EF). In other words, a greater number of charge carriers are induced for transport and the graphene changes from a semiconductor to a semimetal. In addition, it is shown that the dependence of the DOS at EF on the contact area ratio has a bimodal distribution which responses to the experimental observation, a pinnacle curve. The maximum number of induced carriers is expected to occur at contact area ratios of 40% and 60%. In general, the present results indicate that modulating the EDL provides an effective means of tuning the electronic properties of graphene in the presence of liquid.A theoretical investigation is performed into the electronic properties of graphene in the presence of liquid as a function of the contact area ratio. It is shown that the electric double layer (EDL) formed at the interface of the graphene and the liquid causes an overlap of the conduction bands and valance bands and increases the density of state (DOS) at the Fermi energy ( The generation of electric energy by a liquid flowing over a carbon nanotube (CNT) was first reported by Kral and Shapiro in 2001234689101112141516Various experimental studies have confirmed the feasibility for generating electric energy by flowing liquid over a graphene surface1819202122231234181920212223et al.For many substances, an EDL is formed at the interface of the substrate surface when placed in contact with a liquid 252629303132+ ion within a NaCl aqueous solution is around 0.1 eVπ-electronic structures of the graphene44π-electronic structures of graphene.As with many other materials, graphene forms an EDL when brought into contact with liquids3435361819202122233839Accordingly, this study performs a theoretical investigation into the electronic properties of graphene in the presence of liquid for various values of the contact area ratio. To more easily manipulate the contact area ratio, and to increase the total exposure area so as to enhance the generated power, the investigation considers the system shown in ky, but varies only weakly with kxky-dependent energy spectrum. For pristine graphene, the conduction bands and valance bands are symmetric and meet at the Fermi level. In other words, the graphene acts as a zero-gap semiconductor  implies that charge carriers are generated by the EDL and further contribute to the voltage induced by the external field. For a larger contact area ratio of w/L = 0.4, the energy dispersions become more oscillatory, and hence more band-edge states are produced . In other words, the number of induced electron states is equal to the number of generated hole states. For contact ratios away from w/L = 0.5, the energy dispersions exhibit an inversion symmetry distribution. For example, given a contact ratio of w/L = 0.6, the energy bands are inverted with respect to those of w/L = 0.4 about the Fermi level , the eneV0). It is noted that the DOS curves are symmetric about w/L = 0.5 for all values of the field strength due to the inversion symmetry of the energy spectrum. For V0 = 0.05 γ0, the DOS increases rapidly to a maximum value at w/L = 0.1, and then decreases slightly and oscillates as the contact ratio approaches w/L = 0.5. For contact ratios greater than w/L = 0.9, the DOS falls rapidly to a value close to zero. The maximum value of the DOS(ω = 0) indicates the presence of the maximum number of induced charge carriers at the corresponding contact ratio. For a field strength of V0 = 0.075 γ0, the maximum DOS(ω = 0) occurs at a contact ratio of w/L = 0.3~0.4. Furthermore, for a field strength of V0 = 0.1 γ0, the dependence of DOS(ω = 0) on w/L shows a bimodal distribution with maximum values located at w/L = 0.4 and w/L = 0.6, respectively. For the highest considered field strength of V0 = 0.15 γ0, the bimodal curve has an even more pronounced characteristic, with strong peaks once again located at w/L = 0.4 and w/L = 0.6, respectively. In general, the results show that under a higher field strength, the induced charge carriers are more abundant and more densely concentrated at specific contact area ratios. In addition, owing to the asymmetry between electron and hole conductivities4647ω = 0) tendency. Moreover, no induced voltage is detected when the graphene is totally isolated and plunged into water. Furthermore, Lee et al. found that the detected voltage along the flow direction is higher than that along the normal direction to the flow20175051To further explore the dependence of the induced charge carriers on the contact ratio, EF from zero to a finite value. In other words, the EDL induces some free carriers, which contribute to the voltage produced by the external electrical field. As the contact area