Between 6 and 30% of human and mouse transcripts are initiated from transposable elements. However, the promoters driving such transcriptional activity are mostly unknown. We experimentally characterized an antisense (AS) promoter in mouse L1 retrotransposons for the first time, oriented antiparallel to the coding strand of L1 open reading frame-1. We found that AS transcription is mediated by RNA polymerase II. Rapid amplification of cDNA ends cloning mapped transcription start sites adjacent to the AS promoter. We identified >100 novel fusion transcripts, of which many were conserved across divergent mouse lineages, suggesting conservation of potential functions. To evaluate whether AS L1 transcription could regulate L1 retrotransposition, we replaced portions of native open reading frame-1 in donor elements by synonymously recoded sequences. The resulting L1 elements lacked AS promoter activity and retrotransposed more frequently than endogenous L1s. Overexpression of AS L1 transcripts also reduced L1 retrotransposition. This suppression of retrotransposition was largely independent of Dicer. Our experiments shed new light on how AS fusion transcripts are initiated from endogenous L1 elements across the mouse genome. Such AS transcription can contribute substantially both to natural transcriptional variation and to endogenous regulation of L1 retrotransposition. F, GF and A) reside in the mouse genome  are a major class of mammalian retrotransposons, comprising ∼19 and 21% of the mouse and human genomes, respectively s are a m. Ongoinge genome ,14,15. TMet proto-oncogene  in human and mouse appear to depend on their genomic context, their sequence structure and other factors . Endogenoncogene  and otheoncogene .Like other mammalian TEs, L1s are constrained by various cellular defenses including DNA methylation, histone modifications, Dicer-mediated RNA interference (RNAi) and other small RNA-mediated effects . BidirecDicer ex5 knockout cells were provided by Dr Bert Vogelstein (Johns Hopkins).Mice were maintained and euthanized according to approved Institutional Animal Care and Use Committee protocols . Mouse strains and purified genomic DNA were purchased from the Jackson Laboratory . A mouse spermatocyte cell line (CRL2196) was purchased from the American Type Culture Collection. HeLa cells were provided by Dr John V. Moran (University of Michigan). HCT116 Genomic DNA and pooled total RNAs were isolated from CRL2196 cells and from various tissues, ages and lineages of mice as indicated, using standard methods and Trizol (Invitrogen), respectively.Supplementary Table S1.Primer sequences and annotations are listed in F GenBank accession number AF016099; GF, AC068252; A, AY053456 and FIII, AC002406) and a synthetic L1 element smL1 . Amplicons included fragments of L1 TF (represented by L1spa in pTN201), sense promoter (primers DES1212 and DES1213), AS promoter  and AS ORF2 (DES1459 × DES1460). Promoter candidates were cloned directionally upstream of TEM1, a β-lactamase reporter gene, in pBLAK-b, which lacks a promoter (Invitrogen) . They were confirmed by sequencing. One microgram of BglII-digested (linearized) plasmid DNA was transiently transfected into CRL2196 or HeLa cells using FuGENE 6 (Roche). As positive and negative controls, plasmids with and without the SV40 promoter upstream of TEM1 were used .Genomic DNA fragments representing four mouse L1 subfamilies  (TF GenBent smL1  were amp2 well. Cells were incubated in the dark at room temperature (RT) for 1 h with gentle shaking, washed with HBSS and visualized using an Axiovert 200 M inverted fluorescence microscope (Zeiss) with blue/aqua and β-lactamase ratio filter sets (Chroma Technology Corp.) and ORCA-ER high resolution digital camera (Hamamatsu Photonics) using Openlab software . Flow cytometric analysis was performed using a BD LSR II flow cytometer with a 405 nm violet laser, 440/40 nm (blue) and 530/30 nm (green) filters, and FACSDiva software (BD Biosciences). Ratios of blue to green intensities were collected as a linear parameter. Each flow cytometry session included positive and negative controls to normalize output.To quantify β-lactamase protein expression, cells were stained with CCF2/AM substrate (Invitrogen) ,49 by reTEM1 expression also was quantified by quantitative reverse transcriptase-mediated PCR (qRT-PCR). Promoter candidates were linearized by BglII digestion and transfected into HeLa cells using FuGENE 6. Total RNAs were isolated ∼48 h after transfection using RNeasy kit (Qiagen). Standard curves were based on serial dilutions of control plasmids. First strand cDNAs were synthesized using oligo-d(T) (DES2633) primer and the SuperScript double-stranded cDNA synthesis kit (Invitrogen). As further controls, RNAs were treated with and without reverse transcriptase. qRT-PCR was performed on an iCycler (Bio-Rad) or Step One Plus (Applied Biosystems) instrument, using SYBR Green Supermix (Bio-Rad). TEM1 transcript concentrations were calculated by interpolation, after subtracting for input plasmid DNA contamination. Beta-actin transcript levels were calculated for each sample. Each sample was measured in triplicate. Results are presented for each sample as the normalized ratio of TEM1 to beta-actin transcript levels.Anti-mouse RNA polymerase III subunit RPC39 mouse monoclonal antibody was purchased from Santa Cruz . Anti-mouse RNA polymerase II mouse monoclonal antibody (Cat. 39097) was from Active Motif. For chromatin immunoprecipitation (ChIP), the Magna ChIP G Tissue kit (Millipore) was used following the manufacturer’s instructions.Supplementary Table S1) for first strand cDNA synthesis.We performed 5′ RACE cloning using a second-generation 5′/3′ RACE kit (Roche) with an AS L1 ORF1-specific primer , a representative full-length Ttemplate . BacteriSupplementary Table S1). Resulting PCR products were gel purified and radiolabeled by random nonamer priming. Commercial bacteriophage cDNA libraries from mouse testis (Clontech) and thymus (Stratagene) were plated at ∼50 000 plaques per dish, transferred to Hybond-N filters (Amersham) and hybridized with the ORF2 probe. Filters were washed at 65°C in 0.1 × SSC and 0.1% SDS and autoradiographed. This procedure was repeated with the ORF1 probe to identify ORF1+ORF2− clones, which were purified upon additional rounds of hybridization. Phage plaques were converted to plasmids and sequenced using BigDye v. 3.1 (Applied Biosystems) on a 96 capillary sequencer (Transgenomic Spectrumedix) with primers DES886 and DES837  and standard M13R and M13F oligonucleotides . Additional sequences for full-length cDNA sequencing by primer walking are available on request.The primer pairs used to amplify fragments from L1spa ORF2 and ORF1 were DES1165 × DES1166 and DES 1167 × DES1168, respectively  from DNase-treated total RNAs, using the M13F-anchored primer DES1141 paired with DES1256 for mouse L1spa ORF1, AS nucleotides 2011–1991. PCR products (1–3 kb) were isolated using gel purification columns (Qiagen), and cloned for sequencing. Tissue-specific expression patterns of AS L1 RIFTs and corresponding cognate genes were analyzed by RT-PCR using a commercial multiple-tissue mouse cDNA panel (Clontech).F subfamily) ORF1 as query, i.e. AS nucleotides 2225–1838 . Resulting AS RNA was purified; a second round of first strand cDNA synthesis was performed with reverse transcriptase, dUTP and random primers; cRNA was hydrolyzed using RNaseH, and resulting sense strand DNA was purified. Products were fragmented with uracil DNA glycosylase and apurinic/apyrimidinic (AP)-endonuclease I. Terminal labeling was performed with terminal deoxynucleotidyl transferase (TdT), and resulting labeled fragments were hybridized to the exon microarray.To develop a novel assay to identify L1 RIFTs, we modified the manufacturer’s protocol for the Affymetrix GeneChip mouse exon microarray. First strand cDNA synthesis was performed on total RNA isolated from various tissues and lineages, using a primer including both T7 promoter and oligo-d(T) sequences and SuperScript II reverse transcriptase (Invitrogen). Polyadenylated cDNAs containing AS L1 sequences were amplified using a primer for a particular L1 ORF1 AS template sequence paired with the T7 promoter primer. Resulting double-stranded RIFT cDNAs containing T7 sequences were used as templates for 2 intensity >7) as high expression probes and counted the number of consecutive high expression probes per annotated gene. On alignment with the reference mouse genome, candidate fusion transcripts were scored positive if a neighboring AS L1 could be identified within 30 kb of an overlapping RefSeq gene and/or within 100 kb of the upregulated probe(s). We also required five consecutive high expression probe intensities (corresponding to adjacent exons in a given gene) in exon microarray data; length of