Long INterspersed Element-1 (LINE-1 or L1) retrotransposition poses a mutagenic threat to human genomes. Human cells have therefore evolved strategies to regulate L1 retrotransposition. The APOBEC3 (A3) gene family consists of seven enzymes that catalyze deamination of cytidine nucleotides to uridine nucleotides (C-to-U) in single-strand DNA substrates. Among these enzymes, APOBEC3A (A3A) is the most potent inhibitor of L1 retrotransposition in cultured cell assays. However, previous characterization of L1 retrotransposition events generated in the presence of A3A did not yield evidence of deamination. Thus, the molecular mechanism by which A3A inhibits L1 retrotransposition has remained enigmatic. Here, we have used in vitro and in vivo assays to demonstrate that A3A can inhibit L1 retrotransposition by deaminating transiently exposed single-strand DNA that arises during the process of L1 integration. These data provide a mechanistic explanation of how the A3A cytidine deaminase protein can inhibit L1 retrotransposition.DOI:http://dx.doi.org/10.7554/eLife.02008.001 Transposable elements are often referred to as ‘jumping genes’ because they can move between different locations within a genome. These sequences of DNA are found in many organisms and can make up a significant proportion of the genetic material: almost 50% of the DNA in the case of the human genome.Transposable elements are grouped by how they move to new locations in a genome. Some move by a cut-and-paste mechanism—whereby the transposable element DNA is removed from one location and inserted back at a new genomic location. Others, termed retrotransposons, move by a copy-and-paste mechanism: the DNA sequence is transcribed into an RNA intermediate, and then copied back into DNA before being inserted into a new location. Retrotransposons can accumulate to great numbers in genomes: and one retrotransposon, called LINE-1, is present at an estimated 500,000 copies in the human genome.Although most copies of LINE-1 are inactive, the average human genome contains about 80–100 that are predicted to be able to ‘jump’ to new locations. Given that these retrotransposons could insert into, and disrupt, vital genes, it follows that our cells would have evolved ways to limit their movement. An enzyme named APOBEC3A is known to limit the movement of LINE-1 retrotransposons in cells. APOBEC3A can alter the letters, or bases, that make up the genetic code. This enzyme acts on single-strand DNA to change ‘C’ bases to ‘U’ bases, which could explain how APOBEC3A combats LINE-1. However, no evidence for such mutation of LINE-1 sequences by APOBEC3A had been found to date.Now, Richardson et al. recreate the copying of LINE-1 RNA back into DNA in a test tube—and reveal that APOBEC3A can mutate single-strand LINE-1 DNA. Critically, as long as the RNA intermediate and DNA copy remain together, the LINE-1 DNA is protected. However, when LINE-1 inserts into a new location the temporarily exposed single strand of LINE-1 DNA becomes susceptible to mutation by APOBEC3A. Human cells can detect and destroy ‘U’ bases in DNA—and only by inhibiting this ability were Richardson et al. able to observe APOBEC3A mutations in new LINE-1 copies within the genomes of living cells.Richardson et al. speculate that the activity of APOBEC3 enzymes must strike a balance between limiting the spread of retrotransposons and minimizing the mutation of the cell's own DNA. Future work could address important questions, such as: do APOBEC enzymes affect the ‘jumping’ of LINE-1 retrotransposons in human reproductive cells and the early embryo, where new LINE-1 insertions could be passed on to subsequent generations? Also, does a loss of APOBEC3 activity lead to new LINE-1 insertions in cancerous cells? And does this effect how tumors form and/or progress? Since APOBEC3 enzymes can cause mutations in cancers, they have been proposed as new targets for anti-cancer drugs—therefore, it is crucial to uncover any harmful effects of inhibiting APOBEC3 enzymes that might limit the effectiveness of such treatments.DOI:http://dx.doi.org/10.7554/eLife.02008.002 Long INterspersed Element-1 (LINE-1 or L1) derived sequences comprise roughly 17% of human genomic DNA . The vasOngoing L1 retrotransposition events contribute to intra- and inter-individual genetic variation and, on occasion, can disrupt gene function, leading to sporadic genetic diseases . Given tHere, we employ an in vitro assay (L1 Element Amplification Protocol or LEAP) that recapitulates the first strand cDNA synthesis step of the L1 integration to elucidate a mechanism for A3A-mediated inhibition of L1 retrotransposition. In LEAP reactions that include RNase H, an enzyme that specifically degrades the RNA strand of an RNA/DNA heteroduplex, we report that single-strand L1 cDNAs are rendered susceptible to A3A-mediated deamination. These data suggest that newly synthesized L1 cDNAs remain annealed to their mRNA templates after reverse transcription, protecting the L1 cDNA from deamination by A3A. We further demonstrate that engineered L1 retrotransposition events derived from cultured human cells that co-express A3A and the uracil DNA glycosylase inhibitor protein (UGI) contain strand-specific, A3A-mediated C-to-U deamination events. Together, these data indicate that A3A can inhibit L1 retrotransposition by deaminating transiently exposed single-strand DNA that arises during the process of L1 integration. Moreover, our results provide direct evidence that A3A can deaminate single-strand genomic DNA in human cells.We used a cultured cell assay  to deterWe co-transfected HeLa cells with retroelement expression constructs and plasmids encoding WT A3A or a deaminase-deficient mutant, A3A-C106S . As a coSince LINE element retrotransposition proceeds by target-site primed reverse transcription (TPRT), which requires element-encoded endonuclease (EN) and reverse transcriptase (RT) activities , we inveEscherichia coli. As expected rA3A had cytidine deaminase activity on single-strand  or in the presence of rA3A or deaminase-deficient rA3A_C106S. We did not detect significant numbers of C-to-U deamination events in L1 cDNAs derived from LEAP reactions in the absence of rA3A or the presence of rA3A_C106S . In contThe non-uniform pattern of deamination events observed above suggested that the L1 cDNA may remain annealed to L1 mRNA, generating L1 mRNA/cDNA heteroduplexes after reverse transcription, thereby leaving only the single-strand oligonucleotide LEAP adapter susceptible to deamination. Thus, we tested whether inclusion of RNase H into LEAP reactions renders L1 cDNA susceptible to deamination. In the presence of rA3A and RNase H, 228 of 600 (38%) 5′-TCA-3′ substrates within the L1 cDNAs showed evidence of deamination . In contOur results demonstrated that rA3A could deaminate single-strand L1 cDNAs in vitro. We therefore hypothesized that A3A may deaminate transiently exposed single-strand DNA intermediates that arise during L1 TPRT in cultured cells. Uridine residues are efficiently removed from DNA through combined actions of uracil DNA glycosylase (UNG) and apurinic/apyrimidinic endonuclease (APE) . In prinTo test whether UNG contributed to the inhibition of L1 retrotransposition downstream of A3A activity, we conducted L1 retrotransposition assays in the presence of codon optimized bacteriophage-derived uracil DNA glycosylase inhibitor (UGI) that selectively inhibits UNG activity . RemarkaWe hypothesized that UGI expression should allow the detection of A3A-mediated C-to-U deamination events in retrotransposed L1s. Thus, we co-transfected HeLa cells with A3A, UGI, and pJM140/L1.3/Δ2/k7, an engineered L1 vector that contains both an mneoI retrotransposition cassette and a ColE1 bacterial origin of replication , 2005 F. This moTo gain further evidence for A3A-mediated deamination of L1 retrotransposition events, we employed a stable UGI-expressing U2OS cell line  Figure . In contWe next analyzed sequences of engineered L1 retrotransposition events. We used pJM140/L1.3/Δ2/k7 to generate a panel of clonal G418-resistant U2OS_UGI cell lines, isolated the resultant L1 retrotransposition events, and examined them for evidence of A3A-mediated deamination. Characterization of 24 control L1 retrotransposition events did not reveal any G-to-A mutations . SimilarWe propose a straightforward model for how A3A inhibits L1 retrotransposition . In the The over-expression of A3A does not abolish L1 retrotransposition completely, but reduces it to ∼30% of control levels. Moreover, some L1 retrotransposition events generated in the presence of A3A and UGI lacked evidence of deamination. Why and how some L1 retrotransposition events evade A3A-mediated deamination requires further study. However, it is possible that ectopic A3A expression is too low to efficiently target L1 TPRT intermediates in some cells or that L1 second-strand cDNA synthesis occasionally is completed before A3A can gain access to TPRT intermediates to deaminate single-strand L1 cDNAs.In sum, we conclude that deamination of transiently exposed single-strand DNA arising during L1 TPRT is responsible, at least in part, for A3A-mediated inhibition of L1 retrotransposition. It is noteworthy that the mobility of an evolutionarily related bacterial group II intron requires RNase H1 activity . SimilarMechanistic insights gained from studying deaminase-dependent A3A-mediated L1 inhibition could be applicable to retroelement inhibition by other A3 proteins. A3A expression is restricted to peripheral blood lymphocytes , suggestAPOBEC3 activity has been implicated in genome hyper-mutation in numerous cancers , 2013b. lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17  phoA supE44 λ–thi-1 gyrA96 relA1) competent E. coli . Competent cells were prepared in house as described in All plasmids were grown in DH5α  containing a full-length L1.3 element with the pJM102/L1.3 was described previously  and is ispa .pLGCX/UGI was described previously  and consNEO) gene from pEGFP-N1  introduced into the backbone. The multi-cloning site contains the U6 promoter. To generate linearized plasmid for control transfections, pU6i NEO was digested with BglII , which does not disrupt NEO gene expression, and run on an agarose gel to confirm linearization. The restriction digest reactions were purified using the QIAquick Gel Extraction Kit (Qiagen).pU6i NEO is a pBSKS-based plasmid with the neomycin phosphotransferase . The reduction of G418-resistant foci in the presence of A3A was determined to be reliable and reproducible. Approximately eighteen hours after plating, transfections were carried out using the FuGene 6 transfection reagent  and Opti-MEM (Life Technologies), according to the protocol provided by the manufacturer (3 μl FuGene and 97 μl Opti-MEM per one μg of DNA transfected). Media were replaced the following day. Approximately 72 hr post-transfection, cells were subjected to selection with 400 μg/ml G418 (Life Technologies). G418 selection was carried out for 12–14 days and the selection media were replaced every other day. Colonies were washed with 1X phosphate buffered saline (PBS) , fixed with 2% paraformaldehyde/0.4% glutaraldehyde, and stained with 0.1% crystal violet solution.HeLa and U2OS cell retrotransposition assays were carried out as previously