increases, the DOS(EF) does not increase monotonically, but exhibits a bimodal distribution. In other words, the charge carriers do not increase proportionally with the exposure area, but are enriched initially and then decrease as w/L continues to rise.This study has utilized a tight-binding model to investigate the electronic properties of graphene in the presence of fluid for various values of the graphene-fluid contact area ratio. Results have been presented for both the low-energy bands and the density of states near the Fermi level. It has been shown that pristine graphene is a zero-gap semiconductor, and has no charge carriers at the Fermi level. In addition, the results have shown that the electric double layer formed at the interface between the graphene surface and the fluid results in an effective electric potential. The electric field causes the conduction bands and valence bands to overlap, and consequently increases the DOS at For a field strength greater than 0.25 eV, the maximum number of free carriers is induced at contact ratios of 40% and 60%, respectively, and gives rise to corresponding peaks in the induced voltage signal. Notably, the number of induced carriers varies not only with the contact area ratio, but also with the field strength of the EDL, which depends in turn on the type of liquid in contact with the graphene. In addition, the majority-carrier type is tunable by changing contact area ratio. The calculation results obtained in this study are consistent with the experimental observations202123σ-bondsπ electron) is free for transport. As a result, the low-energy electronic properties are governed mainly by the 2Pz orbitals. The eigenstate of the system can be represented using two tight-binding Bloch functions, i.e.,The graphene monolayer shown in Pz orbitals belonging to periodic A (B) atoms.where V0, period length L, and contact ratio w/L  can be obtained from the Slonczewski-Weiss-McClure model53For the arrangement shown in atio w/L . In prevRE carbon atoms  can be considered simultaneously. The energy spectrum of the system can be solved by diagonalizing the band-like Hermitian matrix. In addition, the features of the electronic structures impact directly on the DOS, and hence the density of states can be calculated asIn the above basis, the Hamiltonian is expressed as a 4where Γ is the phenomenological broadening parameter owing to various de-excitation mechanisms, e.g., the electron–electron, electron–phonon, electron–impurity scattering. In this study, Γ is treated as a constant (Γ~ 1.3 meV). Note that in investigating the behavior of the charge carriers, the present study focuses specifically on the low-energy bands and the DOS near the Fermi level.How to cite this article: Tsai, S.-J. and Yang, R.-J. Bimodal behaviour of charge carriers in graphene induced by electric double layer. Sci. Rep.6, 30731; doi: 10.1038/srep30731 (2016).


The assembly of individual two-dimensional materials into van der Waals heterostructures enables the construction of layered three-dimensional materials with desirable electronic and optical properties. A core problem in the fabrication of these structures is the formation of clean interfaces between the individual two-dimensional materials which would affect device performance. We present here a technique for the rapid batch fabrication of van der Waals heterostructures, demonstrated by the controlled production of 22 mono-, bi- and trilayer graphene stacks encapsulated in hexagonal boron nitride with close to 100% yield. For the monolayer devices, we found semiclassical mean-free paths up to 0.9 μm, with the narrowest samples showing clear indications of the transport being affected by boundary scattering. The presented method readily lends itself to fabrication of van der Waals heterostructures in both ambient and controlled atmospheres, while the ability to assemble pre-patterned layers paves the way for complex three-dimensional architectures. The reliable assembly of two-dimensional materials into van der Waals heterostructures is a critical step towards nanoscale electronic integration. Here the authors present a technique for batch fabrication of graphene/boron nitride stacks with clean interfaces and high-yield. The controlled isolation and assembly of single- and few-layer sheets of two-dimensional 2D) materials into van der Waals (vdW) heterostructures has thrown open the doors for the design and fabrication of new devices and functionalities based on 2D materials with an unprecedented flexibility and atomic precision. The perspectives are astounding, with applications ranging from electronics1D materia358I(2D)/I(G) peak ratioClean interfaces