predicted initiating genomic L1 integrant had to be >5 kb; and its subtype had to be L1 TF, A, GF or F as per RepeatMasker (www.repeatmasker.org).Resulting raw data from an Affymetrix microarray chip reader were analyzed for transcript expression, using Partek Genomics Suite software. CEL files (MoEx-1_0-st-v1) were imported using RMA background correction and quantile normalization. Probe intensities were transformed to log base 2. We defined signals with intensity > mean + one standard deviation (i.e. ∼logNotI-XhoI fragment from pTN201 (L1spa)  (NotI-HindIII fragment back into the NotI-HindIII backbone (5.4 kb) from pMK20, resulting in ∼11-kb pMK27, i.e. a marked, full-length L1 donor element. This plasmid was Sanger sequenced , revealing a missense mutation in ORF2, i.e. Ala756Ser, along with two noncoding mutations present in pTN201. The missense mutation was repaired by replacement of a ∼1.4-kb EcoRI fragment in pMK27 with the corresponding fragment from pTN201. The 8.1-kb NotI-XhoI fragment of repaired pMK27 was ligated with a ∼11.7-kb NotI-XhoI pTN201 backbone fragment, yielding the desired final plasmid, i.e. pMK28 or L1spa::smL1-ORF1.To compare retrotransposition frequencies of various L1 donors, we started by replacing native L1 ORF1 sequences with a synonymous fragment of smL1 , by movi (L1spa) , into pBnd ORF1) , resultiSupplementary Figure S2). This fragment (Blue Heron Bio), which also included 50-nt flanking arms on both ends for recombineering, was cloned into pUC MinusMCS, resulting in plasmid pJL2. The recoded L1 ORF1 fragment from pJL2 was amplified by PCR using DES3353 × DES3354 and Platinum Taq DNA polymerase High Fidelity (Invitrogen), gel purified (Qiagen), mixed with PstI-linearized pMK20 and co-transformed into electrocompetent DY380 bacteria, bearing the lambda red recombination system for recombineering , using primer pairs DES2880 × DES2881 (L1spa nucleotides 2150–1286); DES2880 × DES2882 (nucleotides 2150–1636) and DES2879 × DES2881 (nucleotides 2823–1286). Resulting PCR products were digested with NotI and BamHI, electrophoresed on agarose gels, purified and ligated to linearized pCEP4 backbone. Fragments from AS synthetic L1 elements similarly were generated using primers DES3818 × DES3820 , DES3819 × DES3820 (nt 2150–1801), DES3818 × DES3821 (nt 2823–1121) and DES3819 × DES3821 (nt 2823–1801). Products were digested with NheI and BamHI and ligated to similarly linearized pCEP4.Various AS L1 transcript overexpression plasmids were engineered from L1 TR) cells then were grown without selection for several days, prior to selection for NeoR L1 integrants in 0.4 mg/ml G418 (Invitrogen) for 2 weeks. In the transient assay , fixed in 2% formaldehyde/0.2% glutaraldehyde in 1 x PBS, washed and stained using 0.4% Giemsa (Sigma) at RT overnight and then counted.HeLa cells were cultured in DMEM with 10% heat-inactivated fetal bovine serum and 2% penicillin/streptomycin (Gibco). Cells at ∼75% confluence in six-well plates or T25 flasks were transfected with plasmid DNA mixed with FuGENE HD (Roche) at a ratio of 1 µg to 3 µl FuGENE. To quantify transfection efficiency, GFP expression was assessed by fluorescence microscopy in cells transfected with pEGFP-N1 (Clontech). Both stable and transient transfection assays were performed. In the former ,23, cellnt assay , NeoR L1in trans, we co-transfected HeLa cells with smL1 donor plasmid together with AS smL1 fragment-expressing constructs. One µg of pCEP4/smL1/Neo donor plasmid DNA was mixed with 1 µg of various smL1 AS fragment-expressing constructs or empty vector pCEP4, respectively, in FuGENE 6. Two µg of vector pCEP4 was used as another negative control. A transient assay (Supplementary Information).To assess effects of AS L1 transcripts overexpressed nt assay  was perfTEM1 reporter constructs, we conducted qRT-PCR. Plasmids were transfected into HeLa cells, and total RNAs were isolated and treated with DNase (Ambion). First strand cDNAs were synthesized using an oligo-d(T) primer. As a control, samples were treated with no reverse transcriptase. L1 expression was measured by SYBR green PCR master mix (Applied Biosystems) using ORF2 primers DES2784 × DES2790 for pTN201/TEM1, pJL3/TEM1 and pMK28/TEM1; and DES1847 × DES1848 for pCEP4/smL1/TEM1. As a control for transfection, Hygromycin gene expression was measured by qRT-PCR using DES1249 × DES1250.In an independent assay of expression levels from various L1-R   reporter cassette and a portion of pCEP4 backbone were excised from pDES46 (which contains human L1.3) by digestion with NotI and BstZ17I. The resulting ∼13-kb backbone fragment was gel-purified. Native, hybrid or fully synthetic mouse L1 constructs in pTN201, pJL3, pMK28 and pCEP4/smL1 were digested by BamHI, ends were filled in by Klenow and digested by NotI. Each of the resulting ∼6.5-kb L1 fragments was ligated with the NotI - BstZ17I fragment. Positive candidates were confirmed by conventional Sanger sequencing. Resulting L1 donor plasmids, marked by the TEM1-AI reporter, included pTN201/TEM1, pJL3/TEM1, pMK28/TEM1 and pCEP4/smL1/TEM1.To assay L1 retrotransposition in Dicer ex5 −/− HCT116 human colorectal cells, which are constitutively NeoR , we usedron (AI) . The TEMTEM1-AI reporter in HCT116 wild-type versus Dicer −/− cell lines, transfectants were selected for 10 d on 400 mcg/ml hygromycin. Expression levels of spliced TEM1 transcripts were assayed by qRT-PCR using DES3062 × DES3063.To compare retrotransposition with native or hybrid L1 donors marked with TEM1 β-lactamase reporter gene otherwise lacking a promoter . The candidates were derived from mouse L1 subfamilies TF, GF, A and FIII; fully synthetic synonymously recoded smL1  .Previously, we and others identified mouse AS L1 RIFTs ,45. Basepromoter . To assa ORFeus) ; and a nF AS nucleotides 2823–2125, mapped as per L1spa coordinates  >> GF ∼ A (∼10% of control) > F (∼5% of control). For these functional promoter assays, we chose particular elements to represent the subfamilies, i.e. L1spa for L1 TF subfamily; L1 GF62 for the GF subfamily and L1Md_A2 for the A subfamily . Within ORF1, these individual surrogates were 99.8, 99.7 and 99.9% identical to the consensus subfamily sequences, respectively . Differences between the subfamily consensus sequences and the individual surrogates were predicted at 1944A>G and 2261G>C ; 1963C>T, 2687T>A, 2716T>C and 2857A>C (L1_GF>L1 GF62); and 2857G>A (L1_A>L1Md_A2).The highest level of AS promoter activity was found in L1Trdinates A. VariouF AS promoter activity was robust, i.e. again, approximately half that of the L1 5′ UTR sense promoter . By contrast, virtually no promoter activity was detected in various fragments derived from the sense (coding) orientation of ORF1, AS ORF2, L1 3′ UTR, smL1 or a novel recoded ORF1 sequence which we designed to contain A/T content comparable with natural L1 sequences  (A qRT-PCR assay for reporter transcript expression confirmed that L1 Tpromoter . Low butgure S1) .Supplementary Figure S2). Comparison of representative L1 subfamily amino acid sequences encoded by ORF1 indicated that the particular portions comprising the AS promoter were more conserved, but still distinct, between subfamilies, compared with the flanking, proximal and distal portions of ORF1 . By contrast, the L1 subfamily sequences within ORF2, which do not contain this AS promoter, were nearly identical (not shown). These results suggested that ORF2 and the AS promoter segment within ORF1 may have undergone strong purifying selection . A recent analysis of the evolution of mouse and human L1s confirmed that the mouse ORF1 coiled-coil domain has undergone much less adaptive change than that of human elements  II and III, either of which plausibly could bind to and initiate fusion transcription from various endogenous TE sequences. As shown in To confirm the role for RNA pol II in transcribing AS L1 RIFTs, we treated a mouse spermatocyte cell line, CRL2196, with α-amanitin B, a poteF subfamily probe for ORF2 . Sequential hybridization with an L1 TF ORF1 probe  were ORF1+.To find mouse transcripts that included sequences from genomic L1 templates, we screened full-length transcripts represented in bacteriophage libraries. Although 6–30% of all mouse and human transcripts recently were reported to be initiated from TEs including L1s , we obseF1 probe  revealed+ORF2- transcripts would include AS L1 RIFTs. This possibility was prompted by our previous identification of fusion transcripts in adult mouse tissues, mapping to L1 elements  contained AS L1 ORF1 sequences spliced with other exons in the sense orientation, forming AS L1 RIFTs. Additionally, 2 of 13 thymus cDNAs  also were spliced AS L1 RIFTs. Other ORF1+ORF2- cDNAs either were unspliced AS RIFTs, reading antiparallel to ORF1 through the 5′ UTR into flanking genomic sequences , or were prematurely