described . Cells w5 cells were plated per T-75 flask or 2 × 104 cells per well of a six-well dish. 8 hr later, transfections with wild-type (pAD2TE1) and endonuclease mutant (pAD136) retrotransposition indicator plasmids were carried out using the FuGene 6 transfection reagent and Opti-MEM according to manufacturer's protocol (3 μl FuGene and 97 μl Opti-MEM per one μg of DNA transfected). Media were replaced the following day. Approximately 72 hr post-transfection, cells were subjected to selection with 400 μg/ml G418. Selection was carried out for 12–14 days and the selection media were replaced every other day. Colonies were washed with 1X PBS, fixed with 2% paraformaldehyde/0.4% glutaraldehyde, and stained with 0.1% crystal violet solution.CHO cell retrotransposition assays were carried out in 4364a and XR-1 cell lines as previously described . ApproxiFor toxicity control transfections, which were carried out in parallel with each retrotransposition assay, HeLa and CHO cells were plated and transfected as described above, substituting pU6i NEO plasmid for the L1 retrotransposition indicator cassette. As in the retrotransposition assays, selection with 400 μg/ml G418 was initiated approximately 72 hr post-transfection and carried out for 12–14 days. The selection media were replaced every other day. Colonies were fixed and stained as described for the retrotransposition assays. The percent of G418 colonies obtained from A3A/pU6i NEO co-transfections relative to control (β-arrestin/pU6i NEO) co-transfections was calculated and used to normalize the number of G418-resistant colonies obtained in the corresponding A3A/L1 and control β-arrestin/L1 retrotransposition assays. In this way, any nonspecific toxicity caused by A3A expression is accounted for, and the percent inhibition reported for each assay reflects specific inhibition of L1 retrotransposition by A3A.E. coli, strain C43 (F– ompT hsdSB (rB- mB-) gal dcm (DE3)) . Recombinant protein was produced by expression at 37°C in LB plus kanamycin for 3 to 5 hr after induction with 1 mM IPTG. Bacterial cultures were pelleted and cell pellets were frozen prior to lysis. Cell pellets were resuspended in lysis buffer  and lysed using a fluidizer . The soluble (supernatant) fraction was separated by centrifugation at 40,000×g for 1 hr at 6°C. Recombinant protein from the supernatant was purified by binding to Ni-Sepharose Fast Flow , washed with lysis buffer, and eluted with lysis buffer containing 0.5M imidazole. Protein was concentrated using Amicon Ultracell-10K filters , further purified by gel filtration on a HiLoad 16/60 Superdex 200 prep grade column , and dialyzed in Slide-A-Lyzer 10K MWCO cassettes  in dialysis buffer . The proteins then were concentrated using Amicon Ultracell-10K filters (Millipore). Protein purification was monitored by SDS-PAGE, Coomassie blue staining, and immunoblotting with antibodies against His-tag  and APOBEC3A  in buffer containing 20 mM Tris–HCl (pH 8.0) and 1 mM DTT for 1 hr at 37°C. The resultant products were treated with 150 mM NaOH for 30 min at 37°C, followed by incubation at 95°C for 5 min, and chilled at 4°C for 2 min. The samples were separated using 15% TBE/urea-PAGE. Gels were directly analyzed using a FLA-5100 scanner . The PAGE-purified single-strand DNA oligonucleotide (Invitrogen) used for the deaminase assays (FITC-TCA) contains a single cytosine in the A3A specific target trinucleotide, 5′-TCA:To determine rA3A deaminase activity in UDG-dependent deaminase assays, increasing concentrations of rA3A or rA3A_C106S were incubated with a 5′-end fluorescein isothiocyanate (FITC) labeled single-strand deoxyoligonucleotide (0.4 μM) in a final reaction volume of 30 μl containing: 40 mM Tris–HCl (pH 8.0) 10% glycerol, 40 mM KCl, 50 mM NaCl, 5 mM EDTA, and 1 mM DTT. The reactions were incubated at 37°C for 4–8 hr, stopped by heating to 90°C for 5 min, cooled on ice, and then centrifuged at 10,000×CATTTATTTATTTATTTATTT-3′) or non-complementary (ns) oligonucleotide at the ratios indicated in Preparation of L1 RNPs and LEAP assays were carried out generally as previously described in Integrated DNA Technologies  synthesized the oligonucleotides used in the studies below.5′-GATGGATGATGAATAAAGTGTGGGATGATCATGATGTATGGATAGGTTTTTTTTTTTT-3′5np1 outer: 5′-GATGGATGATGAATAAAGTG-3′L1 3′ end: 5′-GGGTTCGAAATCGATAAGCTTGGATCCAGAC-3′ LEAP adapter 5np1, purified by high-performance liquid chromatography (HPLC):6 HeLa cells were plated per T-175 flask (BD Biosciences). Approximately eighteen hours later, they were transfected with 30 μg of L1 plasmid DNA using FuGene 6 transfection reagent and Opti-MEM according to manufacturer’s protocol (3 μl FuGene and 97 μl Opti-MEM per μg of DNA transfected). Approximately 72 hr post-transfection, cells were subjected to selection with 200 μg/ml Hygromycin B (Life Technologies). Hygromycin media were replaced daily for 9–11 days. Untransfected control HeLa cells were plated 2–3 days before harvest day. On harvest day, cells were washed three times with 10 ml cold 1 × PBS and collected by scraping into 10 ml cold 1 × PBS. Cells were pelleted in a 15 ml conical tube (BD Biosciences) at 3000 × g for 5 min at 4°C. The PBS was removed and the cells were lysed for 15 min on ice in 1 ml lysis buffer . Following lysis, cell debris was pelleted by centrifugation at 3000×g for 5 min at 4°C and the whole-cell lysate was transferred to a new tube.Approximately 6 × 102, 20 mM Tris–HCl (pH 7.5), 1 mM DTT, and 1 × Complete EDTA-free protease inhibitor cocktail (Roche). Cells were ultracentrifuged for 2 hr at 178,000×g at 4°C. The resulting pellet was resuspended in 50–100 μl (depending on pellet volume) in 1 × Complete EDTA-free protease inhibitor cocktail (Roche). Protein concentration was determined by Bradford assay  and the sample was brought to a final concentration of 1.5 mg/ml. The portion of the sample intended for LEAP reactions was diluted to 50% vol/vol glycerol, aliquoted, flash-frozen on dry ice/ethanol, and stored at −80°C. Additional portions were reserved for RNA extraction.Whole-cell lysate (1 ml) was centrifuged through a sucrose cushion consisting of 8.5% and 17% wt/vol sucrose in 80 mM NaCl, 5 mM MgClPfuTurbo Cx Hotstart DNA polymerase (Agilent) using the following cycling conditions: 95°C for 2 min, followed by 35 cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C, with a final extension time of 7 min at 72°C. PCR products were visualized on 2% agarose gels.RNA was extracted from RNP preparations using the RNeasy Mini Kit (Qiagen) following the manufacturer's instructions, omitting the cell lysis step and including on-column DNase treatment. RNA was quantified using a Nano-Drop spectrophotometer and diluted to 0.5 μg/μl. RT-PCR was carried out on 0.5 μl of purified RNA, using MMLV reverse transcriptase  and 0.8 μM LEAP adapter (5np1) at 42°C for 30 min. Reactions containing 100–300 ng of rA3A or rA3A_C106S protein in 10% glycerol were included in the MMLV RT reaction. For control reactions containing ‘heat-killed’ rA3A, rA3A samples were incubated at 100°C for 15 min prior to inclusion in the MMLV RT reaction. Following the MMLV RT reaction, reaction mixtures were incubated at 100°C for 15 min to denature rA3A protein. An aliquot (0.5 μl) of the MMLV-RT reaction product was PCR amplified with 2, 10 mM DTT, 0.4 μM LEAP primer, 20 units of RNasin (Promega), 0.2 mM dNTPs (Invitrogen), and 0.05% vol/vol Tween 20 in a final volume of 50 μl for 30 min at 37°C. A ‘no RNP’ reaction was prepared as a negative control. For A3A containing reactions, 5–100 ng of WT rA3A or deaminase-deficient rA3A_C106S protein in 10% glycerol were included in the LEAP reaction. For RNase H-containing reactions, 2 units of recombinant RNase H (Invitrogen) were included in the LEAP reaction. For control reactions containing ‘heat-killed’ rA3A, rA3A samples were incubated at 100°C for 15 min prior to inclusion in the LEAP reaction. Following the L1 RT reaction, reactions were incubated at 100°C for 15 min to denature rA3A protein. An aliquot (1 μl) of LEAP product was then included in the PCR amplification step using PfuTurbo Cx Hotstart DNA polymerase (Agilent) under the following conditions: 95°C for 2 min, followed by 35 cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C, with a final extension time of 7 min at 72°C. PCR products were visualized on 2% agarose gels.One μl of 0.75 μg/μl (50% vol/vol glycerol) RNP sample was incubated with 50 mM Tris–HCl (pH 7.5), 50 mM KCl, 5 mM MgClPCR products were excised from agarose gels and purified using the QIAquick Gel Extraction Kit (Qiagen). Products were cloned into ZERO Blunt PCR cloning vector (Invitrogen), transformed, and plasmid DNA recovered by mini-prep using the Wizard Plus SV Minipreps DNA Purification System (Promega). Individual clones were then sequenced. Sequence alignments were produced using MegAlign software from the Lasergene DNASTAR suite.NEOasReco: 5′-TCTATCGCCTTCTTGACGAG-3′Rescue3seq: 5′-ACTCACGTTAAGGGATTTTGGTCA-3′PolyAseq: 5′-AAAAAAAAAAAAAAAAAAAABN-3′Integrated DNA Technologies (IDT) synthesized the oligonucleotides used in the studies below:5 HeLa cells were plated per 15 cm dish (BD Biosciences) and transfected using FuGene 6 and Opti-MEM with 3 μg each of pLGCX/UGI, an A3A expression vector, and pJM140/L1.3/Δ2/k7, according to manufacturer's protocol (3 μl FuGene and 97 μl Opti-MEM per one μg of DNA transfected). For U2OS_UGI cells, approximately 5 × 105 cells were plated per 15 cm dish and transfected with either 2 μg of an A3A, A3A_C106S, or β-arrestin expression plasmid and 8 μg of pJM140/L1.3/Δ2/k7, using FuGene 6 and Opti-MEM according to manufacturer's protocol (3 μl FuGene and 97 μl Opti-MEM per one μg of DNA transfected). Selection with 400 μg/ml of G418 was initiated approximately 72 hr post-transfection and carried out for 14 days. G418 media were replaced every other day. On day 14, individual colonies were manually picked into individual wells of 12-well culture dishes (BD Biosciences). These colonies were expanded for 2–3 weeks to establish clonal cell lines. Cells from confluent T-75 flasks (HeLa) or 15 cm dishes (U2OS) of each cell line were harvested by trypsinization and genomic DNA was prepared using the Qiagen Blood and Cell Culture DNA Midi Kit, according to manufacturer's instructions.Approximately 2 × 10HindIII, SspI, BglII, or BamHI: New England Biolabs; BclI: Promega) overnight at 37°C. The following morning, an additional 2 μl of the respective restriction enzyme were added to the digest, and the reactions were incubated at 37°C for 2 hr. Digestion reactions were heat-inactivated . In the case of BglII digestions, the Wizard DNA Clean-Up Kit (Promega) was used to inactivate the enzyme. The entire digest was then ligated with 3200 units of T4 DNA ligase (New England Biolabs) under dilute conditions  overnight at 16°C. The next morning, an additional 2 μl of T4 DNA ligase were added to the reaction and incubated at room temperature for 4 hr. Ligations were concentrated on an Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane (Millipore) at 8000×g for 5 min. The membrane then was washed by applying 500 μl dH2O, and spinning for 5 min at 8000 × g. An aliquot (10–100 μl) of concentrated DNA was recovered with a 10 s spin at 8000 × g. The entire ligation then was added to 500 μl of XL-10 gold ultra-competent E. coli  and gently resuspended in 500 μl fresh LB. About 400 μl of this resuspension were plated on a 15 cm Kanamycin plate (30 μg/ml) and the remaining ∼100 μl were used to seed a 2 ml liquid culture (30 μg/ml Kanamycin). Plates and liquid cultures were incubated for 18–24 hr at 37°C. DNAs were prepared from 2 ml cultures using the Wizard Plus SV Minipreps DNA Purification System (Promega). Individual colonies were picked from 15 cm plates to inoculate 2 ml cultures (30 μg/ml Kanamycin), grown overnight at 37°C, and plasmid DNA was prepared by mini-prep using the Wizard Plus SV Minipreps DNA Purification System (Promega) the following day. Plasmid DNA recovered from mini-preps was digested with the original enzyme used for recovery to confirm intramolecular ligation. To characterize flanking genomic DNA, recovered insertions were sequenced using primers annealing to the 5′ (NEOasReco) and 3′ (Rescue3seq) ends of the NEO_ColE1 recovery cassette, as well as an oligo dA primer (polyAseq). The genomic locations of insertions were determined by aligning flanking sequence to the human genome , using the BLAT function of the UCSC genome browser . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , and Harmit Malik and Nina Papavasiliou (peer reviewers). A third reviewer remained anonymous.The reviewers noted: 'Well (and cleverly) done – no experimental concerns'. 'I find the data to be compelling and very clearly presented. This is a short, crisp and high impact study'. 'The manuscript is very comprehensive and well written. Materials and methods are well described, and the figures very clear. I was a real pleasure to review this nice paper'.eLife. We have made some minor comments for your consideration, which we feel would clarify some points and improve the structure and readability of the manuscript.We all agree that the manuscript is suitable for publication and we are delighted to accept it for Minor comments:Although the choice of format is at the discretion of the authors, perhaps it is not in the best interest of the paper to have a combined Results and Discussion section. Indeed, although several small discussion points motivate the next experiment, the true Discussion begins with the paragraph “We propose a straightforward model....” and the model In addition, the Discussion might include three further points:(a) The few L1 retrotransposition events that occur in the presence of A3A that are devoid of obvious deamination - is the model that these reflect cases where the TPRT events were beyond the 'saturation limit' or restriction capacity of A3A?(b) Are there differences in A3A and A3B (which has recently become quite prominent in cancer mutagenesis) which have similar preferences (and anti anti-L1 activity) but perhaps slightly different cellular localization? Finally, it may bear mentioning that whereas A3B is expressed in germline cells, A3A is not (I think).(c) Is this something that actually happens in real life? For instance, if A3A (and other A3's for that matter) have anti-retrotransposon activities in unmanipulated systems, then it follows that their absence would correlate with increased transposition. Mouse has a single A3 and mA3 deficiency, though possibly anti-retroviral, has not led to appreciable defects in transposable element restriction . Perhaps the authors can comment on this. Although the choice of format is at the discretion of the authors, perhaps it is not in the best interest of the paper to have a combined Results and Discussion section. Indeed, although several small discussion points motivate the next experiment, the true Discussion begins with the paragraph “We propose a straightforward model....” and the model. So, please consider splitting the Results and Discussion.We agree with the reviewers’ suggestion. As requested, we have now separated the Results and Discussion sections. We agree that this change was in the best interest of the paper.In addition, the Discussion mightinclude three further points:(a) The few L1 retrotransposition events that occur in the presence of A3A that are devoid of obvious deamination - is the model that these reflect cases where the TPRT events were beyond the 'saturation limit' orrestriction capacity of A3A?We thank the reviewers for their scholarly comments. As requested we have now included a new paragraph that discusses possibilities for why some L1 retrotransposition events are devoid of deamination. Specifically, we state the following in the Discussion section of the manuscript:“The over-expression of A3A does not abolish L1 retrotransposition completely, but reduces it to ∼30% of control levels. Moreover, some L1 retrotransposition events generated in the presence of A3A and UGI lacked evidence of deamination. Why and how these L1 retrotransposition events evade A3A-mediated deamination requires further study. However, it is possible that ectopic A3A expression is too low to efficiently target L1 TPRT intermediates in some cells or that L1 second-strand cDNA synthesis occasionally is completed before A3A can gain access to TPRT intermediates to deaminate single-strand L1 cDNAs.”(b) Are there differences in A3A and A3B (which has recently become quite prominent in cancer mutagenesis) which have similar preferences (and anti anti-L1 activity) but perhaps slightly different cellular localization? Finally, it may bear mentioning that whereas A3B is expressed in germline cells, A3A is not (I think).Again, we thank the reviewers for their comments. As requested, we have highlighted differences between the expression patterns of A3A and A3B in the Discussion section of the manuscript. We also have highlighted that both A3A and A3B can localize to the nucleus. Specifically, we have added text to our Discussion (paragraph starting” Mechanistic insights gained from studying deaminase-dependent A3A-mediated L1 inhibition could be applicable to retroelement inhibition by other A3 proteins”).We further agree with the statement that A3B has become quite prominent in cancer mutagenesis and we have added the text starting “APOBEC3 activity has been implicated in genome hyper-mutation in numerous cancers .”(c) Is this something that actually happens in real life? For instance, if A3A (and other A3's for that matter) have anti-retrotransposon activities in unmanipulated systems, then it follows that their absence would correlate with increased transposition. Mouse has a single A3 and mA3 deficiency, though possibly anti-retroviral, has not led to appreciable defects in transposable element restriction . Perhaps the authors can comment on this.Again, we thank the reviewers for their comments. Because A3A expression is largely restricted to peripheral blood lymphocytes, we agree that it may not restrict germ line L1 retrotransposition events in “real life.” However, we posit that mechanistic insights gained from studying deaminase-dependent A3A-mediated L1 inhibition could be applicable to retroelement inhibition by other A3 proteins. For example, and as detailed in our response above, we highlight that the related A3B cytidine deaminase protein , and can inhibit the retrotransposition of engineered L1s in hESCs) may restrict germline L1 retrotransposition events. Moreover, we have provided a brief discussion about how differences in A3B expression levels may, in part, contribute to differences in L1 activity reported in human and non-human primate induced pluripotent stem cells. Finally, we raise the possibility that differences in the presence/absence of A3B in humans may contribute to differences in L1 retrotransposition activity.To the best of our knowledge, no one has conducted an in depth study of L1 retrotransposition activity in A3 deficient mice; thus, we did not discuss this point in our study.


Reststrahlen band in the MIR. This allows us to present IR reflectance data exhibiting a continuous evolution from the regime of intraband to interband charge carrier transitions. A consistent and simultaneous analysis of the contributions from both transitions to the optical response yields precise information on the carrier dynamics and the number of layers. The properties of the graphene layers derived from IR reflection spectroscopy are corroborated by other techniques . Moreover, we also present MIR microscopy mapping, showing that spatially-resolved information can be gathered, giving indications on the sample homogeneity. Our work paves the way for a still scarcely explored field of epitaxial graphene-based THz and MIR optical devices.Graphene has emerged as a promising material for infrared (IR) photodetectors and plasmonics. In this context, wafer scale epitaxial graphene on SiC is of great interest in a variety of applications in optics and nanoelectronics. Here we present IR reflectance spectroscopy of graphene grown epitaxially on the C-face of 6H-SiC over a broad optical range, from terahertz (THz) to mid-infrared (MIR). Contrary to the transmittance, reflectance measurements are not hampered by the transmission window of the substrate, and in particular by the SiC  Versatile is probably the most appropriate word to qualify the properties of graphene, this one-atom-thick crystal made of carbon121435−1) and around 200 meV (1613 cm−1), due to the phonon-related absorption bands in the SiC substrate10121415161767891011As a prerequisite for harnessing the peculiar properties of graphene in such applications, it is mandatory to devise accurate methods to perform IR spectroscopy of graphene on different types of substrates of interest. For epitaxial graphene, i.e. graphene obtained by decomposition of SiC at high temperature878900 meV 16 cm−1, du−1, associated with the longitudinal optical (LO) phonon in SiC, and vertically shifted for clarity. The Raman scattering spectra confirm that graphene was successfully grown in all samples, as shown by the presence of the characteristic graphene bands D, G, and G’ located around ~1362 cm−1, ~1581–1596 cm−1 and 2704–2714 cm−1, respectively202122−1, less pronounced for the thicker MEG samples. In the MEG samples, the G and G’ bands are more intense, indicating a larger number of layers. In all samples (except samples #6 and #7 - see below), the shape of the G’ band is well fitted by a single Lorentzian curve, as shown in 82425−1 and 28–52 cm−1, in good agreement with previous studies202122et al. for a thick graphene stack with 70–90 layersD’ band appears (~1623 cm−1) in the spectrum of sample #6, which indicates a larger density of defectsD band varies among the samples, reflecting different disorder densities in the epitaxial graphene layers. Moreover, within the same sample, the relative intensity of the D, G and G’ bands and the FWHM of the G’ band varies (not shown), an indication of local variations in the number of layers and/or doping level and mechanical constraints. Furthermore, some dark inclusions are present in the MEG samples #6 and #7. Two representative spectra measured at the location of these inclusions are shown in the inset of D peak is more prominent, together with a D’ band appearing as a shoulder around 1623 cm−1. By comparing the intensity of the graphene features with the LO phonon of the SiC substrate we can infer that these inclusions are a stack of multilayers, thicker in sample #6 than