between 2D materials result in the best device performance1011121314I(2D)/I(G) ratio in graphene due to plasma treatment of the cleavage substrate is fully reversed when the crystals are lifted from this substrate, meaning that plasma pre-treatment is a viable way to obtain large and pristine 2D material flakes for integration into vdWs heterostructures. By actively tuning the interfacial adhesion and cleanliness through temperature whilst completely avoiding any contact with liquids in the stacking procedure, we are able to controllably pick up and drop-down 2D materials, including single-layer crystals that have been pre-patterned using electron-beam lithography (EBL). This method enables us to produce a statistically significant data set of field effect mobility measurements from 22 mono-, bi- and trilayer encapsulated graphene devices with >280 contacts. Seven of the 16 monolayer devices and 55% of the measurements display carrier mean-free paths comparable or exceeding the channel width, with carrier mean-free paths limited by boundary scattering.2 V−1 s−1, respectively. No annealing at high temperatures is necessary to obtain this high performance1321We present here room temperature mobility measurements from a batch-fabricated set of 22 mono-, bi and trilayer blister-free encapsulated graphene field effect devices – assembled in ambient atmosphere–with over 280 individual metal-graphene contacts. We find that blisters of trapped interfacial contamination commonly observed in such samples by optical and atomic force microscopy can be completely eliminated by stacking individual 2D crystals into vdWs heterostructures at temperatures of 110 °C, even in ambient atmosphere. We prove that the reduction of the 2, are produced on oxidized silicon substrates by micromechanical cleavage. A combination of oxygen plasma pre-treatment of the substrate and thermal release of the adhesive tapeI(2D)/I(G) peak ratios of graphene flakes cleaved on plasma-treated oxide show a noticeably higher doping than for graphene on untreated oxide (I(2D)/I(G) peak ratio=1.1 on plasma-treated versus 1.8 on untreated–I(2D)/I(G) peak ratio increases from <2 to 4.2 and 3.3 for graphene from plasma-treated and untreated SiO2, respectively – Mono-, bi- and trilayer graphene and thin (thickness in the range 20±10 nm) hexagonal boron nitride (hBN) flakes, all with areas of over 2,000 μmx, y and z using a micromanipulator (We use a polypropylene carbonate (PPC) coated polydimethylsiloxane (PDMS) block mounted on a glass slide to capture (pick-up) and release (drop-down) 2D materials . For fulipulator  fixed toipulator , 2a–c. H2 using PPC, regardless of the temperature, but we are unable to pick up graphene using PPC in the same way – graphene adheres more strongly to the oxidized silicon surface than the PPC, leading to the tearing of edges that can be seen in 2 surface dominates. For graphene on SiO2 surfaces that have not been plasma treated, hBN can be used to pick up the graphene flakes regardless of temperature, as previously reportedTemperatures above 110 °C favour the vdWs adhesion between hBN and graphene, whereas a temperature of 40 °C is sufficient to lift hBN from oxidized silicon cleavage substrates using the PPC/PDMS block. We note that assembly at high temperatures produces adhesion forces between graphene and hBN which are strong enough to tear the graphene flake, leaving behind graphene regions which are not covered by hBN adhering to the oxidized silicon substrate . Such hiA key feature of this technique is the ability to produce large numbers of encapsulated graphene samples in parallel, by covering many graphene flakes on their production substrate with dropped down hBN flakes . This en−1 by controlling the z-height of the stage.We compare the number and size of blisters formed during stacking at 110 versus 40 °C using optical and atomic force microscopy, and find that the cleanliness of the interface critically depends on both the speed of the approach of the flakes and the temperature. One point of the PPC/PDMS block touches the target substrate first, due to the slide being tilted by a few degrees in the micromanipulator. Full contact between these two surfaces is made controllably in all cases, limiting the speed of the contact area front to <1 μm sStacking at 40 °C results in apparently blister-free heterostructures, but on heating above 70 °C these blisters become visible and mobile, increase in size, agglomerate and then stabilize within a few seconds . These bIn attempt to detect the presence of trapped contamination within stacks, we transferred graphene adhered to hBN to silicon nitride transmission electron