polyadenylated, sense-strand transcripts   . Some RI+ORF2- transcripts initiated across the genome in a variety of chromosomal and tissue contexts, as illustrated by schematics of their genomic templates including the initiating L1 elements  (Supplementary Table S2) expressed in cultured mouse spermatocyte cells or adult testes. Twelve (29%) aligned to genomic regions lacking a previously annotated gene, and two (5%) were initiated from polymorphic L1s absent from B6 mice (Supplementary Table S2). Fifty-seven EST clones contained AS L1 sequences in their 5′ ends. Of these, 22 were spliced, but no splicing was observed within L1 sequences per se. Many of the AS L1 RIFTs identified by bioinformatics analysis were found in testis and embryonic cells at certain developmental stages, again suggesting a high level of tissue specificity. This search identified >80 EST clones with AS alignment ≥300 nt and >90% identity with L1 at their 5′ ends, of which 15 were full-length RIKEN cDNAs. In some cases, 3′ paired ends of other EST clones were identified from the EMBL/EBI database using 5′ clone IDs; 57 clones were sequenced from both ends.We identified diverse spliced ORF1gure S3) . To dete and two % were in B6 mice , reveali B6 mice  as splicSupplementary Figure S4). As expected, almost all of these RIFTs were confirmed in testis. Relatively few were expressed in other tissues assayed, but we did recover clones 1ASII1, additionally expressed in 11-day embryos; L1-5AS1-1, additionally expressed in brain; and CRL2196C10, widely expressed in most tissues assayed. We also assayed for overlapping spliced transcripts from cognate genes. Although AS L1 RIFTs that were spliced to downstream exons of Erbb2ip, Usp29 and Arhgap15 each were expressed in testis, the corresponding conventional transcripts of these genes (i.e. lacking sequences from L1s) were not detectable there.To compare RIFT expression levels in different tissues, we re-assayed 17 RIFTs identified initially in adult testis or from a spermatocyte cell line (Supplementary Table S2), of which many were also identified in phage cDNA libraries (Supplementary Table S2). Thus, many transcripts were corroborated by independent methods.To identify genes whose expression levels may be affected by AS L1 RIFTs, we probed Affymetrix mouse exon microarrays conventionally with total RNAs. As commercial exon microarrays typically exclude probes for repetitive elements such as L1 retrotransposons, we also developed a novel, unconventional assay using the arrays to screen specifically for AS L1 RIFTs that include downstream exons. In this assay, hereafter called the RIFT assay, we prepared cDNAs from several tissues and mouse lineages by RT-PCR, using an AS L1-specific primer paired with an oligo-d(T) primer. At least 130 unique spliced AS L1 RIFTs were identified in adult testis . In addition, the 5′ ends of 24 RIKEN cDNA clones, most of which were reported previously  analysis on fusion transcripts expressed in testis, kidney and brain. A primer specific for the L1 Tarby TSS . A largeeviously , mapped eviously .Figure F ORF1 , but it is likely too distant from the RIFTs’ 5′ ends, identified by RACE  of 65 RIFTs, was GATGgtgag , whereas the highest levels were seen for full-length smL1 . Intermediate levels were seen for the novel hybrid element containing recoded ORF1, harboring no AS promoter activity and neutral changes in A/T content, engineered upstream of native L1spa (TF) ORF2. Somewhat higher expression was seen for the second hybrid L1 element, i.e. smL1/L1spa in pMK28, which has lower A/T content in ORF1 . Th. Th13). trans, first we engineered AS smL1 fragments to overexpress them in the desired orientation. Four AS fragments from smL1, corresponding to AS L1spa coordinates 2119–1120, 2800–1120, 2119–1812 and 2800–1812, each were cloned downstream of the CMV promoter and were co-transfected with marked smL1 in a transient retrotransposition assay . As a positive control, where smL1 could mobilize in the absence of overexpressed AS L1 transcripts in trans, empty pCEP4 was co-transfected with smL1. A negative control consisted of cells transfected with no smL1 donor and pCEP4 alone. The overexpression of AS smL1 transcripts in trans significantly suppressed smL1 retrotransposition, i.e. by ∼50–75%  were overexpressed . These AS L1 transcripts overlapped in part with endogenous AS L1 RIFTs .To determine whether overexpressed AS L1 transcripts could inhibit retrotransposition in on assay  . Thus, Dicer played a modest role in suppressing native L1 retrotransposition, mediated by AS L1 transcription; most of the suppression by AS L1 transcripts occurred independent of Dicer. Previous experiments showed a similar ∼2-fold level of suppression of human L1 retrotransposition on knockdown of Dicer in cultured cells. That result was interpreted as showing the role for Dicer-dependent RNA interference in regulating human retrotransposition  RNAs. In turn, these dsRNAs could trigger formation of short interfering RNAs or microRNAs through a Dicer-dependent pathway ,61, therR , we engil intron . Either sertions . The retposition .Supplementary Figure S3 and Supplementary Table S3) (Supplementary Table S2) .A recent analysis of human and mouse transcriptomes suggested that 6–30% of all transcripts are initiated from repetitive elements . Here, wable S3) ,45. AS LThe co-existence of a protein-coding sequence together with an antiparallel promoter activity in opposite overlapping orientations is unusual, but is not unprecedented, in mammalian genomes .i.e. TF, GF and A elements . Several putative transcription factor binding sites in the AS promoter sequence of L1spa . Although members of each subfamily retrotransposed recently , consistent with a complete lack of AS promoter activity observed in those elements . Thus, we concluded that AS L1 promoter activities ranked as L1 TF >> GF ∼ A > F. Notably, the latter subfamilies possessed modest, but detectable, AS promoter activities.The various AS promoter activities associated with each L1 subfamily  were rouF elements range from 0.25 to 1.23 million years old , in this study we have not tested whether sense and AS L1 promoters may be active simultaneously in single cells. If they are not, the resulting unbalanced expression of sense versus AS L1 transcripts in distinct cells or tissues could allow particular L1 elements to evade this putative defense mechanism. Moreover, individual mouse and human L1 elements can mobilize over a wide range of frequencies, despite similar ORF sequences shared by ‘hot’ versus ‘cool’ elements . These included screens of phage cDNA libraries, RT-PCR followed by cloning and sequencing, bioinformatics surveys of transcript sequence databases, Northern blots .The presence of a particular full-length L1 element was necessary, but not sufficient, to initiate a locus-specific AS L1 RIFT. We found that only 13 (19%) of 68 polymorphic full-length L1s initiated AS L1 RIFTs in testis, as assayed by RT-PCR. Moreover, some RIFTs only were expressed in embryonic, newborn or adult mouse testis, whereas smaller numbers were expressed in other organs such as brain and kidney . A few Ain situ   may play several distinct biological roles including possible protein translation. In some cases, the predicted protein-coding ORF sequences of AS L1 RIFTs match the cognate ORF in transcripts from the associated native genes, suggesting that although they may encode identical proteins, their expression patterns may be added to, or modified by, the AS L1 promoter. Other AS L1 RIFTs may modify or replace cognate protein structures or expression, generate novel proteins or long noncoding RNAs  and mobilization on synonymous recoding of the AS L1 promoter in ORF1 in cis and upon overexpression of AS L1 RIFTs in trans . Hybrid L1s, containing either a recoded synonymous ORF1 segment from smL1 with decreased A/T content , the only elements capable of triggering dsRNAs that were tested here. By contrast, Dicer appeared to be a crucial component in RNAi-mediated regulation of L1 expression and mobilization in mouse ES cells . For this reason, we speculate that the RNAi pathway is not likely to be the predominant suppressive mechanism of mouse L1 elements, and that other suppressive mechanisms are involved, at least in the differentiated somatic cells tested here . Thus, we conclude that AS L1 transcripts act mostly independent of Dicer in decreasing L1 expression and retrotransposition.We showed that Dicer played a modest <2-fold role in suppression of endogenous mouse L1 elements ; federal funds from the National Cancer Institute, NIH to SAIC-Frederick [contract N01-CO-12400]; and Ohio State University Comprehensive Cancer Center. Funding for open access charge: Ohio State University Comprehensive Cancer Center.Funded by the Conflict of interest statement. None declared.