in sample #7. Nevertheless, the G’ peak is almost single Lorentzian, showing no clear signature of graphitic residues as in earlier studies21The micro-Raman spectroscopy results on FEG and MEG samples are summarized in pears ~16 cm−1 in σ1L = σintra + σinter, which includes both intraband and interband processes92728EF, carrier scattering time τ and thermal broadening Γ) on graphene optical conductivity in the THz-MIR range can be visualized in σ(ω) and of both σintra(ω) and σinter(ω) are shown for varying EF. The computed curves are normalized by σ0 = e2/4ħ. The interband threshold is clearly observed at ħω ~ 2EF, and the total conductivity σ(ω) exhibits significant variations over the full THz and MIR ranges, except above the interband threshold, where σ(ω) ~ σ0. The inset shows σ(ω) in the THz range, dominated by the intraband Drude-like absorption. In σ(ω) was computed for a fixed Fermi level (EF = 100 meV) while varying τ and Γ, respectively. Increasing τ impacts σ(ω) in the THz-MIR range, as shown in et al.i.e. a value close to the thermal energy kBT at room temperature.The optical properties of monolayer graphene can be fully described by its complex dynamical in-plane optical conductivity σinter and σintra can have significant contributions over the full spectral range.Note that in most previous studies on the optical response of epitaxial graphene the intra- and interband contributions were treated separately, mostly due to the difficulty to gather data over the full spectral range, e.g. due to the substrate absorption in particular ranges89σ1L(ω)The optical reflectance of graphene layers on a SiC substrate was computed in the framework of the thin film approximation, as shown schematically in the inset of EF, N, τ . In the MIR region, the optical response depends both on intraband and interband transitions, in particular in the Reststrahlen band. For a scattering time τ = 20 fs , the reflectance presents a different trend, as detailed in the upper right inset of EF and considering the Reststrahlen region, R first increases (for EF < 150 meV) and then decreases (for EF > 150 meV). This is due to the intraband transition in the low frequency range, and the interband threshold at higher frequencies: below 2EF, the main contribution to the optical conductivity of graphene arises from the carriers absorption. The latter effect is better visualized in R gradually decreases in the MIR range (in the Reststrahlen region), when increasing EF up to EF ~ 150 meV and rises again when EF increases above this value. The inset of R in the vicinity of the interband threshold (~2EF) when EF is varied in the range 25–400 meV. The effect of increasing τ is shown in Reststrahlen region and in the low frequency limit of the THz range due to the intraband transitions.To investigate on the impact of graphene on the overall optical response, we have computed the reflectance of air/SiC and air/graphene/SiC for varying EF, N, τ . In eachR for increasing N. From monolayer (N = 1) to FEG/MEG, R increases in the THz and MIR regions, with an opposite trend in the Reststrahlen band. The optical contrast increases with increasing N, with respect to the SiC substrate, leading to remarkable changes within this region. The latter feature alone can be used to assess the formation of graphene and evaluate the number of graphene layers. As reported recentlyN, analogous to transmission measurements. For highly doped graphene, this should be performed in the NIR region due to the interband threshold , although the contrast increases with the number of layers. Therefore slight variations in EF, N and τ will result in a unique optical spectrum that can be distinguished from those obtained using different sets of parameters, as will be demonstrated below.R changes markedly with varying N. Moreover, the shape of the curves in the THz range is qualitatively different for each sample, an indication that the carriers properties are distinct. The analysis of the experimental data was accomplished by fitting the reflectance model to the given spectra over the full THz-MIR ranges. We present the obtained fitting curves in N from XPS for samples #6 and #7. Nevertheless, it corroborates with the fits to the IR reflectance data: we indeed find N > 35.To compare with the theoretical predictions, we present in 173233Reststrahlen band are closely related to the properties and number of graphene layers. This can be demonstrated by examining local IR reflectance data from a FEG sample with a heterogeneous number of layers N. Due to its trapezoidal shape, sample #3 was subject to an intentional temperature gradient during graphene growth. We could therefore anticipate that this would give rise to a gradient in N along the sample length. N. We now show that this gradient can be measured thanks to a mapping of reflectance spectra in the MIR range, a rapid and non-invasive method. The number of graphene layers is usually estimated from data in wavenumber range above 3200 cm−1 Reststrahlen band, can be used to estimate N.As shown above, the remarkable changes in the Reststrahlen band: the reflectance is decreasing when going from the left- to the right-hand side of the sample. Fitting the data to the reflectance model yields the spectra presented in N is gradually varying from 2.2 to 14 layers between the two ends of the measured region (see Methods and 2) performed on the same sample, as shown in 2) to approach the experimental conditions of the XPS measurements (1 mm2 spot). Note that for the spectral region analyzed here and our experimental setup configuration, the spatial resolution could be improved to 25–30 μm.IR spectra taken at the positions indicated by the numbered colored squares on EF can be estimated from the threshold in the MIR region. The same kind of analysis can be performed on CVD graphene transferred to other opaque or transparent substrates e.g. Si/SiO2 or quartz. Note that, in our case, the detector used in the IR mapping has a higher sensitivity than the one used in the classical IR spectroscopy. We can thus observe that when N increases, two features appear: a peak at 1584 cm−1, and a broad band centered around 3200 cm−1 (inset of Eu1 (not IR active in monolayer graphene)γ1 ~ 0.37 eV (~2984 cm−1) in bilayer graphene81531Besides being a rapid and non-invasive tool to differentiate between epitaxially grown FEG or MEG samples, it also offers perspectives for mapping the homogeneity of the electronic properties across graphene samples. inset of  The firsEF and τ was obtained through magnetotransport experiments on the FEG sample #1. The Hall coefficient was measured in a Hall bar geometry at low temperature (4.2 K) to avoid parallel conduction through the substrate37EF = 172 meV (with n-type charge carriers), i.e. in excellent agreement with EF = 170 ± 5 meV found from the analysis of IR spectra. In addition, measurement of the sheet resistance at zero magnetic field yielded τ = 12.5 ± 3 fs using the Einstein relationIR = 16 ± 2 fs), considering the differences in experimental conditions (the electron transport experiment probed a few micrometers-squared area).For the sake of comparing the results obtained from the IR analysis, an independent measurement of R , where 1 THz = 33.3 cm−1 = 300 μm = 4.1 meV. The IR beam aperture diameter on the sample was set between ~1.25 and ~2.5 mm2, depending on sample dimensions, and optimized to obtain the best signal-to-noise ratio. All measurements were performed at near-normal incidence (θ ~ 8°). A bare Al mirror was used as reference. In addition, IR spectroscopy mapping was employed to investigate sample homogeneity. In this case, the measurements were performed under ambient conditions using an IR microscope (Bruker Hyperion 3000) coupled to a FTIR spectrometer (Bruker Vertex 80v) under vacuum (~1 mBar). A liquid nitrogen-cooled mercury cadmium telluride (MCT) detector, a KBr beamsplliter and a globar source were used, covering the MIR and NIR regions, between 400 and 7000 cm−1 (12–210 THz or 25–1.4 μm). The IR beam was focused on the sample by a Cassegrain 15x IR objective (NA = 0.4) with a square beam-aperture of 170 × 170 μm2. An Au-coated mirror was used as reference. In all measurements the spectral resolution was set to 4 cm−1. The systematic error in the relative value of the reflectance (for R = 0.985) is around 1% and 2–3% for the IR spectroscopy and microspectroscopy, respectively. Micro-Raman measurements were performed in a LabRam HR spectrometer (Jobin Yvon - Horiba), in a confocal back-scattering setup with a 514 nm (2.41 eV) laser beam focused by a 100× optical objective (NA = 0.95). A high-resolution diffraction grating of 1800 grooves/mm was used. The laser power was kept below 1 mW to avoid damaging of the graphene layers.IR spectroscopy was performed from FIR or THz up to the MIR range in a Fourier-transform infrared spectrometer (FTIR) Bruker IFS 113v under vacuum (~1 mBar) equipped with a liquid helium-cooled Si bolometer, mylar  beamsplitters, a deuterated-triglycine sulfate (DTGS) detector, a KBr beamsplitter, and a silicon carbide light source (globar). The measurements covered the spectral range between 35 and 4500 cmα line. The number of graphene layers was estimated by the integrated intensity ratios of the C1s core level for graphene (~284.5 eV) and SiC (~282.7 eV), as described in Biederman et al.Angle-resolved X-ray photoelectron spectroscopy (XPS) was performed using an Escalab 250Xi instrument from Thermo Scientific and the Al KR between the two media separated by the graphene layer is given by:30σ1L is the conductivity of monolayer graphene, c is the speed of light in vacuum, ε0, ε1 and ε2 are, respectively, the permitivity of vacuum, and the relative dielectric functions of vacuum (ε1 = 1) and of the SiC substrate. We have included the number of graphene layers N in the model by considering that rotational disorder between layers is large enough so there is no Bernal (AB) stacking811354142811354141σFEG/MEG = Nσ1L.The optical response of SiC is described by a Drude-Lorentz model:4445ε∞ is the high-frequency dielectric constant. The first term in the above equation is the well-known four parameters damped oscillator model, where ωLO(ωTO) and ΓLO(ΓTO) denotes respectively the frequency and damping rate of longitudinal (LO) and transverse (TO) optical phonons. The second term describes the Drude absorption due to the free carriers in the n-doped 6H:SiC. All the main features of the reflectance spectrum of SiC were well reproduced using the following fixed parameters: ε∞ = 6.55, ωTO (ωLO) = 797.7 (970.7) cm−1, ΓTO(ΓLO) = 2.3 (5.6) cm−1, ωP = 103 cm−1 and ΓP = 90  cm−1, in good agreement with previous reports323334454749n and average mobility μ are respectively given by n = (N/π)(EF/ħvF)2 and μ = evFτ/ħ(πn)1/2 813F is the Fermi velocity in graphene (~106 m/s). For epitaxial graphene, it was demonstrated that only the first few graphene layers, closer to the substrate, and the outermost layer in MEG are doped, whereas the remaining ones are quasi-neutralsIn the case of two dielectric media 1 (vacuum) and 2 (SiC substrate), as shown schematically in the inset of How to cite this article: Santos, C. N. et al. Terahertz and mid-infrared reflectance of epitaxial graphene. Sci. Rep.6, 24301; doi: 10.1038/srep24301 (2016).


