisible due to the strong interaction with substrate phonons. In this paper, we use the ratio R between the first maximum of the electric field (close to graphene edge) and the field magnitude in the inner part of the flake, (Es)pl/(Es)bg, to represent the damping rate of GPs. Moderately damped GPs at λ2 = 11.2 μm (out of SiO2 phonon line) is characterized by R ≈ 1.5, while for λ1 = 10 μm, the ratio R ≈ 1. Thus, different damping values are characterized by different R. In the following study we will experimentally analyze a possible enhancement of the parameter R for the case of excitation at λ1, by inserting a spacing layer between graphene and the SiO2 substrate.We investigate on samples which initially (when no spacer is applied) possess strongly damped GPs. Experimentally this is realized at an excitation wavelength λ2 spacer we apply the following fabrication processes: (1) thermally oxidized Si-SiO2 wafer is covered with a photoresist by spin-coating, followed by softbaking (2) the photoresist is partially cross-linked by dry-etching, (3) the sample is sonicated in a stripper and rinsed with 2-propanol (see Methods section for more details). This leads to the formation of few-nm thick nano-dots layer of novolac-based polymer2 roughness and the damping-related factor R in the graphene  than it actually presents in the sample. It is well-known that AFM records the tip trajectory as it goes along the surface, rather than the true topography of the sample during measurements4445Graphene is then mechanically exfoliated on top of the fabricated spacer. A typical atomic-force microscope (AFM) image and the height profile of a graphene flake above NS are shown in 2 without any continuous base-layer of polymer beneath. Zoomed-in AFM topography inside the cyan dotted square in 2 surface is formed by randomly placed nano-dots with an average height of 1.2 nm, a typical size of 20 – 40 nm and a typical spacing of 10 – 100 nm.Additional scratching and AFM measurements of NS close to the flake location, as well as presented further optical s-SNOM data, show that nano-dots have been grown directly on SiO2 (at λ1 = 10 μm). The image corresponds to the bottom part of the tapered ribbon displayed in 2 substrate  with randomly distributed black spots which represent the near-field signal of polymer dots. Therefore, we conclude that the roughness in topography observed within NS beneath the flake does not lead to considerable reflections/scattering of GPs field. This agrees with previously reported observationsgp/2) of propagating GPs reflected from the dot. Additionally, as it can be seen from 1 = 10 μm. Additional, study of the same graphene flake at λ2 = 11.2 μm, where SiO2 phonon damping effects can be excluded ; the refraction index of the polymer n = 1.45. The GP is launched in the graphene from the left-hand side. The evolution with the distance of the absolute value of vertical component of plasmonic electric field is plotted in the graph in 2, that represents a significant suppression of damping and agrees with the experiment. Our model describes only the effect of spacing and partial suspension of graphene sheet from SiO2, while the value of the mobility is fixed.Finally, the physics of mid-infrared plasmon damping and propagation in graphene on NS-SiOIn summary, we demonstrate that lifting exfoliated graphene from the silicon dioxide surface with an ultra-thin nanostructured polymer spacer, helps with the control of mid-infrared plasmon damping and propagation. Polymer nano-dots result in spacing and partial suspension of graphene that is beneficial for remote phonons screening. Owing to the ultra-small thickness of the spacer, the nanoscale roughness does not lead to strong chaotic reflections of GPs at the polymer nano-dots, that is one of requirements for unperturbed performance of possible devices fabricated in graphene on top of NS. Numerical simulations of plasmons propagation in graphene placed above silicon dioxide covered with polymer nano-dots show an increase of the propagation length and a suppression of damping that is in agreement with the experiment. This work contributes to understanding of mid-infrared GPs damping mechanism, and gives insight into the fundamental problems of interaction of the plasmons with deeply subwavelength nanostructures.2 wafer (285 nm oxide thickness) was covered with AZ 5214E photoresist by spin-coating at 4000 rpm for 30 s. The thickness of the resulting photoresist film is about 1.6 μm. (2) The wafer was then softbaked for 100 s at 105 °C. (3) The resulting sample was dry-etched for 4.5 min in the Plasma-Therm 790 series RIE . The dry-etching process leads to a higher degree of cross-linking, within the novolac-based polymer component of the photoresist due to a possible overheating/UV exposure from the plasma. The dry-etching does not remove the photoresist film completely with the chosen etching time. (4) Then the sample was sonicated in NI555 stripper for 5 min at room temperature, and finally, it was rinsed thoroughly with 2-propanol and dried with nitrogen.To fabricate the NS, we use the polymer which is, essentially, self-organized as a highly nanostructured film during the process of ultrathin (nanometric) films fabrication. The following process was performed. (1) Virgin thermally oxidized Si-SiO2 laser. Background-free near-field imaging is given by the demodulation of recorded optical signal at higher harmonics of the tapping frequency . We note that, as commonly known, near-field optical images recorded with the QCL laser (λ1 = 10 μm) typically show lower signal to noise ratio compare to the CO2 laser (λ2 = 11.2 μm). With reference to Neaspec GmbH, this difference in performance is mainly attributed to intrinsic laser specific aspects in the sense how the mid-infrared lasing is realized in the two lasers.Our setup is based on a commercial s-SNOM (Neaspec GmbH) performing measurements with a Pt-coated Si tip at the tapping frequency Ω of about 250 kHz and the tapping amplitude of about 60 nm. We use Daylight Solutions tuneable QCL laser and temperature-tuneable Access Laser CONumerical simulations are based on the finite-difference time domain method presented in the reference1 = 10 μm we can define the first field maxima close to the graphene edge and the field magnitude in the inner part of the flake, while due to strong damping, it is not possible to clearly define multiple maximums of GPs field over the flake. Therefore we implemented another damping-related factor, (Es)pl/(Es)bg, which compares the near-field magnitude in the first maximum and the inner part of the flake where a plasmon, reflected from the edge, has been already completely damped. As it was verified above, on the example of strong and weak damping, this factor can be used for qualitative comparison of GPs damping strength.A.M.D. proposed the idea of the paper, performed samples fabrication and near-filed measurements, analysed data and prepared the main manuscript text. J.T. performed numerical simulations. J.T. and X.C.Y. contributed to samples fabrication. Q.J.W. and N.I.Z. supervised the project and contributed to the manuscript text. All authors discussed the results and commented on the manuscript.How to cite this article: Dubrovkin, A. M. et al. The reduction of surface plasmon losses in quasi-suspended graphene. Sci. Rep.5, 09837; doi: 10.1038/srep09837 (2015).Supplementary Figure S1-S5