ARPES and XAS data of the H2O (NH3)/graphene/Ni(111) system give an information regarding the kind of interaction between the adsorbed molecules and the graphene on Ni(111). The presented experimental data are compared with the results obtained in the framework of the DFT approach.In this article, an angle-resolved photoelectron spectroscopy (ARPES), X-ray absorption spectroscopy (XAS), and density-functional theory (DFT) investigations of water and ammonia adsorption on graphene/Ni(111) are presented. The results of adsorption on graphene/Ni(111) obtained in this study reveal the existence of interface states, originating from the strong hybridization of the graphene π and spin-polarized Ni 3 These bands have a filled shell and, hence, form a deep valence band. The unaffected 2z porbital, which is perpendicular to the planar structure of the graphene layer, can bind covalently with neighboring carbon atoms, leading to the formation of a π band. Since each 2z porbital has one extra electron, the π band is half filled. The π and π* bands touch in a single point at the Fermi energy (EF) at the corner of the hexagonal graphene's Brillouin zone, and close to this so-called Dirac point, the bands display a linear dispersion and form perfect Dirac cones. Thus, undoped graphene is a semimetal ("zero-gap semiconductor"). The linear dispersion of the bands results in quasi-particles with zero mass, namely, the so-called Dirac fermions.Graphene is a single layer of carbon atoms arranged in a honeycomb lattice with two crystallographically equivalent atoms C1 and C2) in its primitive unit cell , and theoretical methods [density-functional theory (DFT) calculations]. XAS and ARPES studies of graphene/Ni(111) reveal the existence of the interface states, originating from the strong hybridization of the graphene π and Ni 3d valence band states with partial charge transfer of the spin-polarized electrons on the graphene π* unoccupied states. This leads to the appearance of induced magnetism in the carbon atoms of the graphene layer as confirmed by X-ray magnetic circular dischroism (XMCD). ARPES and XAS data of the H2O-NH3/graphene/Ni(111) systems enable us to discriminate between different strengths of interactions (physisorption or chemisorption), which appear between the adsorbed molecules and graphene on Ni(111). DFT calculations were used to model different geometries of the adsorbed molecules on top of graphene/Ni(111), and electronic structure calculations were performed for them. The results thus obtained and those of the previous theoretical studies are compared with the present experimental results.In this article, an attempt to modify the electronic structure of graphene via contact of this material with metal (ferromagnetic Ni substrate) and via adsorption of polar molecules (H3H6) according to the procedure described elsewhere , as well as intensity profiles as a function of water deposition time (t) taken at particular binding energies  are shown in the upper and right panels, respectively. The behavior of the water-related photoemission features, I and II, allows us to conclude that island-type growth of water on graphene/Ni(111) takes place: (i) These features start to grow simultaneously at t = 130 s, but slopes of the intensities growth are different; (ii) After t = 170 s, the intensity of feature I decreases via the exponential law, and there is a small plateau for the feature II (first ML is complete); (iii) At t = 230 s, when the thickness of deposited water is more than 2ML, probably, the structural phase transition takes place--formation of ice. Since ice is an insulator, the rapid decrease of the photoemission intensities of the Ni-related features and the shift of some states to higher binding energies can be explained by the formation of an insulating thin film of ice on top of the graphene/Ni(111) system. The delay in starting of the growth of the water-related photoemission features is somewhat puzzling , but this delay could be because some clustering centers on the graphene/Ni(111) surface are necessary to allow water growth process to start. As soon as sufficient numbers of such centers are formed, the process of growth is accelerated. The general trend in the observation of the ammonia-related photoemission features in the similar experiments is the same. In subsequent XAS and ARPES experiments, the thicknesses of water and ammonia layers were chosen to be 1/3-1/2 of the ML (as discussed above with regard to the structure).In order to study the growth modes of water or ammonia, the time sequences of the photoemission maps around the Γ point of the Brillouin zone  were recorded. The extracted photoemission intensity map showing the modification of the valence band at the Γ point of the graphene/Ni(111) system upon adsorption of water molecules (K-edge XAS spectra of (a) graphene/Ni(111) and this system after adsorption of 1/2 of the ML of (b) H2O and (c) NH3, respectively.The effects of the possible orbital mixing of the valence band states of the graphene layer on Ni(111) and orbitals of water and ammonia molecules were studied by XAS Figure . This fis core level to the interface state above the Fermi level , which originates from the C z p- Ni 3d hybridization and corresponds to the antibonding orbital between a carbon atom C-top and an interface Ni atom. The second peak in the XAS spectrum at 287.1 eV of photon energy is due to the dipole transition of an electron from the C 1s core level to the interface state above the Fermi level  which originates from C z p- Ni xp,yp,3d hybridization and corresponds to a bonding orbital between C-top and C-fcc atoms, involving a Ni interface atom. As was found in the experiment, the observed hybridization leads to the orbital mixing of the valence band states of graphene and Ni and to the appearance of the effective magnetic moment of carbon atoms in the graphene layer. This moment was detected in the recent XMCD measurements of this system [B per atom.The XAS spectra of the clean graphene/Ni(111) system Figure  were anas system , which a2O/graphene/Ni(111) and the NH3/graphene/Ni(111) systems measured at the C K absorption threshold are shown in Figure s → π* transitions; the photon energies in the region of 290-320 eV correspond to the C 1s → σ* transitions). In this context, it is worth emphasizing that the presented XAS measurements were recorded at the C K absorption edge and that they reflect (to some extent) the partial density of states of the carbon atoms in the system [2O and NH3 molecules on graphene/Ni(111).The XAS spectra of the He system , and thecenter or in the on-bond position on graphene/Ni(111)  with the lone-pair toward carbon atoms and N-H bonds along C-C bond of the graphene layer.The interpretation of the XAS spectra measured after water or ammonia adsorption can be performed on the basis of the peak-assignment, which has been presented above. For the water adsorbate, the new structure in the XAS spectra appears at the photon energy range corresponding to the hybrid state in the electronic structure of graphene/Ni111) involving both carbon atoms in the unit cell of graphene and interface Ni atom. This leads to the assumption that water molecules are adsorbed either in the 11 involvhν = 75 eV along the Γ-K direction of the Brillouin zone for the graphene/Ni(111), H2O/graphene/Ni(111), and NH3/graphene/Ni(111) systems. In all the series, one can clearly discriminate the dispersions of graphene π- and σ-derived states in the region below 2 eV of the binding energy as well as Ni 3d-derived states near EF. The binding energy difference of ≈2.4 eV for the π states and ≈1 eV for the σ states in the center of the Brillouin zone (in the Γ point) between graphite and graphene on Ni(111) is in good agreement with previously reported experimental and theoretical values [d states. The effect of hybridization between Ni 3d and graphene π states can be clearly demonstrated in the region around the K point of the Brillouin zone: (i) one of the Ni 3d bands at 1.50 eV changes its binding energy by ≈150 meV to larger binding energies when approaching the K point; (ii) a hybridization shoulder is visible in photoemission spectra which disperses from approximately 1.6 eV to the binding energy of the graphene π states at the K point. The full analysis of the electronic band structure and magnetic properties of the graphene/Ni(111) system were performed in Ref. [Figure l values , and it  in Ref. .2O-3a1 and NH3-1e states, respectively. As can be clearly seen from the photoemission spectra, the adsorption of H2O or NH3 on graphene/Ni(111) leaves the electronic structure of graphene π- and Ni 3d-states almost intact. This observation can be taken as an indication of the inertness of the graphene layer on Ni(111) as was earlier demonstrated in Ref. [K point, there is a shift of this band to the higher binding energies of about 50 and 70 meV for the water and ammonia adsorptions, respectively.The adsorption of 1/2 of ML of water and ammonia molecules on graphene/Ni(111) leads to the appearance of the additional photoemission signal in the spectra at 6.5 and 7.3 eV, respectively Figure . In thes in Ref. . There a6R-6 potentials  system as physisorption. From a theoretical point of view, physisorption can be considered as weak interaction arising due to two types of forces, namely, dispersion forces and/or classical electrostatic ones. The dispersion interactions are long-range electron correlation effects, which are not captured in DFT because of the local character of common functionals. Consequently, DFT often fails to describe physisorption correctly. For a correct and consistent treatment of physisorption interaction, it is necessary to use high-level wave-function-based post-Hartree-Fock methods like the Møller-Plesset perturbation theory 1 system . One prog., Ref. ).2O molecule adsorbed on freestanding graphene were performed by O. Leenaerts et al. [Eint) are listed in Table d0).  supercell yield similar values). One can observe very low interaction energies and no energetic preference regarding the adsorption site or orientation of the adsorbate. We have repeated the calculations taking into account the dispersion correction as proposed by Grimme [2O and graphene are significantly shorter. In addition, DOWN orientation is clearly more preferred in this case as compared to the opposite one . It can be noted that the obtained results are in reasonable agreement with the recent CCSD(T) data evaluated for the H2O/graphene system. Thus, for further consideration of the systems of interest, the PBE-D2 approximation will be used.Recently, studies based on first principles for single Hs et al. . For comy Grimme  (DFT-D2 R30° overstructures of adsorbed molecules on graphene/Ni(111) are presented in Table on-top positions have to be considered in these cases. The difference between adsorption behaviors of water on graphene and graphene/Ni(111) indicates the effect of the substrate underneath of the graphene layer, and can be explained by the fact, that in the latter case, the electron charge density is shifted to the interface between the graphene layer and the Ni(111) support. At the same time, similar to the case when free-standing graphene is used as a substrate, for the H2O/graphene/Ni(111) system, DOWN orientation is the energetically most favorable one and the preferable adsorption site is the center of the carbon ring. This theoretical observation confirms our prediction based on the interpretation of XAS spectra. It can be noted that during these calculations, structural optimization of the system was not preformed, and only the distance between graphene and adsorbate is relaxed. Full optimization of H2O geometry in the case of C_DOWN configuration leads to d0 = 2.51 Å, that is a deviation of 2% with respect to the non-relaxed value. The corresponding interaction energy is lower by 4%, than Eint given in Table The results obtained for the (√3 × √3)2O on top of graphene/Ni(111), the band structure calculations were performed. One finds the H2O-related states at the following binding energies: 3.97, 5.96, and 9.85 eV, which satisfactorily match the APRPES data.For the most stable arrangement of H2O/graphene/Ni(111) system, which is also in good agreement with the experimental results, where the modification of the XAS C K spectra was observed. In this context, the UP orientation is preferable for any adsorption position. Although on-top (T_C1) adsorption yields the highest interaction energy, one has to be aware that the present calculations cannot give exact answer regarding the energetically most favorable adsorption position since the obtained interaction energies are very close to each other (within 3%). Geometry optimization can make this difference more pronounced, especially when taking into account stronger interaction between ammonia and the considered substrate.One can see, when looking at data listed in Table 2O and NH3 on top of graphene/Ni(111) have to be considered. Optimization of molecular geometry as well as relaxation of interlayer distances within the substrate has to be performed. Furthermore, parameter-free way of accounting for dispersion corrections is preferable. The latter is possible via van der Waals density functional, developed by Dion et al. [Overall, from a theoretical side, one can see good agreement between the experimental data and the ones obtained by means of DFT calculations. However, further investigations are required before making the final conclusion regarding the position and orientation of the adsorbate with respect to the substrate under study. First, all possible arrangements of Hn et al. .K-edge spectra indicating the orbital mixing of the valence band states of graphene and adsorbates. For the occupied states, the small shifts of the graphene π states were detected in both cases with overall shift of the graphene π states to the lower binding energies reflecting the effect of p-doping  after adsorption of water and ammonia on graphene/Ni(111). Analysis of experimental results leads us to the idea of the site-selective adsorption: water is adsorbed either in the center of carbon ring or on the bond between two carbon atoms; ammonia molecules are adsorbed on the carbon atom, which is located above the Ni interface atom. This assumption is supported by the results obtained via DFT calculations.In conclusion, the authors have studied the modification of the electronic structure of the graphene/Ni(111) system upon adsorption of water and ammonia molecules at low temperature. Adsorption of both types of adsorbates leads to the modifications of the XAS C Ab Initio Simulation Package; XAS: X-ray absorption spectroscopy; XMCD: X-ray magnetic circular dischroism.ARPES: angle-resolved photoelectron spectroscopy; DFT: density-functional theory; PBE: Perdew-Burke-Ernzerhof; VASP: Vienna The authors declare that they have no competing interests.SB, MW and YSD carried out the experiment and perform treatment of experimental data. SB and ENV performed the calculations. YSD conceived of the study, and participated in its design and coordination. KH participated in design and coordination of the experimental part of this study. BP coordinated the theoretical part of this study. YSD and ENV prepared the manuscript initially. All authors read and approved the final manuscript.