Selective filtration of gas, water, and liquid or gaseous oil is essential to prevent possible environmental pollution and machine/facility malfunction in oil-based industries. Novel materials and structures able to selectively and efficiently filter liquid and vapor in various types of solutions are therefore in continuous demand. Here, we investigate selective gas-water-oil filtration using three-dimensional graphene structures. The proposed approach is based on the adjustable wettability of three-dimensional graphene foams. Three such structures are developed in this study; the first allows gas, oil, and water to pass, the second blocks water only, and the third is exclusively permeable to gas. In addition, the ability of three-dimensional graphene structures with a self-assembled monolayer to selectively filter oil is demonstrated. This methodology has numerous potential practical applications as gas, water, and/or oil filtration is an essential component of many industries. Water-oil separation is a key step in a number of processes, namely oil drilling in exhausted oil fields, tertiary oil extraction, oil spill cleanups, and oil-bearing industrial wastewater purification. In this context, many studies have recently focused on the separation or absorption of water-oil123The volume capacity of existing 2D meshes is however limited owing to the weight of larger mesh substrates. Two-dimensional meshes are furthermore susceptible to damage to any part of the mesh, do not readily filter vapor oil, and separate oil less efficiently than three-dimensional (3D) meshes. Indeed, large volumes are readily filtered through thicker 3D meshes without increasing the size of the substrate. Moreover, in terms of reliability, surface damage does not compromise the filtering ability of 3D meshes because of the underlying connected structure. Finally, the larger reaction surface presented by 3D meshes traps significantly larger volumes of oil vapor. Thus, in order to overcome many drawbacks of 2D filters, 3D structures for water-oil separation were investigated5673O4 supercapacitor electrodes have been prepared by creating 3D graphene networks through chemical vapor deposition14Graphene has superior mechanical, thermal, and electrical characteristics than inorganic materials and its antibiotic properties inhibit the formation of bacteria101112via surface treatments .In this study, 3D graphene structures were employed to selectively filter gas, water, and oil. To that end, three forms of graphene foams were prepared by adjusting their hydrophobicity 2-plasma treatment, the hydrophobic pristine graphene foam, and the omniphobic foam with self-assembled trichlorosilane (HDF-S). As synthesized, 3D structural graphene is hydrophobic but the O2-plasma treatment generates defects on the graphene surface, rendering it omniphilic. For the omniphobic structure, the hydroxyl groups (−OH) on the graphene surface and the active group (trichlorosilane–SiCl3) of HDF-S form covalent bonds from which a strong siloxane network is built up. These three different types of graphene foam allow selective filtering of gas and/or liquids.A schematic diagram and a photograph of a piece of 3D graphene foam 25.4 mm in diameter and 2 mm thick are shown in 2-plasma-treated, pristine, and with self-assembled HDF-S) to water, gasoline, kerosene, and olive oil. The average and standard deviation of five measurements performed for each sample are shown. The contact angle of all the liquids on the O2-plasma-treated graphene foam is 0°, which indicates omniphilic behavior. The contact angles of water on pristine graphene is 107.9 ± 1.1°, which contrasts with the 0° measured for gasoline, kerosene, and olive oil. The (hydrophobic) pristine graphene therefore blocks water only. On the other hand, the 3D graphene covered with self-assembled HDF-S is not only superhydrophobic , but also oleophobic, with gasoline, kerosene, and olive oil contact angles of 78.1 ± 1.4°, 105.6 ± 3.8°, and 121.7 ± 1.7°, respectively. It is thereby constitutes an omniphobic filter.i.e. ~0.22 N/m) for our systems. Based on this criterion, sp2 carbon is the important factor governing the filtration of gasoline, kerosene, and olive oil.The adsorption strength between each solvent and the substrate was investigated from a molecular perspective to elucidate different filtration behavior. First of all, the theoretical balance between surface tension and the gravitational force was calculated in a model pore system to estimate the maximum height of solvent that can be maintained by surface tension alone on a single pore see , suggestIn summary, 3D graphene structures were fabricated whose wettability was controlled using a self-assembled monolayer. The hydrophobicity of the 3D graphene structures is substantially increased by the self-assembled HDF-S surface layer, with the water contact angle increasing from ~107.9° to ~143.2°. Raman analysis shows that the graphene in these structures is uniformly formed and X-ray photoelectron spectroscopy (XPS) measurements demonstrate that HDF-S self-assembles as desired on the graphene. The static apparent contact angle was measured on the graphene foams for liquids with different values of surface tensions, namely water, gasoline, kerosene, and olive oil. Water is selectively filtered by pristine (hydrophobic) graphene, while the HDF-S functionalized (omniphobic) graphene filter blocks all solvents. The porous structure of both filters (with 150–200 μm diameter pores) means, however, that they are both permeable to gas. Molecular dynamics simulations show that this selective filtration stems from the effects of different functional groups grafted onto the graphene. Indeed, while a high-percentage coverage of −OH and −COOH blocks kerosene, water is not filtered out with low-percentage epoxide coverage. This suggests a topic for further investigations, that the surface functionalization of graphene can be tailored to solve particular separation problems involving oil and water mixtures. This approach has numerous potential industrial applications. This study also provides a basis for future advances in gas-oil-water separation methods.2 gas (100 sccm); the substrate temperature was then raised from room temperature to 1020 °C and kept at this temperature, first for 90 min under H2 gas and then for further 30 min under CH4 gas (20 sccm). The CH4 gas flow was then interrupted and the CVD chamber was cooled at a rate of 150 °C/min in H2 environment.Ni foam with a 3D porous structure (pore size: 150–200 μm (120 pores per 25.4 mm), diameter: 25.4 mm, thickness: 2 mm, purity: 99.9%) was used as a catalyst and supporting frame for the fabrication of 3D structural graphene foams. The Ni foam was soaked in acetone and ulrasonicated for 3 min for cleaning and to remove organic contaminants from its surface. The clean foam was then annealed at 600 °C under flowing Ar (100 sccm) for 1 h. To grow graphene, the annealed Ni foam was placed in a chemical vapor deposition (CVD) chamber under H2-plasma treatment . Graphene is hydrophobic as grown. In order to render it omniphobic, HDF-S (Gelest) was used to produce a trichlorosilane monolayer that self-assembles. The fabricated 3D graphene foam was immersed in a mixed solution of anhydrous toluene (99.8%) and silane precursor (3.0 mM), and trichlorosilane was left to self-assemble on the graphene surface in N2 ambient for 1 h. The graphene substrate covered with self-assembled HDF-S was then rinsed three times in anhydrous toluene and twice in isopropyl alcohol, before being left to dry for 1 h under ambient conditions.Omniphilic graphene was obtained by OThe surface topology of the foams was observed by FE-SEM  and the graphene was characterized by Raman spectroscopy. The presence of HDF-S and the surface chemical state of the graphene were verified by X-ray photoelectron spectroscopy ). Contact angles  were measured by placing droplets (5 μL) of water, gasoline, kerosene, and olive oil on the surface of the samples under ambient conditions. For the gas penetration tests, the graphene foams were placed above dry ice in a closed bottle. To reveal the penetration of the different solvents, a water-soluble dye  or an oil-soluble dye  was mixed into appropriate samples.NVT  ensemble at room temperature with 1 fs time steps. The systems were left to relax for 200 ps ahead of the 1 ns production runs used to calculate the adhesion energies. The values reported are averages from two independent simulations and the COMPASS force field was used throughout1718Simulations were performed with four different solvents, namely water, gasoline , kerosene (consisting of n-hexadecane and n-eicosane), and olive oil  .


We investigated single-layer graphene on SiC(0001) by atomic force and tunneling current microscopy, to separate the topographic and electronic contributions from the overall landscape. The analysis revealed that the roughness evaluated from the atomic force maps is very low, in accord with theoretical simulations. We also observed that characteristic electron scattering effects on graphene edges and defects are not accompanied by any out-of-plane relaxations of carbon atoms. Graphene epitaxially grown on a substrate differs in many aspects from free-standing graphene or graphene exfoliated onto insulating surfaces. The influence of the substrate hinders applications of epitaxial graphene in the nanoelectronics . The two× 6 quasi-periodic corrugation (q-6 hereafter) [Understanding the interplay between atomic and electronic structure of graphene grown on SiC substrate is a crucial factor for device construction. In this respect structural properties of graphene were extensively studied by atomic force microscopy (AFM) –9 and screafter) ,13–15. Ireafter) –18. Howereafter) –21.Until now, the morphology of SLG on SiC(0001) has been studied by STM and AFM separately. Therefore, an investigation using the combined STM–AFM technique, that has simultaneous access to the electronic and topographic channels, is needed.×R30°  modulations, characteristic for the areas of graphene edges and point defects. Our results complement the previous findings about the origin of scattering at the defects and also serve for a comparison with properties of different kinds of epitaxial graphene.For the first time we bring experimental data, that can distinguish the topographic landscape from the local electronic structure of SLG on a 6H-SiC(0001) substrate. At room temperature we employed a combined STM and dynamic atomic force microscopy (dAFM) based on the Q-plus sensor working under UHV conditions. We observe the −10 mbar. N-doped Si-face 6H-SiC(0001) wafers purchased from CREE Inc., were cut into 3 mm × 10 mm stripes and mounted onto sample holders constructed for a direct-current sample heating. After inserting into the UHV, the samples were degassed at 600 °C and annealed to 800–850 °C under a Si flux for 30 min, which resulted in a perfect Si type 3 × 3 reconstruction and typical terrace widths above 50 nm. As the source of the Si flux we used a Si wafer, which was annealed up to 1200 °C by passing the direct current. The deposition rate was 1 mL per 5 min. The temperature of the sample was measured using an optical pyrometer with spectral emissivity set to 0.6, operating at 1.6 μm wavelength, focused onto a spot on the sample with a diameter <2 mm. Buffer layer and graphene growth were achieved by further annealing of the sample up to 1150 °C, repeated in 10 min increments, until the desired coverage of graphene was achieved (about 2/3 of the surface) [−1 [f), is the possibility of doing measurements in the region of a negative frequency shift gradient (repulsive regime), even at room temperature, without enhanced risk of losing the tip apex. This approach is specifically chosen for the conditions when the graphene contrast provided by the Δf is not giving atomic resolution in the attractive regime. Kelvin probe force measurements (KPFM) were also done in the constant-height mode, with a slow feedback between the measurement points, to compensate the tilt of the sample. KPFM parabola was measured by sweeping the bias voltage and measuring the Δf value [The experiments were performed in an ultra-high vacuum (UHV) environment with a base pressure not exceeding 1 × 10 surface) –14 and tace) [−1 . The conace) [−1 –23. The Δf value .ss* basis set was used for the H atoms, a sp basis set for the Si and C atoms. The cutoff radii of the pseudo-atomic basis functions were as follows: R = 4.5 a.u., R = 4.5 a.u., R = 4.5 a.u, R = 4.5 a.u., R = 6.0 a.u., R = 6.00 a.u. Local density approximation is used for the exchange correlation functional. Our atomic models of the SLG-SiC(0001) surface consisted of a slab of 1648 atoms, 10 atomic layers thick (including the buffer and SLG layer) with an additional passivating hydrogen layer on the underside. The lateral size of the supercell was 6× 6. The calculations were restricted to the Γ point of the first surface Brillouin zone. The bottom Si atomic layer as well as the hydrogen layer were kept fixed during the geometry optimization while all other atoms were allowed to relax freely into their equilibrium positions. The criterion for terminating the relaxation was that maximal forces on free atoms had to be below 0.05 eV/Å and the change of total energy between subsequent iterations had to be smaller than 10−4 eV per unit cell.In order to estimate the SLG corrugation we carried out large scale total energy density functional theory (DFT) calculations. We used local-orbital FIREBALL code –26. FIRE) modulation corresponding to the 6× 6R30° quasiperiodic structure detected by low-energy electron diffraction [ periodicity. This characteristic feature has been observed before, not only near to the graphene boundaries, but also around single-point defects in graphene and graphite [fraction . Figure graphite ,28–29 angraphite . We alsographite . In contf, time-averaged current (<It>) are measured vs the tip–sample relative distance (Z) during an approach and retraction of the tip. Voltage is kept at 100 mV. The <It> grows exponentially with Z and the Δf has a minimum near to −80 Hz. The value of Z = 0 corresponds to the tip–sample separation at <It> setpoint of 0.46 nA. In the region of the set point, the Δf is rising at a rate of (0.98 ± 0.04) Hz/pm, as determined by the least-square fitting. Force (F) is calculated from the Δf signal, using the approach proposed by Sader et al. [The set of curves in r et al. . Maximum structure of graphene a Kelvin-probe measurement [ modulation) in the Δf image, at 0.5 nm from the setpoint, further from the sample. In To identify any charge transfer effects within the q-6surement  has beenf and <It> maps are presented for the same area of the sample, taken in a constant height regime with a slow feedback setpoint at 0.46 nA, producing a tip–sample separation close to the Z = 0 in It> map shows an image resembling the STM topography in  periodicity. The Δf signal shows the honeycomb structure and the q-6 modulation as well. In this case, the slow feedback control of the tip–sample distance allows us to stay in the repulsive regime, achieving atomic resolution. This would be otherwise possible only at very stable conditions, e.g., at low temperatures. The lower part of the <It> image in  ripples on graphene visible in the <It> channel. In contrast, the Δf image shows only a flat honeycomb structure of graphene.In  modulation of graphene in STM has, apart from a significant electronic contribution, a topographic component. It is a question how strong is the topographic effect and whether it can be evaluated from the semi-constant height data. Neglecting the effect of the slow feedback and by using the Δf(Z) curve from Z height modulation of graphene from the Δf images in f region, which has a of 1.0 Hz/pm, we use the Δf roughness corresponding to the q-6 modulation (5.7 Hz RMS), to obtain an average value of about 6 pm RMS, corresponding to 16 pm corrugation (peak-to-peak). This is significantly lower than the roughness obtained by standard topographic mode of STM, pointing out the dominant electronic contribution of the buffer layer to the STM topography of SLG. In our fully relaxed theoretical model of SLG on the buffer layer , which corresponds well to the experimental estimation and is comparable to a similar calculation in [The experimental results reveal that the q-6er layer , which iation in . The valation in –39. pattern . We can conclude that variation at this scale detected by previous STM measurements is caused mainly by electronic contributions arising from the buffer layer. In the case of the characteristic  rippling of graphene, which occurs due to presence of defects or boundaries, any relaxations of carbon atoms perpendicular to the surface can be excluded. These results are consistent with recent findings and show the single-layer graphene on SiC(0001) as a morphologically very flat substrate.In summary, we successfully probed the epitaxial graphene on SiC(0001) by a combined STM and dAFM technique, gathering extended information on its atomic structure near defects. By atomic force microscopy, we detected a topographical corrugation of the q-6The research was funded by GACR grant no.14-02079S, GACR grant no. EXCELENCE 14-374527G.