microscopy (TEM) aperture grids using the same drop-down methods described above. An optical image of the hBN-graphene heterostructure adhered to PPC/PDMS is shown in 2521I(2D)/I(G) ratio of >5, approaching 6, as previously shown for hBN-encapsulated graphene heterostructures, and a G peak full width at half maximum of 14±0.5 cm−1 over 5–10 μm areas of the heterostructures. Raman maps showing the spatial variation of these values are provided in the Raman data for a representative hBN-encapsulated graphene sample in 6 plasma is used to etch, which we find shows significantly improved selectivity for hBN relative to Poly(methyl methacrylate) , graphene (>90:1) and SiO2 (>90:1), rather than the more commonly used CHF3 etch10We batch fabricate hBN-encapsulated graphene field effect devices with edge contacts in both Hall bar and van der Pauw geometries from blister-free vdWs heterostructures stacks via EBL, plasma etching and metal deposition . SF6 pla12 cm−2 at zero gate voltage for all monolayer, bilayer and trilayer graphene devices  for single-layer devices kFλmfp, where kF=n is the carrier density, e is the elemental charge and h is Planck's constant. The high mobilities we calculate indicate that in the majority of cases in our monolayer devices the transport is limited by boundary scattering since the calculated mean-free path is comparable to or larger than w, the device widthλmfp≥w/2  geometries of 3, 5 and 10 μm side length with four contacts placed at the corners of the device edges. For such devices we find hole and electron mobilities of 38,000±15,000 cmTo prove the versatility of our technique, top-gated samples were fabricated by the drop-down of an additional hBN flake to cover the entire device area . This flThe presented method for stacking 2D materials allows the preparation of clean heterostructures with a high throughput due to a higher yield of large exfoliated single-layer crystals, the parallelization of the different fabrication steps enabling batch production and with close to 100% pick-up yield.I(2D)/I(G) peak ratio in graphene flakes micromechanically cleaved on plasma-treated oxidized silicon is fully reversed on subsequent pick-up with hBN shows that oxygen plasma treatment of the substrate does not introduce point defects or charged contaminants attached to the exfoliated graphene. As a result, oxygen plasma pre-treatment is a suitable means of producing large numbers of appropriately sized exfoliated 2D materials on oxidized silicon for the fabrication of prototype vdWs heterostructures, which consistently exhibit clean interfaces and high carrier mobilities.The fact that the observed reduction of I(2D)/I(G) ratio and G peak full width at half maximum of the encapsulated graphene in limited areas of the surface /I(G) ratio approaching 6 and a G peak FWHM of 14 cm−1, which serve as evidence of a lack of doping of the graphene within the stack – results which are also confirmed by our electrical measurements of the low doping of our samples: all show residual carrier densities <1012 cm−2 at zero gate bias.Atomic force microscopy shows that any residual contamination between layers in bubble-free regions must be either uniform or completely absent, since we are able to detect changes in thickness of the graphene encapsulated in hBN even down to a single monolayer . Dark fiThe presented techniques also enable us to pick up pre-patterned 2D materials, despite the presence of PMMA residues on top of the patterned flakes, an inevitable by-product of EBL. This means both that PMMA residues are largely excluded from the surface via this technique, and also that it is not a prerequisite of this technique to have atomically flat and clean surfaces before carrying out the stacking procedure, as presented previouslyThe devices produced using this hot stacking technique display very high room temperature mobilities, with 55% of the measurements in monolayer devices exhibiting mean-free paths larger than the half of the device width as well as uniformly low contact resistance. This high consistency is achieved without any annealing101216While little data is available in literature concerning the yield of heterostructure device fabrication, we believe that our stacking yield of nearly 100% and contact fabrication yield of 88% represent significant progress towards consistent, reproducible device fabrication of vdWs heterostructures. We find that statistically significant figures for these yields is unfeasible to obtain without batch fabrication, and that the high yield across many samples is a direct consequence of the complete exclusion of interfacial contamination during stacking at high