Group I and II introns can be considered as molecular parasites that interrupt protein-coding and structural RNA genes in all domains of life. They function as self-splicing ribozymes and thereby limit the phenotypic costs associated with disruption of a host gene while they act as mobile DNA elements to promote their spread within and between genomes. Once considered purely selfish DNA elements, they now seem, in the light of recent work on the molecular mechanisms regulating bacterial and phage group I and II intron dynamics, to show evidence of co-evolution with their hosts. These previously underappreciated relationships serve the co-evolving entities particularly well in times of environmental stress. One of the most intricate relationships in biology is that between a host and a parasite. Almost all organisms studied so far harbor mobile genetic elements and/or their derivatives. At the genomic level, the traditional view of mobile elements is that they provide seemingly little or no benefit to the host while parasitizing the host's cellular machinery to promote element mobility through complex molecular pathways ,2. The hOne group of mobile genetic elements comprises the group I and II introns. These sequences interrupt protein-coding and structural RNA genes in all domains of life and can be considered as molecular parasites. When the gene is transcribed into RNA, the intron sequence acts as a ribozyme (an RNA with enzymatic activity), which removes the intron sequence from the primary RNA transcript, thus limiting the phenotypic cost associated with insertion of the element into a host gene and promoting their maintenance in the genome. In the case of group I and II introns, the host-parasite relationship is enriched by the fact that the introns themselves have been invaded by smaller parasitic elements - genes that encode mobility-promoting activities that enable the DNA element to move within and between genomes . Thus, aGroup I introns commonly inhabit bacterial, organellar, bacteriophage and viral genomes, and the ribosomal RNA genes (rDNA) of eukaryotes, and produce a self-splicing RNA . Group Iin trans to promote splicing of other group I introns in the same genome [ND5 gene of the mushroom coral Discosoma that encodes 15 mitochondrial genes in the P8 loop of the intron [cox1 gene, which is interrupted by another self-splicing group I intron.Many group I introns move by a DNA-based transposition mechanism known as 'homing'. Such introns harbor genes encoding so-called homing endonucleases, site-specific but sequence-tolerant DNA endonucleases that introduce double-strand breaks (DSBs) in cognate alleles that lack the intron, initiating intron mobility via a DSB-repair process  Figure . The oute genome ,18. Intee genome ,20. Groue genome ,22, as ee intron ,24. InteClostridium that contain an IS605-like element inserted at the 3' end of the intron [IS605 machinery. Intriguingly, another unusual clostridial group I intron arrangement was recently found by a bioinformatic search for riboswitches [Some bacterial group I introns have been invaded by mobile elements other than those that encode homing endonucleases. Notable among these are the chimeric intron/insertion sequence (IS) elements (IStrons) of e intron . It is nswitches , RNA strtd intron, the 3' end of an ORF called I-TevI  contributes 20 nucleotides that form part of the P6a, P6.0 and P7 structures that are essential for splicing of the intron [While many ORFs embedded within group I introns are entirely located in loop regions, a surprising number of ORFs extend beyond peripheral loops to contribute nucleotides to more distant regions of the intron that form key structural elements needed for splicing . The exte intron  . In the crystal structure of the I-TevI DNA-binding domain bound to DNA representing either its homing target site or its target operator site, only one of the carboxy-terminal residues (Tyr242) makes a hydrogen bond to the phosphate backbone of the DNA substrate, clearly not providing any specificity to the interaction of I-TevI with its DNA substrate [Importantly, this hypothesis gives rise to a number of testable predictions. First, the length of the 5' or 3' extension should be variable for each independent case of endonuclease invasion, and the position of the initiation or termination codon should be influenced by the GC content of the intron because termination and initiation codons are slightly more AT rich than GC rich and the GC content of the intron will therefore influence the probability of mutation of a sense codon into a nonsense (stop) codon. The second prediction is that the sequence at the 5' or 3' ends of an endonuclease ORF that extends into the intron may not be essential for endonuclease function. In the case of I-TevI, the 20 nucleotides that extend into the ubstrate . BioinfoThe successful spread and retention of mobile introns depends on expression of the mobility-promoting protein from within the intron, and on accurate splicing-out of the introns from the flanking exon sequences. For most group I introns, mobility and splicing are independent of each other, whereas for group II introns, and some organellar group I introns, these processes are not mutually exclusive. In these cases, translation of the IEP from the pre-splicing intron transcript is necessary for splicing because the IEP acts as an RNA maturase, in addition to facilitating mobility (reviewed in ). FurtheOne potential barrier to efficient intron splicing in bacterial and organellar genomes is the coupled nature of transcription and translation, which raises the possibility that ribosomes translating the RNA transcript could encounter the 5' exon-intron junction before the 3' splice site of the intron is transcribed, thus preventing the folding of critical intron structures and recognition of the correct splice sites by the intron. Ironically, a number of studies with bacterial group I introns have shown that translation of the exon upstream of the 5' splice site is necessary for efficient splicing, probably because a ribosome at this position acts as a 'chaperone' to prevent nonproductive interactions between exon and intron sequences that would disrupt the intron-folding pathway -51. MostLactococcus lactis [More complicated types of regulation are implied by numerous examples of ribosome-binding sites in introns that are sequestered by RNA secondary structure -58. Mutas lactis ,61. LtrDidymium [Structured group I introns interrupt the nuclear rDNA of many eukaryotes, in which coupled transcription and translation is not an issue, but they nonetheless face a different set of problems connected with intron-encoded ORFs. The well-studied group I introns in rDNA genes in the slime mold Didymium  contain Didymium . I-DirI Didymium , and hasDidymium . This isAs noted earlier, many proteins encoded within introns in organellar genomes are initially translated as fusions with upstream exon sequences, requiring subsequent proteolytic processing to provide an active protein with an amino terminus in domain IV ,41,66. L2+ is sufficient for efficient folding and splicing. The requirement for host-encoded proteins is especially evident for many organellar group II introns: at least 14 nuclear gene products promote efficient splicing of the two group II introns in the chloroplast-encoded psaA gene of Chlamydomonas reinhardtii [2+ transporter Mrs2p, and the chaperone activity of three DEAD-box proteins, Mss116p, Ded1p and Cyt19p, to promote group II intron splicing in the mitochondria of fungi [Host-encoded proteins function to stimulate the splicing of group I and II introns. In the case of group II introns, host-function-assisted splicing is also crucial for mobility, as the spliced intron RNA is an active intermediate in the mobility pathway . Detailenhardtii ,69. Anotof fungi -74.Escherichia coli where host factors, including the major replicative polymerase Pol III, repair polymerases Pol II, Pol IV and Pol V, the endonuclease RNase H1, and DNA ligase, all function to complete a retromobility event [In terms of mobility, the primary response of a host genome to the presence of mobile elements is repressive, as unregulated mobile element activity will lead to an unbearable mutational load. In recent years, a number of studies have uncovered host proteins that downregulate the activities of mobile introns, many of which (not unexpectedly) are involved in aspects of RNA processing. These proteins include RNase E and RNase I, which negatively regulate group II intron mobility by reducing the steady-state level of intron RNA -77. At tty event . Thus, tUp to this point we have considered the dynamic interplay between introns, their intramolecular inhabitants and their hosts, without considering evolutionary and environmental factors that might influence these partnerships. One traditional view of introns is that they are purely selfish DNA elements, imparting neither benefit nor burden to the host genome in which they reside. Recent evidence, however, has forced a re-evaluation of this viewpoint, particularly in the light of experimental data showing that introns can mobilize in response to stress-induced conditions ,83, as hFor instance, the group I intron endonuclease I-TevI (described in Figure E. coli is not only regulated by RNase E [Group II introns respond to metabolic stress with a burst of retrotransposition to new sites by a mechanism different from that used by group I introns. Retrotransposition of the lactococcal Ll.LtrB group II intron in  RNase E , but is  RNase E . These gStaphylococcus aureus, the ORF orf182 is interrupted by three similar group I introns, and analysis of spliced products revealed that some transcripts lack one exon, suggestive of programmed exon skipping [trans-splicing between highly similar group II introns in organellar genomes also has the potential to generate novel transcripts [Clearly, the mechanisms whereby these variant introns respond to oxidative and nutritional stresses in order to disseminate are different, but with similar outcomes - the 'abandoning of ship' for more hospitable genomic environs. For the group I intron, the mobility machinery itself, the intron endonuclease I-TevI, transduces the signal . Whereasskipping . Similarnscripts . It is tRecent results have challenged our perceptions regarding self-splicing introns, from the notion that they represent simple genomic parasites imparting neither cost nor benefit to the host genome, to that of sophisticated mobile DNA elements fully integrated into host-cell metabolism in ways that could be viewed as molecular mutualism. Host organisms devote considerable resources, whether by design or accident, to both positively and negatively influence intron behavior, and elucidating the molecular basis of host-factor involvement in the regulation of intron splicing and mobility is one area ripe for future investigation. In particular, the mechanism underlying the processing of intron ORFs that are initially translated as fusion proteins with upstream exons in organellar introns represents an obvious gap in our knowledge, but this is a technically daunting problem to address. However, it is questions of an evolutionary slant that will challenge intronologists for years to come. Foremost among these is the possibility that introns could provide some benefit to hosts by generating genetic diversity as a consequence of transposition events brought on by cellular stress.