This paper identifies trends within and relationships between the amount of participation and the quality of contributions in three crowdsourced surveys. Participants were asked to perform a collective problem solving task that lacked any explicit incentive: they were instructed not only to respond to survey questions but also to pose new questions that they thought might-if responded to by others-predict an outcome variable of interest to them. While the three surveys had very different outcome variables, target audiences, methods of advertisement, and lengths of deployment, we found very similar patterns of collective behavior. In particular, we found that: the rate at which participants submitted new survey questions followed a heavy-tailed distribution; the distribution in the types of questions posed was similar; and many users posed non-obvious yet predictive questions. By analyzing responses to questions that contained a built-in range of valid response we found that less than 0.2% of responses lay outside of those ranges, indicating that most participants tend to respond honestly to surveys of this form, even without explicit incentives for honesty. While we did not find a significant relationship between the quantity of participation and the quality of contribution for both response submissions and question submissions, we did find several other more nuanced participant behavior patterns, which did correlate with contribution in one of the three surveys. We conclude that there exists an optimal time for users to pose questions early on in their participation, but only after they have submitted a few responses to other questions. This suggests that future crowdsourced surveys may attract more predictive questions by prompting users to pose new questions at specific times during their participation and limiting question submission at non-optimal times. Crowdsourcing  holds thet al.. Tab. Tab29].R2 values). We hypothesized that these poor results were due to the one participant who submitted 318 of the 591 questions to the survey. Due to one individual submitting so many questions, many of the eight variables described above had nearly identical values for all 318 observations in our dataset. For this reason, this participant was removed from the dataset and only the remaining 273 questions were considered.However, we did eventually find useful insight from the EnergyMinder dataset. Initially, we did not find consistent trends in the modeling results  In addition, the finding that some users submit many more questions than others without a measurable increase in quality suggests that it may be beneficial to limit question submission once a user has already participated a great deal.We obtained permission to conduct human subjects research from the Institutional Review Boards at the University of Vermont. Our IRB proposal was entitled “Wikinnovation: Automating and Democratizing Online Survey Construction.” Participants granted written consent by clicking on a button to proceed to the online survey after watching a video outlining the purpose of the crowdsourced surveys.https://github.com/BobFromUVM/crowdsurvey2014. All images used in this paper are also accessible from the above URL under the MIT license.The data obtained from our three crowdsourced surveys is freely accessible under the MIT license from S1 File(PDF)Click here for additional data file.


Long INterspersed Element-1 (LINE-1 or L1) is the only autonomously active, transposable element in the human genome. L1 sequences comprise approximately 17 % of the human genome, but only the evolutionarily recent, human-specific subfamily is retrotransposition competent. The L1 promoter has a bidirectional orientation containing a sense promoter that drives the transcription of two proteins required for retrotransposition and an antisense promoter. The L1 antisense promoter can drive transcription of chimeric transcripts: 5’ L1 antisense sequences spliced to the exons of neighboring genes.The impact of L1 antisense promoter activity on cellular transcriptomes is poorly understood. To investigate this, we analyzed GenBank ESTs for messenger RNAs that initiate in the L1 antisense promoter. We identified 988 putative L1 antisense chimeric transcripts, 911 of which have not been previously reported. These appear to be alternative genic transcripts, sense-oriented with respect to gene and initiating near, but typically downstream of, the gene transcriptional start site. In multiple cell lines, L1 antisense promoters display enrichment for YY1 transcription factor and histone modifications associated with active promoters. Global run-on sequencing data support the activity of the L1 antisense promoter. We independently detected 124 L1 antisense chimeric transcripts using long read Pacific Biosciences RNA-seq data. Furthermore, we validated four chimeric transcripts by quantitative RT-PCR and Sanger sequencing and demonstrated that they are readily detectable in many normal human tissues.We present a comprehensive characterization of human L1 antisense promoter-driven transcripts and provide substantial evidence that they are transcribed in a variety of human cell-types. Our findings reveal a new wide-reaching aspect of L1 biology by identifying antisense transcripts affecting as many as 4 % of all human genes.The online version of this article (doi:10.1186/s12864-016-2800-5) contains supplementary material, which is available to authorized users. Our genome is replete with L1 retrotransposon-derived sequences that can affect the transcriptome . GenomicIt is estimated that there are approximately 90 full-length, retrotransposition competent L1HS in the human genome with intact internal promoters and open-reading frames . AdditioActivity of the L1 antisense promoter (ASP) was first demonstrated by Speek who described the discovery of four chimeric L1 ASP transcripts, comprised of L1-derived 5’-UTR and spliced exons from neighboring single-copy genes . Speek aIn another approach to identify chimeric L1 ASP transcripts, Cruickshanks and Tufarelli applied L1 chimera display to identify eighteen novel chimeric L1 ASP transcripts, some of which were selectively detected in breast and colon cancer specimens but not in matched normal tissues . These iThe human-specific subfamily L1HS and the primate specific subfamilies L1PA2-8 were identified to contain an open reading frame termed ORF0 that is transcribed downstream of the L1 ASP on the antisense strand . The ORFAlthough the existence of L1 ASP transcripts is well-documented, many questions remain. The extent to which L1 chimeric antisense transcripts impact the human transcriptome and what other genes may be under the influence of L1 ASP is unknown. The importance of these L1 ASP transcripts is further highlighted by the recent identification of ORF0, which suggests chimeric L1 gene fusion transcripts might be expressed. Herein, we report the identification and comprehensive characterization of 988 putative L1 ASP transcripts.We queried human expressed sequence tag (EST) genome alignments to identify ESTs incorporating part of an L1 sequence in the ASP. First, we required that the transcription start site (TSS) of a spliced human EST begins in the antisense orientation relative to an annotated L1 element and second, that a spliced exon of the EST must also overlap an annotated exon of a gene. Third, we removed putative L1 ASP transcripts where an independent EST supported L1 exonization, rather than transcription starting from within the L1 element. We implemented the third criterion because ESTs are not full-length transcripts and gene transcripts can retain intronic L1s on the opposite strand because L1s contain cryptic splice sites , 18. We Of the L1 ASP transcripts, 609 were represented by only one EST, which suggests that many L1 ASP transcripts are transcribed at relatively low levels. The remaining 379 transcripts were represented by multiple ESTs Fig. . The topNext, we reviewed the primary tissues 1531 ESTs) and cell lines (355 ESTs) that were sources of ESTs, and categorized them as cancerous (578 ESTs) or normal (1308 ESTs) (Table S1). Some ESTs could not be characterized due to a lack of supporting information. Interestingly, 69.4 % of ESTs supporting an L1 ASP transcript originated from non-cancerous tissues Fig. . For EST1 ESTs an−16, chi-squared test); on the contrary, only 182/2316 or 7.9 % were in the antisense orientation. We examined the distance from the EST transcription start site (TSS) to the TSS of the cognate gene in these two categories. The L1 ASP transcripts sense to their cognate genes was typically downstream of the gene TSS. Thus, the L1 provides an alternative start site and produces a transcript in which the annotated 5’UTR or first coding exon is not included  sequenced using a Pacific Biosciences (PacBio) instrument . The RNANext we sought to address the coding potential of L1 ASP transcripts because the majority was sense to the cognate gene and could potentially contain open reading frames (ORFs). We applied TransDecoder software to identify putative peptides encoded by ESTs with at least 100 amino acids (aa) . We alsoNext we compared the L1 ASP transcription start sites to the L1 consensus sequences. We aligned the 2015 ESTs supporting L1 ASP transcripts to full-length consensus sequences in RepBase and those reported by Khan et al. . We diviWe further interrogated ESTs from recent primate L1PA2-8 subfamilies and the human L1HS subfamily for transcription factors that might drive transcription from the ASP . YY1 is To test whether TSS locations of L1 ASP transcripts were bound by YY1 and marked by histone modifications, we examined ENCODE ChIP-seq alignments to the human genome (build hg19). We reviewed ChIP data for YY1, H3K4me2, and H3K4me3 histone modifications in the K562, H1-ESC, and Hela cell-lines . The hisWe selected two genes associated with L1 ASP transcripts for Sanger sequence-based confirmation. We successfully detected L1 ASP transcripts associated with both KIAA1324L and UVRAG genes Fig. . The valThe method to identify L1 ASP transcripts implemented a criterion to remove ESTs where an independent EST suggested L1 exonization . We examined this category in more detail and report these putative transcripts separately  RNA –52. HoweWe also describe L1 ASP transcripts incorporating gene sequences in the antisense orientation. Such transcripts are rare, comprising less than 8 % of our total, but may be biologically important. Where expressed, these have the potential to produce double-stranded RNAs because they encode a transcript with reverse complementarity to a portion of the cognate mRNA  format. First, we downloaded the locations of L1 elements by extracting the coordinates of all LINE-1 family repeats from the Repeatmasker (hg19) annotation . Second,Next, we defined criteria to identify putative L1 ASP transcripts. The 5’ TSS of the EST was required to originate from within an annotated L1 element, and the transcriptional direction strand of the EST was required to be antisense to the L1. Second, an exon coordinate of the EST was required to overlap with an annotated exon coordinate of a gene. Third, we excluded L1 ASP transcripts where an independent EST supported L1 exonization . We selected ESTs that met this criteria and represented L1 ASP transcripts using sequential intersections performed with bedtools . To veriThe long-read PacBio RNA-seq data from human embryonic stem cells (H1-ESCs) was annotated as transcript isoform predictions by Au, K. et al. . We downhttp://www.ncbi.nlm.nih.gov/sites/batchentrez), which was then added to Additional file http://transdecoder.github.io/) of Trinotate Transcript Annotation, which is a part of the Trinity package [Additional information on tissue of origin, cell line of origin, and normal/cancer status were obtained by extracting and parsing associated information on ESTs downloaded from the Batch Entrez portal  [We examined L1 ASP transcripts in four categories: human-specific L1HS, primate-specific L1PA2-8, ancient primate L1s, and ancient mammalian L1s. The EST sequences were aligned to full-length consensus sequences of L1s in RepBase and those reported by Khan et al.  using thbrc.jp/) . For eacbrc.jp/) . To creabrc.jp/) . The logTo build TSS profiles of L1 ASP transcripts, we obtained the TSS coordinate for L1PA2-8 and L1HS antisense EST transcripts for plus-strand or