temperatures. In comparison, stacking at lower temperatures lead to blisters occurring over an interface area fraction of 20% or more which strongly decreases the fabrication yield.2 V−1 s−1, with peak values of 37,000 and 23,000 cm2 V−1 s−1, are consistent with the highest values reported at room temperature in the literature for bilayersThe mean carrier mobilities measured in bilayer and trilayer graphene devices of 21,000±7,000 and 16,000±4,000 cmWe note that in all but one instance, the electron mobility exceeds the hole mobility for bilayer and trilayer samples . This trIn 16% of the measurements the calculated room temperature mean-free path exceeds the sample width102010202 V−1 s−1. The majority of monolayer Hall-bar devices exhibit transport limited by the edges, with mean-free paths exceeding half of the channel width. This stacking technique enables the pick-up and drop-down of flakes of 2D materials at desired locations with nearly 100% yield, and with a yield of 88% for subsequently fabricated electrical contacts with uniformly low contact resistance.In conclusion, we have presented a facile and robust technique for the batch fabrication of vdWs heterostructures, demonstrated by the controlled production of 22 mono-, bi- and trilayer encapsulated graphene devices. Stacking at elevated temperatures (higher than 110 °C) results in high mean carrier mobilities for bilayer and trilayer samples of 21,000±7,000 and 16,000±4,000 cmThe absence of trapped contamination in our samples, manifesting as blisters between the stacked flakes, indicates that adsorbants are largely or completely excluded from between the flakes during the drop-down procedure, being pushed out in front of the proceeding vdWs contact region. In addition, the high temperature used for drop-down may also allow a more conformal contact between 2D materials during stacking as a result of reduction of viscosity of the PPC. The presented method readily lends itself to fabrication of any desired vdWs heterostructures, completely avoiding contact with liquids, whether in ambient conditions or in controlled atmospheres. The technique even permits the pick-up of lithographically patterned 2D materials and integration into vdWs heterostructures, or for additional layers to be dropped down onto pre-existing devices. By picking up device layers such as graphene with encapsulating layers such as hBN, the need for multiple separate encapsulation steps is avoided allowing efficient batch fabrication of heterostructure devices.The ability to produce a large number of devices with high yield is a key advantage of this process, and paves the way for the statistical studies of device performance. Such studies are essential to gain an understanding of vdWs heterostructure based device performance in a technological perspective, for fundamental research and for further progress towards real-world device applications of 2D materials.2, 120 W), and the 2D material loaded tape is immediately applied to the silicon oxide surface. The tape is subsequently released from the surface by heating to 85 °C on a hot plate.Graphene and hBN are cleaved on 100 nm Si oxide thermally grown on standard 4 inch Si wafers. Natural graphite crystals (NaturGrafit GmbH) and hBN bulk crystals (HQgraphene) were mechanically exfoliated with Nitto Denko SWT 20+ die sawing tape. Oxidized silicon is treated in oxygen plasma for 3 min  piece of PPC coated PDMS to a glass microscope slide.The PDMS is prepared from SYLGARD 184 by mixing 10 parts base and 1 part curing agent and cured at 70 °C overnight, leading to a 1-mm-thick layer. The PDMS is treated with oxygen plasma as above for 10 min and then a PPC layer is spun on top of it , resulting in a thickness of 5 μm. Double-sided tape is used to attach a 1 × 1 mmRaman spectra are taken with a Thermo Fisher DXR Raman spectrometer using a 455-nm laser source with a power of 1 mW and 20 s duration multiple exposures. Raman spectra and maps for encapsulated graphene were acquired in a Thermo Fisher DXRxi Raman spectrometer using a 455-nm source, with a power of 10 mW and 20 s duration to provide adequate signal to noise ratio from the graphene within the heterostructure.