Here we present a facile technique for the large-scale production of few-layer graphene flakes. The as-sonicated, supernatant, and sediment of the graphene product were respectively sprayed onto different types of silicon wafers. It was found that all devices exhibited current rectification properties, and the supernatant graphene devices have the best performance. Schottky junctions formed between graphene flakes and silicon n-type substrates exhibit good photovoltaic conversion efficiency while graphene/p-Si devices have poor light harvesting capability. Tremend cm2/Vs). A monol cm2/Vs), which m cm2/Vs). Graphen cm2/Vs).J-V characteristics of graphene/silicon interfaces has not been studied.Previous use of graphene in organic solar cell applications was mainly confined as flexible transparent electrodes to replace transparent indium tin oxide or fluorine-doped tin oxide for collecting charge carriers. RecentlIn this work we present a facile technique for the large-scale production of few-layer graphene flakes. Next, as-sonicated (So), supernatant (Su) and sediment (Se) of the graphene product were respectively sprayed onto n-/p-type silicon wafers. The current rectification properties of the formed Schottky junctions between different graphene sources and silicon substrates were compared.3)3·9H2O and Co(NO3)2·6H2O were dissolved in ethanol under agitation. Subsequently, the MgO powder with a surface area of 130 m2/g was mixed the solution and followed by drying at 60°C overnight. The catalyst was obtained by calcinating the resulting mixture in air at 500°C for 2 h. Graphene sheets can grow on the catalyst system from pyrolysis of acetylene at 1,000°C with the argon flow as carrier gas. The mixture product can be collected after 30 min of reaction and cooled under argon flow for about 10 min. Impurities like catalystsupport MgO and Fe-Co metal particles can be removed by washing the mixture product with hydrochloric acid under sonication. The purified graphene sheets can be obtained after filtration and washing.The graphene flakes were synthesized through chemical vapor deposition of acetylene on a MgO-supported Fe-Co bimetallic catalyst (Fe-Co/MgO with a stoichiometric composition of 2.5:2.5:95 wt%). The catN-methyl-2-pyrrolidone  under sonication. The G-NMP solution can be directly utilized to make G/n-Si devices. Part of the G-NMP solution was centrifuged; the supernatant (Su) and the sediment (Se) were taken out respectively and used for the fabrication of G/n-Si devices. The three different solutions were sprayed respectively on n-type silicon wafer , p-type silicon substrate  and glass substrates (for reference) by airbrushing. A silicon wafer with a window of pre-deposited insulating layer and a glass substrate were placed on a heating platform side by side (so that the resulting graphene flake films on Si and glass substrates should have the same thickness) and heated up to 150°C in order to evaporate the NMP solvent from the coatings. The schematic diagram of graphene-on-silicon Schottky device was displayed in FigurePurified graphene flakes were first dispersed in pure e NMP, 0. mg/mL unKα radiation line and general area detector diffraction system were used as an excitation source and detector, respectively. The experiments were carried out in Bragg-Brentano geometry.To characterize the morphological properties of graphene nanosheets, several techniques such as microscopy and X-ray diffraction (XRD) were utilized. Atomic force microscopy (AFM) images were obtained on Veeco Dimension 3100 AFM system . Scanning electron microscopy (SEM) images were obtained using a JEOL 7000 F high-resolution scanning electron microscope . This microscope has a resolution of 1.2 nm at an accelerating voltage of 15 kV and a working distance of 10 mm. The final products were mounted on aluminum pins with double-sided carbon tape and their corresponding SEM images were obtained. Elemental analysis was performed with Genesis energy dispersive spectrometer system . The X-ray powder diffraction profiles of graphene sheets were recorded in θ-2θ mode on Bruker D8 Discovery diffraction system . The monochromatic Cu J-V) characteristics were investigated in the dark and under illumination using a solar simulator at air mass coefficient 1.5 (approximately 100 mW/cm2) inside a glove box in a nitrogen environment. The illumination was on the graphene flake side. The devices were irradiated in an area of 1 × 1 cm2 and data were recorded using a Keithley 2400 source meter .To understand how a different graphene source affect the G/n-Si Schottky junction properties, current density-voltage , G band  and the 2D band . The D band arises due to the breathing modes of sp2 atoms in rings and its intensity is usually associated with defects in the carbon-based material[G band is often referred to the E2g mode at the Brillouin zone center due to the bond stretching of sp2 atoms in both ring and chains[D band is the second order mode and its shape as well as its position is used to identify a single layer from bilayer and few (less than five) layer graphene[As shown Figurematerial, in the material. The G bd chains. The 2D graphene. Figured-spacing) between two subsequent graphene sheets can be calculated from the (002) diffraction peak position[B cos θ), where λ is the x-ray wavelength, B is the full width at half maximum of the diffraction peak, and θ is the Bragg angle)[d-spacing and the size of crystallite, the graphene sheets in this work were estimated to have in average of about four layers.The crystallinity and number of layers in the graphene nanosheets can be analyzed by XRD technique. The XRD profile of the graphene sheets grown by radio frequency catalytic chemical vapor deposition method was shown in Figureposition. The widg angle). Based oThe top view morphologies of a typical G/Si device made of supernatant were displayed in SEM and AFM images Figure. Both SE3 at ±1 V. As seen from FigureJ-V curve. The Shockley ideal diode equation gives the J-V characteristic of an ideal diode in either forward or reverse bias (or no bias). The equation is as follows:The typical dark current–voltage characteristic of G/n-Si heterojunction device is displayed in the top inset of FigureJ is the diode current, JS is the reverse bias saturation current , VD is the voltage across the diode, VT is the thermal voltage , and n is the ideality factor, also known as the quality factor, or sometimes emission coefficient. The ideality factor n varies from 1 to 2 depending on the fabrication process and the semiconductor material and, in many cases, is assumed to be approximately equal to 1. As shown in Figuren(Su) > n(So) > n(Se) . The saturation current density Js can be expressed with thermionic emission model[2)where on model in the fA is the Richardson constant, which is 112 A/(cm2K2)for n-Si and 32 A/(cm2K2) for p-Si substrates[eϕSB is the zero-bias Schottky barrier height. By combining equations 1 and 2 and fitting the J-V curves of the devices in dark, the Schottky barriers are estimated to be 0.52 to 0.67 eV on average for the graphene/n-Si devices and 0.61 to 0.73 eV for the graphene/p-Si devices at 300 K, which is consistent with the higher current densities observed in n-Si devices versus p-Si devices. These values are comparable to the values of the bilayer graphene-on-silicon devices[where bstrates, and eϕS devices, but con devices, and the devices.Usually graphene flakes, generally known to exhibit weak p-type conductivity, and difon/ioff ratio of >102, suggests that the electron–hole pairs could be efficiently generated and separated in the G/Si solar cells, which helps facilitate the Schottky junctions to harvest solar light more effectively. The schematic energy band diagrams of G/n-Si and G/p-Si were displayed in FigureTo investigate the underlying physics of the G/Si Schottky junction solar cells, photoresponse characteristics of both G/n-Si and G/p-Si devices were shown in Figure2 or nitric acid in G/n-Si devices[Although the photovoltaic conversion efficiency of the G/Si solar cells is relatively low, the photovoltaic performance could be improved by doping graphene with nitrogen in G/p-Si configuration, which could be attributed to the enhanced n-type conductivity of the graphene film at higher N doping concentration, or treating graphene with a strong acid such as SOCl devices. AdditioWe developed chemical vapor deposition approach to the synthesis of graphene at large scale and low cost, which makes the wide applications of graphene possible. The graphene coating on n-Si wafer forms Schottky junction with rectification behavior. The fabrication of Schottky junctions has the merits of low cost and simplicity. By comparing different graphene sources, it was found that the supernatant graphene is the most desirable material for the fabrication of G/n-Si junction with excellent rectifying capability and good photovoltaic conversion efficiency. Although G/p-Si also exhibit rectification behavior, they demonstrate poor photovoltaics conversion efficiency due to the weak p-type conductivity of our graphene flakes.The authors declare that they have no competing interests.MM and ZL contribute equally in this work. MM carried out the experiments and measurements; ZL offered idea, performed data analysis and drafted the manuscript. JC and CT participated in the discussion. All authors read and approved the final manuscript.MM is Ph.D. candidate connected with the Department of Physics and Astronomy, University of Arkansas at Little Rock. ZL is a visiting professor at the Department of Physics and Astronomy, University of Arkansas at Little Rock, JC and TC are the professors at Department of Physics and Astronomy, University of Arkansas at Little Rock.