minus-strand ESTs. We downloaded alignment files from the ENCODE data repositories in BAM format  was resolved on a 2 % agarose gel to confirm the presence of a singular PCR product. The remainder of the reaction was cloned using a TOPO TA Cloning Kit according to the manufacturer’s specifications  and sequenced via Sanger sequencing.We originally yielded very small RNA amounts  from fibrous organs, such as skin and muscle. Therefore, we developed an in-house RNA isolation method by modifying the protocol of an RNAeasy Plus Mini Kit . First, a razor and forceps were used to finely mince one small tissue fragment, weighing up to 50 mg, on dry ice. The minced tissue fragments were suspended in 900 ml of PBS, to which 100 μl of collagenase/dispase solution (stock at 10 mg/ml) was added. The solution was incubated at 37 °C for 1 h. Then, the lysate was centrifuged for three (3) minutes at full speed using a bench-top centrifuge. The pellet was collected and mixed with 10 μl of β-mercaptoethanol and 1 ml of TRIzol reagent. The specimen on ice was homogenized using a hand-held homogenizer for 5 takes, each entailing continuous homogenization for 1 min followed by rest for 30 s. The specimen was incubated for 5 min at room temperature. Proteinase-K was then added to a final concentration of 250 μg/ml (which came out to 12 μl of 20 mg/ml stock) and incubated at 56 °C for 10 min . The lysWe estimated the relative abundance of targeted RNAs by resulting traditional Cq  interval values in a StepOnePlus™ Real-Time PCR System . Each experiment was performed in technical triplicates. Actual PCR products were quantified with a FastStart Universal SYBR Green probe . The relative abundance of experimental RNA—specifically, the arithmetic means of the Cq values—was normalized to that of an internal control RNA (GAPDH) to relative PCR efficiencies and pictorially represented as Delta Cq (ΔCq) means ± standard deviation. The PCR primers used in this report are in Additional file We identify 988 putative L1 antisense chimeric transcripts, the vast majority of which are novel. We independently verify some L1 antisense chimeric transcripts using both bioinformatics analysis and empirical evidence. Interestingly, some L1 antisense chimeric transcripts are associated with evolutionarily ancient L1 subfamilies, suggesting exaptation of these evolutionarily-older L1 sequences. We conclude that L1 antisense promoters contribute to the transcription of up to 4% of all human genes and may potentially have wide-ranging effects in health and disease.AS, Antisense; ASP, Antisense promoter; CAGE-seq, Capped analysis of gene expression sequencing; ChIP, Chromatin immunoprecipitation; EST, Expressed sequence tag; GO, Gene ontology; GRO-seq, Global run-on sequencing; L1, Long Interspersed Element-1; LINE-1, Long Interspersed Element-1; lncRNA, Long noncoding RNA; ORF, Open reading frame; TSS, Transcription start site; UTR, Untranslated region


Hepatocellular carcinoma (HCC) accounts for approximately 80% of liver cancers, the third most frequent cause of cancer mortality. The most prevalent risk factors for HCC are infections by hepatitis B or hepatitis C virus. Findings suggest that hepatitis virus-related HCC might be a cancer in which LINE-1 retrotransposon, often termed L1, activity plays a potential role. Firstly, hepatitis viruses can suppress host defense factors that also control L1 mobilization. Secondly, many recent studies also have indicated that hypomethylation of L1 affects the prognosis of HCC patients. Thirdly, endogenous L1 retrotransposition was demonstrated to activate oncogenic pathways in HCC. Fourthly, several L1 chimeric transcripts with host or viral genes are found in hepatitis virus-related HCC. Such lines of evidence suggest a linkage between L1 retrotransposons and hepatitis virus-related HCC. Here, I briefly summarize current understandings of the association between hepatitis virus-related HCC and L1. Then, I discuss potential mechanisms of how hepatitis viruses drive the development of HCC via L1 retrotransposons. An increased understanding of the contribution of L1 to hepatitis virus-related HCC may provide unique insights related to the development of novel therapeutics for this disease. Hepadnaviridae family, which has a relaxed circular DNA (rcDNA) as a viral genome , accounts for 9% of all cancer deaths worldwide  retrotransposons are genetic elements that constitute approximately 17% of the human genome : preC/C (core and Hepatitis B e-Antigen [HBeAg]), P , preS/S (Hepatitis B surface Antigen [HBsAg]) and X  genes Figure . In the Type I interferons (IFNs) play a major role in anti-viral immunity  and non-structural  proteins Figure . The repAlthough it is thought that non-retroviral RNA viruses are not integrated into host genomic DNA, we and others have demonstrated that they do become integrated into the host genome via host retrotransposon machineries (Geuking et al., Most HCV-infected patients develop a chronic infection, suggesting that HCV has developed successful strategies to evade host immune responses (Gale and Foy, The HCV core protein has an oncogenic potential (Moriya et al., de novo insertions identified in cancer tissue occurred at genes commonly mutated in cancer (Lee et al., de novo insertions can affect the expression of nearby genes and the genes in which they inserted (Lee et al., de novo insertion (Ding et al., Although a definitive role for L1 activity in contributing to HCC etiology has not been established thus far, investigating a possible link between L1 activation and the development of HCC would be of considerable interest for a number of reasons Figure . FirstlyTaken together, I conclude two potential roles for L1 elements in the development of hepatitis virus-related HCC. The first relates to a chimeric transcript specific to HBV-related HCC, HBx-L1, which can be detected in more than 20% of HBV-related HCC and correlates with a poor outcome (Lau et al., Presented lines of evidence suggest potential links between hepatitis virus infection and L1 retrotransposon activity. Especially, L1 hypomethylation or some L1 chimeric transcripts are associated with poor prognosis of HCC, suggesting that it can have a significant effect on HCC phenotypes and supporting the idea that HCC is a cancer in which L1 plays a role. However, knowledge of how L1 activation by chronic hepatitis virus infection enhances the development of HCC is still limiting. Further accumulation of examples of recurrent L1 insertion sites in the host genome or recurrent chimeric transcripts specific to hepatitis virus-related HCC will be promising ways to understand L1 involvement in HCC etiology. Single cell analyses of L1 retrotransposition events and expression in tumor cells and surrounding normal cells may enhance these processes. Understanding the potential roles of L1 in HCC may open avenues to developing novel therapeutics, such as RNA interference against HCC-specific L1 chimeric transcripts.TH wrote the manuscript and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this context, the control of the charge transfer between the constituting layers is of crucial importance. Compared to bulk or 0D system, 2D materials are characterized by a large exciton binding energy (0.1–1 eV) which considerably affects the magnitude of the charge transfer. Here we investigate a model system made from colloidal 2D CdSe nanoplatelets and epitaxial graphene in a phototransistor configuration. We demonstrate that using a heterostructured layered material, we can tune the magnitude and the direction (i.e. electron or hole) of the charge transfer. We further evidence that graphene functionalization by nanocrystals only leads to a limited change in the magnitude of the 1/f noise. These results draw some new directions to design van der Waals heterostructures with enhanced optoelectronic properties.Two dimensional layered ( One of the promising next steps is their integration in optoelectronic devices such as photodetectors or photovoltaic cells128 and responsivity of 107 A.W−1 can be achieved−14 W.cm−2), thanks to the high mobility of the graphene channel and the long carrier lifetime of the photogenerated carriers in the CQD layer.As an alternative, van der Waals heterostructures can be used to decouple the photogeneration from the transport. Such devices can be built by coupling CQD with efficient transport layer such as graphene1011132 or WS2, we use colloidally grown CdSe nanoplatelets (NPL). In addition to their 2D character2527In spite of all these progresses, the performances of photoconductors are limited by several parameters. Some of them have already been addressed212223Colloidal metal-chalcogenides nanoplatelets (NPL) such as CdSe offer excellent optical properties originating from the control of their thickness at the atomic level2914 cm−2 which will be of utmost interest to evidence a strong charge transfer. We use a fast sweep rate (50–100 mV.s−1) only allowing the gating of graphene why leaving the charging state of the NPL unaffectedAs a probe for the charge transfer, we choose to use an electrolytic transistor configuration33n doping of the channel where C is the surface capacitance of the electrolyte estimated to be 2 illumination, the negative Dirac point shift corresponds to a Fermi level change from −4.5 eV to −4.3 eV, that is to say that about 1012 electrons.cm−2 are transferred from the NPL to the graphene channel. As a comparison, under the same illumination condition, 109 charges.cm−2 are generated in a film of NPL  in the conduction band  of the NPL and the graphene, λ the reorganization energy and Ea the activation energy. The latter can be writtenwhere EExc being the exciton binding energy and ΔE = sign(charge)×,NPL) the difference between graphene Fermi level and the conduction  band of NPL. Faster charge transfer consequently requests to combine a weaker exciton binding energy with almost resonant coupling of one type of carrier to graphene. The second band of the semiconductor has to stay out of resonance to avoid charge recombination within the graphene. As proposed for quantum well structures, we demonstrate that the use of a heterostructure is well suited to achieve charge displacement within the semiconductorCdSe NPLs with their 2D aspect and limited static dielectric (ε = 10) constant55727EExc and the oscillator strengthfthanks to the relation 65859To quantify the reduction of the exciton binding energy, we use time resolved photoluminescence (PL) spectroscopy, see ε0 the vacuum permittivity, m0 the rest mass of the electron, n the optical index of the medium. The oscillator strength can be estimated aswith Ep being the Kane parameter and Ψe/h the electron or hole wavefunction. Definingas the overlap integral, the radiative lifetime is now given byEExc value is twice smaller than for core only NPL  noise nor by the shot noise because graphene possesses 1/f excess noise at frequencies below a few kHzV between the middle of the two branches are the averaged noise coming from the 4 graphene channels, i.e. VI and presented in equation 14where R = 8kΩ. The value of the power exponent is γ = 1.05 ± 0.05 as expected for 1/f noise without generation-recombination bulges. The magnitude β of the noise is plotted versus the applied voltage bias in the VDS = VI/2 is the voltage applied on each graphene channel. This gives an exponent value c = 1.9 ± 0.1 and a coefficient A0 = 6.5 ± .5×10−9, consistent with previous report of 1/f noise in grapheneN, we can calculate the α parameter characterizing the intrinsic noise of a sample:is performed on each measurement. The obtained thermal noise 2 and the carrier density of bare graphene on SiC being 8.1012 cm−2  choose the availability of one or the other type of carrier toward charge transfer. Applied to a photodetector made from CdSe-based nanoplatelets decorated on a graphene channel, CdS-shell covered NPL induce a more efficient n-type photogating of the graphene while CdTe-crown surrounded NPL produce a p-type photogating. We expect this strategy to be profitable to other 2D materialsHeterostructures grown at the nanocrystal scale is a practical strategy to 1) lower the exciton binding energy by delocalizing one type of carrier How to cite this article: Robin, A. et al. Engineering the Charge Transfer in all 2D Graphene-Nanoplatelets Heterostructure Photodetectors. Sci. Rep. 6, 24909; doi: 10.1038/srep24909 (2016).