Before the deposition, optical microscopy and Raman spectroscopy were utilized to identify the number of the graphene layers. After the deposition, scanning electron microscopy was used to observe the morphologies of the particles. Systematic analysis revealed that the average sizes of the nanoparticles increased with the number of graphene layers. The density of nanoparticles decreased as the number of graphene layers increased, revealing a large variation in the surface diffusion strength of nanoparticles on the different substrates. The mechanisms of formation of these layer-dependent morphologies of silver on n-layer graphene are related to the surface free energy and surface diffusion of the n-layer graphene. The effect of the substrate such as SiO2/Si was investigated by fabricating suspended graphene, and the size and density were similar to those of supported graphene. Based on a comparison of the results, the different morphologies of the silver nanoparticles on different graphene layers were theorized to be caused only by the variation of the diffusion barriers with the number of layers of graphene.The distributions of sizes of silver nanoparticles that were deposited on monolayer, bilayer, and trilayer graphene films were observed. Deposition was carried out by thermal evaporation and the graphene films, placed on SiO It, therefore, has various unique electrical and optical properties of interest to scientists and technologists[2 and Al2O3 with special thickness between graphene and the substrate are typically used to make graphene optically visible[A single atomic layer of graphene is the thinnest spologists-3. Graph visible-7. The e visible. Raman a visible-14. Rece visible-20. The  visible,22.In this work, layers of graphene were fabricated in a sample by micromechanical cleavage. The number of layers of graphene was determined by micro-Raman spectroscopy and optical microscopy. After silver nanoparticles were deposited on the sample using a thermal deposition system, the distribution and sizes of the particles on flakes with different numbers of layers were systematically analyzed. To analyze the effect of the substrate, suspended graphene was fabricated, and the size and density thereof were found to be similar to those of supported graphene. The different results for the mono-, bi-, and tri-layer graphene are theorized to be caused only by the variation among the diffusion barriers of the various graphene layers, which provides a method of determining the number of graphene layers and provides information that can be utilized to elucidate the interaction between a graphene flake and its substrate.2 over a Si substrate. Graphene flakes that contained different numbers of graphene layers were distributed on different areas on the same sample. The variation in the color contrast with the number of graphene layers is observed under an optical microscope, as presented in FigureA graphene flake was fabricated by micromechanical cleavage with scotch tape. It was capped with 300-nm-thick layer of SiO2/Si substrate and the monolayer, bilayer, and trilayer graphene flakes were obtained, and shown on right-hand sides of Figure2/Si substrate.Silver nanoparticles were deposited on graphene flakes at a rate of 0.5 nm/min using a thermal deposition system to a thickness of 5 nm at a fixed temperature of the deposition system of 300 K. To elucidate the surface diffusion, the sample after deposition was maintained at 373 K in a vacuum for 1 h. To study the surfaces of the graphene flakes, scanning electron microscopy (SEM) images of the distribution of nanoparticles on the SiO2/Si substrate, monolayer, bilayer, and trilayer graphene. The sizes of the nanoparticles on the monolayer graphene flake are distributed in the range of 0 to 50 nm, whereas those of the nanoparticles on the trilayer graphene flake were distributed in the range of 10 to 70 nm. Whereas the sizes of the nanoparticles on the SiO2/Si substrate were distributed in the range of 0 to 50 nm, the majority of them were in the range of 10 to 30 nm.To investigate the density and size of the nanoparticles, average size and density were determined by histogram analysis. The histograms, on the left-hand sides of Figure2/Si substrate without any graphene flake. The nanoparticles on the trilayer graphene are largest, with a mean size of approximately 36 nm; as the number of graphene layers decreased, the average size fell to 32, 25, and 18 nm. The density of nanoparticles on the trilayer graphene was also the lowest, at around 3.1 × 1014/m2. Reducing the number of graphene layers increased the density from 3.1 × 1014 to 9.1 × 1014/m2. The density of nanoparticles decreased as the number of graphene layers increased, revealing a large variation in the surface diffusion strength of nanoparticles on the different substrates. The mechanisms of formation of these layer-dependent morphologies of silver on n-layer graphene are related to the surface free energy and surface diffusion of the n-layer graphene. Different morphologies of silver on the differently layered graphene are obtained when silver particles are deposited on the graphene surfaces because of the differences of surface free energy. The variation in surface free energy with number of layers is responsible for the variation in the morphologies of silver on the differently layered graphene. The particles are randomly deposited on the graphene surface, and surface diffusion causes randomly arranged particles to combine to form relative large islands. The surface diffusion coefficient D ∝ exp(−En/kT) indicates the interaction between particles and the substrate surface, where En and k denote the diffusion barrier and the Boltzmann constant, respectively. The density of the particles on the substrate (N) can be expressed as N ∝ (1/D)1/3 for isotropic surface diffusion. The diffusion barrier specifies the interaction between particles and the substrate surface. The difference between the diffusion barriers of the substrate and monolayer can be calculated as ΔE = E0 − E1 = 3kT ln(N0/N1) = 61.7 mev by combining the above equations. The difference between the barriers of the substrates with other numbers of layers can be similarly obtained, and they are 39.1 meV (E1-E2) between the first and second layers and 3.1 meV (E2-E3) between the second and third. The difference between the diffusion barriers of silver decreased as the number of graphene layers increased. According to one review[2/Si substrate and different graphene layers, which the most is trilayer, is in the range of 9 × 1013 to 1.25 × 1013/m2, and the difference between diffusion barriers of different layers is in the range 500 to 291 meV. The area density of the silver nanoparticles herein is approximately ten times this value, indicating that silver nanoparticles can be more easily prevented from moving along the graphene surface, revealing a stronger interaction between silver and graphene than between gold and graphene.In Figuree review, the are2/Si is responsible for the variation in morphology, suspended graphene was fabricated by mechanical exfoliation of the graphene flakes onto an oxidized silicon wafer. First, ordered squares with areas of 6 μm2 were defined by photolithography on an oxidized silicon wafer with an oxide thickness of 300 nm. Reactive ion etching was then used to etch the squares to a depth of 150 nm. Micromechanical cleavage of HOPG with scotch tape was then used to deposit the suspended graphene flakes over the indents. The deposition conditions and silver nanoparticles were the same for both types of graphene. Figure2 does not affect the distribution of silver nanoparticles on the surface of graphene.To determine whether the substrate effect of SiO2, suspended graphene was also fabricated. The size and density of suspended graphene were found to be similar to those of the supported graphene. According to these results, only variations in the interactions between n-layer graphene and the silver nanoparticles were responsible for the variation in their distribution.In this work, the distribution of sizes of silver nanoparticles that were deposited on graphene films with different numbers of atomic layers of graphene was investigated. A systematic analysis revealed that the average size of the nanoparticles increased, and the area density and the difference between diffusion barriers of the nanoparticles decreased as the number of graphene layers increased. To analyze the effect of a substrate such as SiOThe authors declare that they have no competing interests.C-WH carried on the experimental parts: the acquisition, analysis, and interpretation of data. C-WH was also involved in drafting the manuscript. H-YL and C-HH took the analysis and interpretation of data. They also had been involved in revising the manuscript. R-JS and W-HW  prepared the graphene samples using micromechanical method and obtained the OM images. C-YL has made substantial contributions to the conception and design of the study, and in the critical revision of the manuscript for important intellectual content. H-CC, the corresponding author, had made substantial contributions to conception and design, and had been involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript.C-WH received his BS degree in Electrical Engineering from the National University of Kaohsiung, Kaohsiung, Taiwan, in 2008. He studied his MS degree in 2008 and Ph.D. degree directly in 2009. Currently, he is a Ph.D. candidate in the Department of Photonics, National Cheng Kung University, Tainan, Taiwan. He focuses on the property of graphene and surface plasmon resonance of nanoparticles. H-YL received her doctoral degree from the Institute of Biomedical Engineering, National Cheng Kung University in 2010. Her current research interest focuses on the SERS properties of graphene and possible sensing application. C-HH received his doctoral degree from the Institute of Electro-Optical Science and Engineering, National Cheng Kung University in 2009. His current research interest focuses on the Raman and SERS properties of graphene incorporating with metal nanoparticles.


In natural conversation, the minimal gaps and overlaps of the turns at talk indicate an accurate regulation of the timings of the turn-taking system. Here we studied how the turn-taking affects the gaze of a non-involved viewer of a two-person conversation. The subjects were presented with a video of a conversation while their eye gaze was tracked with an infrared camera. As a control, the video was presented without sound and the sound with still image of the speakers. Turns at talk directed the gaze behaviour of the viewers; the gaze followed, rather than predicted, the speakership change around the turn transition. Both visual and auditory cues presented alone also induced gaze shifts towards the speaking person, although significantly less and later than when the cues of both modalities were available. These results show that the organization of turn-taking has a strong influence on the gaze patterns of even non-involved viewers of the conversation, and that visual and auditory cues are in part redundant in guiding the viewers’ gaze. Behind the apparent ease of conversation, whether a formal or a more casual one, lies a tight organization of speaking turns According to the organization of turn-taking, one party talks at a time, and the change of the turn is allowed in transition-relevance places Gaze, as an important nonverbal gesture, has a role in coordinating conversation and turn-taking as well as in informing about the person’s target of attention. The speaker’s gaze direction may signal that the turn is approaching completion: the speaker makes eye contact with the listener around turn exchange, whereas the eye contact is less common when the speaker is still continuing the turn In addition to face-to-face conversation, gaze coordination occurs in many other situations involving social interaction. For example when subjects are listening to a previously recorded description of the same TV scene they are viewing, their eye movements follow the eye movements of the speaker with an average delay of 2 s In the present study we showed a group of volunteers a short video of a real conversation to find out how people follow a two-party conversation with their gaze. As a control, the same video was presented without sound and the same soundtrack with a still frame captured from the original video. To be able to assess the effects of turn transitions and the turn-transition cues to followers of a natural conversation, we developed quantitative methods for analysing gaze behaviour of a group of subjects. This experimental setup with passive viewing of a conversation presented from video enabled us to expose each subject to an identical natural conversation, to evaluate the effect of the verbal and nonverbal cues separately, and to focus our analysis on any conversational events of interest to assess the similarity of the viewers’ gaze behaviour. Although our subjects were non-involved viewers of the conversation, their gaze was expected to be informative of the most attention-capturing aspects of the conversation.We hypothesized that accumulation of the syntactic, pragmatic and prosodic cues of turn transition would prompt a shift of gaze even in a viewer of the conversation. We further expected to see synchronous gaze behaviour between subjects, directed by the organization of turn-taking. We also studied the timing of the gaze patterns with respect to turn transitions.Nineteen Finnish-speaking young adults  with normal or corrected-to-normal vision volunteered in the main experiment. Nine subjects participated in the two control experiments, video with no sound  or still image with sound .The subjects gave their written informed consent prior to the experiment after the course of the study had been explained to them. The study had prior approval by the Ethics Committee of the Hospital District of Helsinki and Uusimaa.(i) the viewer would be guaranteed an access to the facial and posture orientation of the participants of the conversation and (ii) to the topic talked about, (iii) the speaker should change rather rapidly and one participant should not hold the turn for long, and (iv) the scene should be clean of any other possibly interesting items.In the main experiment, we presented an audiovisual stimulus: a 5.5-min video clip of two women  having a conversation. As a control, (i) the same video was shown without sound (later referred as ‘video only’) and (ii) the same soundtrack was played with only a still frame captured from the original video (later referred as ‘sound only’). The criteria for choosing the original video clip were that The video was presented with ClearView 2.7.1 software  on a grey background, and its size was 20 deg×16 deg (16 deg×12 deg for 4/19 subjects because of different display resolution settings) when viewed from the distance of 60 cm. The sound was presented through stereo loudspeakers, located on both sides of the screen 3 P2: mt. Mh >Kuinka paljon< siel oli ihmisiä.Mt. Mh >How many<people were there.[1∶17∶3]The example illustrates syntactic (S), pragmatic (Pra), and prosodic (Pro) cues for turn transition. Syntactic cues mark places where the utterance could be syntactically complete notwithstanding the preceding context. Pragmatic cues mark places where the utterance can be understood as complete in its conversational context. Prosodic cues refer to places where the intonation of the utterance sounds as if the speaker is finishing his/her turn of talk In addition to the turn-transition cues, routinely annotated in conversation analysis, we also defined the ends and the starts of speech, followed or preceded by a minimum of ∼0.5 s of silence. We defined ‘fast turn transition’ (fTT) as an event type in which the speaker change (from the end of one speaker’s speech to the start of the other’s) occurred in less than 300 ms and without overlap of the two speakers; altogether 41%  and ClearView 2.7.1 software, with a sampling frequency of 50 Hz. The eye tracker was calibrated before the experiment by asking the subjects to fixate at five points that covered the screen. The tracking was done binocularly and based on video-oculography by dark-pupil–corneal-reflection method; the infrared camera and the infrared light sources were integrated into the computer screen.As the faces of the conversation participants in the stimulus video stayed relatively still during the whole recording, positions of two regions of interest (ROIs), one ROI for each face, were defined as stationary rectangles see .The gaze data were further analysed in Matlab 7 (MathWorks). Gaze direction changes were defined as gaze moving from one ROI to the other in less than 70 ms, later referred as ‘ROI change’. This limit was chosen because a 10-deg saccade lasts for about 40 ms For the analysis of the group-level effects, we computed the mean ROI change rate over all subjects separately for all different conversational events, in 200-ms windows across a 5-s (from –2 to 3 s) epoch centered on each event; the same analysis was made for the main experiment and for the two control conditions. For comparison, the baseline level of ROI change rate was estimated in the same manner around “events” placed equidistantly once every 6 s throughout the video.The effect of visual and auditory cues to the subjects’ responses to turn transitions was assessed by comparing the cumulative distributions of ROI changes around fTTs in the three experimental conditions with Kolmogorov-Smirnov test in Matlab.p<10–9; see The data of p  = 0.5, between the two ROIs. The colour codes refer to p-values  for obtaining the observed sample from such an underlying distribution. We noted that for 68% of the total time at least 14 out of the 19 subjects viewed the same ROI.The statistical significance of the similarity vs. randomness of the subjects’ gaze behaviour is indicated with coloured dots along the x  = 0 line of The effects of various turn completion cues and of other events in the conversation were assessed by examining the gaze-direction changes around each cue. Our goal was to understand, by tracking the eye gaze, how uninvolved viewers follow a conversation between two other persons. The gaze patterns across our 19 subjects showed similarity that differed statistically significantly from random viewing. Turns at talk directed the subjects’ gaze so that the current speaker received most of the gazes. The time spent looking at the current speaker was on average 74%, which closely resembles the behaviour of participants in a two-party conversation in which the listener looks at the speaker on average 75% of the time The fTTs induced gaze-target changes also in the control experiments when either only the verbal (sound only) or only the nonverbal (video only) cues of turn transition were present. In human speech, speech-sound envelope and mouth opening are highly correlated, and they are both modulated at 2–7 Hz Although we found–as we had expected–that the stronger the cue for turn transition, the more gaze-direction changes it elicits, very similar behaviour was seen also in the video-only condition, in which the subjects could not hear the verbal cues. Thus this result had to arise from the strong temporal correlation of turn transitions and the different turn-transition cues. In addition, because the cue strength is inversely related to the temporal distance to turn transition, it is obvious that the stronger is the cue for turn transition, the more its effects are contaminated by the effects of the close-by turn transition. Consequently, we had to abandon any further attempts to analyse the effects of different verbal cues of turn transition on the viewer’s gaze behaviour. Our results suggest a need for further research on visual cueing–by means of participants’ gaze as well as by body posture and kinesics–of turn transition, and its coordination with the verbal cueing.We did not observe anticipation of turn transition in the viewers’ gaze behaviour; instead, most of the gaze-target changes occurred about 0.5 s after fast turn transition, and about 0.3 s after start of speech. This lag equals the lag of a saccade towards a simple auditory target Our experimental setup is in line with the currently dominant tradition of social neuroscience, where the effects of social stimuli to an uninvolved viewer are assessed. However, this “spectator stance” has been criticized see e.g. , as it n