Exfoliation of graphite is a promising approach for large-scale production of graphene. Oxidation of graphite effectively facilitates the exfoliation process, yet necessitates several lengthy washing and reduction processes to convert the exfoliated graphite oxide  to reduced graphene oxide (RGO). Although filtration, centrifugation and dialysis have been frequently used in the washing stage, none of them is favorable for large-scale production. Here, we report the synthesis of RGO by sonication-assisted oxidation of graphite in a solution of potassium permanganate and concentrated sulfuric acid followed by reduction with ascorbic acid prior to any washing processes. GO loses its hydrophilicity during the reduction stage which facilitates the washing step and reduces the time required for production of RGO. Furthermore, simultaneous oxidation and exfoliation significantly enhance the yield of few-layer GO. We hope this one-pot and fully-scalable protocol paves the road toward out of lab applications of graphene. The major differences between them are the yield and the defect content of their products. Although the use of less-defective graphene is essential in some applications (e.g. electronic devices), in some other cases, the defects and imperfections of graphene are less important or even desirable9Due to the lack of a reliable method to supply the large demand for graphene, we have developed a fast and fully-scalable method for mass production of RGO. The consecutive stages of RGO production are shown in The main intent of this article is to propose a general approach for large-scale production of RGO so that the main idea, reduction of GO prior to washing, can be extended to most of oxidation-based synthesis methods of RGO. Ascorbic acid as a green alternative for conventional reducing agents of GO (e.g. hydrazine hydrate) is an inexpensive and abundant substance which can reduce GO to an acceptable extent{1100}/I{2110} > 1). Diffracted intensity profile along the passing line from  was characterized by various chemical and physical methods. A sample was also taken from the oxidized graphite (AGO) prior to the reduction step with a great exertion for washing [see Methods]. The morphology and microstructure of ARGO sheets were investigated using field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) techniques. Well-exfoliated but crumpled and aggregated RGO sheets are observable in the SEM image , which i1522X-ray diffraction (XRD) patterns of pristine graphite, AGO, and ARGO are represented in 2O, CO2, and CO gases. The weight loss with a slower rate at >300 °C is assigned to the removal of more stable OFGsThermogravimetric analysis TGA,  demonstr−1 (G band) which is due to the first-order scattering of the E2g mode and two weaker peaks located at 1340 cm−1 and 2687 cm−1  originated from the second-order double resonant process between non-equivalent K points in the Brillouin zone of graphene−1 in the Raman spectrum of AGO. The blue shift of the G band has been attributed to the activation and merging of the Raman-inactive D′ band with the G band, the exfoliation of graphite to a single graphene sheet, and the resonation of isolated double bonds at higher frequencies than the G band of graphite−1 was observed that can be ascribed to the reduction of AGO. Such a shift back has been reported in both chemical and thermal reductions of GO2D/IG), which is an estimation of the disorder level in graphene, exhibits a non-monotonic behavior. In a low concentration of defects, the introduction of new defects creates more elastic scattering, resulting in an increase of ID/IG. Further addition of defects leads to a more amorphous structure, attenuating all Raman peaks and decreasing ID/IG . The peak at 1622 cm-1 is attributed to the C = C stretching vibration. Other prominent signals in the AGO’s spectrum which have been attenuated in the ARGO’s spectrum are 1399 cm-1, 1240 cm−1, and 1071 cm-1 originating from the O–H deformation, the C–O (epoxy) stretching vibration, and the C–O  stretching, respectivelySince the attachment of the OFGs to the graphite layers during the oxidation dramatically affects the structure and properties of graphene, their removal is a great concern for a successful RGO production. Although numerous models for the chemical structure of graphene oxide have been proposed to date, there is still a considerable vagueness. Nevertheless, it is widely accepted that the hydroxyl and epoxide groups attached to the basal plane of the graphene and carboxyl and carbonyl groups located at the edges are the dominant functional groups21281S peaks were deconvoluted using Gaussian-Lorentzian functions  is a widely accepted criterion for evaluation of the reduction process. According to the XPS survey spectra , the C/Ounctions . The ass spectra . The cheAs an important characteristic of the synthesis method, the powder yield was determined by performing a complete process, starting with 5 g graphite and obtaining ~6.37 g ARGO powder, which indicated an extremely high powder yield of ~127%. The yield of higher than 100% is attributed to the remaining of some functional groups as described earlier. In order to evaluate the purity of the final product, the metallic ion contents of the ARGO, especially Mn, K, and Na ions which had been introduced to the system during the synthesis procedure were determined using inductively coupled plasma optical emission spectrometry (ICP-OES) and the results are given in In summary, the oxidation of graphite significantly increases its interlayer spacing and effectively facilitates the exfoliation process, but necessitates various post-treatments to remove the chemical oxidants which extremely prolongs the production time. While the use of filtration and centrifuge systems in typical oxidation-based methods are indispensable, they limit the production yield to few grams a day! Although the main idea behind the present method is simple, it removes a huge obstacle from conventional synthesis methods of graphene. GO loses its hydrophilicity which makes the whole process up-scalable and one-pot oxidation-exfoliation-reduction process, considerably reduces the overall production time from several days to few hours, hence kilogram or even ton scale production of RGO is simply attainable just by increasing the capacity of the reaction vessels. The SEM, HRTEM and SAED analyses clearly demonstrate that this method can produce crystalline two-dimensional graphene platelets. The FT-IR, Raman, TGA and XPS analyses verify the successful reduction of GO even in the presence of oxidative agents and the ICP-OES analysis confirms the high purity of the final product. The characteristics of the synthesized product are comparable with other high quality chemically-synthesized RGOs.In a typical procedure, 1 g of graphite flakes  was added to 50 mL concentrated sulfuric acid  while stirring in an ice-water bath. 3 g potassium permanganate  was gradually added by maintaining the temperature under 10 °C. Then, the suspension was stirred at room temperature for 25 min followed by 5 min sonication in an ultrasonic bath . After repeating the stirring-sonication process for 12 times, the reaction was quenched by the addition of 200 mL distilled water. An extra 2h ultrasonic treatment was carried out before dividing the suspension into two equal parts; one washed to obtain AGO (described later) and the other was further processed for preparation of ARGO. After adjusting the pH at ~6 by the addition of 1M sodium hydroxide  solution, the suspension was further sonicated for 1 h. 10 g L-ascorbic acid  was dissolved in 100 mL distilled water and then was slowly added to the exfoliated graphite oxide suspension at room temperature. The reduction was performed at 95 °C for 1 h. The resultant black precipitates were simply filtered by cellulose filter paper and further were washed with a 1M hydrochloric acid solution  and distilled water to neutral pH. Finally, the filtrate was freeze-dried to obtain ARGO powder.The washing of AGO was adapted from a modified Hummers methodα radiation (λ = 1.5406 Å). The investigation of thermal properties was carried out using TGA  under air with a ramp rate of 1 °C min−1. Raman spectroscopy measurements were taken using a Renishaw InVia Reflex Raman microscopy system with a 532 nm laser. FT-IR spectra were collected using a Bruker Tensor 27 instrument. The XPS spectra were recorded using a VG Scientific-ESCA Lab 250 XPS spectrometer with a monochromatic Al Kα radiation source (1486.8 eV). The Spectral Data Processor  software was employed for curve fittings and atomic percent calculations of XPS spectra. The ICP-OES tests were performed using a Varian Vista-Pro Axial. The ICP-OES samples were digested in 2 mL 35% H2O2 and 4 mL 65% HNO3 using a microwave digestion system .SEM images were taken by a Leo Supra 35VP field emission scanning electron microscope. A JEOL JEM 2100F transmission electron microscope was used for TEM imaging, acquiring SAED patterns, and HR-TEM analysis. XRD patterns were recorded with a Bruker AXS advance powder diffractometer equipped with a Siemens X-ray gun, using Cu KS.A. and H.A. conceived and designed the research. S.A. and H.S.K. carried out the experiments. S.A. and H.A. interpreted the results and wrote the manuscript. All authors reviewed and approved the final version of the manuscript.How to cite this article: Abdolhosseinzadeh, S. et al. Fast and fully-scalable synthesis of reduced graphene oxide. Sci. Rep.5, 10160; doi: 10.1038/srep10160 (2015).