−1.The AFM scans are done in a NTEGRA scanning probe microscope from NT-MDT with a Smena measuring head. The scans are performed in tapping mode with typical parameters of a driven frequency of 340 Hz, a magnitude of 10 nA, a set point of 5 nA and scan speed of 10–30 μm s−1 in the diffraction plane. Selected area diffraction (SAD) patterns were taken in the same instrument with a 200-nm-diameter SAD aperture.Tilted beam dark-field images were acquired in a Tecnai T20 G2 operated at 200 kV by selecting a first-order graphene reflection with an objective aperture with a diameter of 2 nm−1) , followed by a post-bake (10 min at 150 °C).The EBL is performed in a JEOL JBX-9500FS, with an acceleration voltage of 100 kV. PMMA is used as resist in the EBL process, a 4-wt% PMMA 996K in anisole solution is spun on the chips .The etching of the stack is performed in a SPTS ICP Etch, OThe data that support the findings of this study are available from the corresponding author on request.How to cite this article: Pizzocchero, F. et al. The hot pick-up technique for batch assembly of van der Waals heterostructures. Nat. Commun. 7:11894 doi: 10.1038/ncomms11894 (2016).Supplementary Figures 1-14 and Supplementary Methods2 assembled at 40°C and heated to 70°C.Trapping and migration of blisters in hBN/graphene/hBN. Mobile blisters within an hBNgraphene-hBN heterostructure on SiOTrapping and migration of blisters after pick-up. Mobile blisters within an hBN-graphene heterostructure on PPC/PDMS before drop-down on hBN, assembled at 40°C and heated to 70°C.2 before pick-up assembled at 40°C and heated to 70°C.Trapping and migration of blisters after drop-down. Mobile blisters within an hBNgraphene heterostructure on SiO2. Control of the contact front forces any contamination between the faces of the 2D materials out of the heterostructure towards the edges.Blister-free drop-down. Drop-down of an hBN flake adhered to PPC/PDMS onto a graphene layer on SiO2. In this instance, the contact front is not well controlled, resulting in the visible formation of blisters and inhomogeneous Raman I(2D)/I(G) and G peak FWHM values.Blister formation during drop-down. Drop-down of an hBN flake adhered to PPC/PDMS onto a graphene layer on SiO


Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements. Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. L1s comprise ~17% of human DNA and it is estimated that an average human genome has ~80–100 active L1s. L1 moves throughout the genome via a “copy-and-paste” mechanism known as retrotransposition. L1 retrotransposition is known to cause mutations; thus, it stands to reason that the host cell has evolved mechanisms to protect the cell from unabated retrotransposition. Here, we demonstrate that the zinc-finger antiviral protein (ZAP) inhibits the retrotransposition of human L1 and Alu retrotransposons, as well as related retrotransposons from mice and zebrafish. Biochemical and genetic data suggest that ZAP interacts with L1 RNA. Fluorescent microscopy demonstrates that ZAP associates with L1 in cytoplasmic foci that co-localize with stress granule proteins. Mechanistic analyses suggest that ZAP reduces the expression of full-length L1 RNA and the L1-encoded proteins, thereby providing mechanistic insight for how ZAP may restricts retrotransposition. Importantly, these data suggest that ZAP initially may have evolved to combat endogenous retrotransposons and subsequently was co-opted as a viral restriction factor. Despited ORF2p) –6. It isd ORF2p) , Duchennd ORF2p) , and cand ORF2p) ,11. Inded ORF2p) .A full-length human L1 is ~6 kb in length and encodes a 5' UTR that harbors an internal RNA polymerase II promoter that directs transcription from at or near the first base of the element –15. The cis-preference [e.g., 5'-TTTT/A, 5'-TCTT/A, 5'-TTTA/A, etc.) [in trans to retrotranspose other cellular RNAs such as Short Interspersed Elements (SINEs)  to feference ,26,37,38eference , althougeference ,41. Onceeference ,42,43. BA, etc.) ,44,45. TA, etc.) ,44. The A, etc.) . Notably.g., Alu  and SINE.g., Alu –52). L1 U6 snRNA ,53,54, s3 snoRNA ), and me3 snoRNA ,36).Since L1 retrotransposition can be mutagenic, it stands to reason that the host cell employs multiple mechanisms to restrict L1 mobilization [reviewed in To gain a more complete understanding of the interplay between the L1 retrotransposition machinery and the host cell, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins that co-immunoprecipitate with L1 ORF1p in HeLa cells, reasoning that some of these proteins may affect L1 retrotransposition. We next analyzed the effects of ORF1p-interacting proteins on L1 retrotransposition by overexpressing a subset of them in a cultured cell retrotransposition assay ,71. HereTo identify proteins that interact with L1 ORF1p, we transfected HeLa cells with a human L1 construct, pJM101/L1.3FLAG, which expresses a version of ORF1p containing a FLAG epitope at its carboxyl-terminus (ORF1p-FLAG) . The pJMBriefly, HeLa cells were transfected with pJM101/L1.3FLAG or pJM101/L1.3, a similar construct that lacks the FLAG epitope sequence . Whole cTo determine the identity of cellular proteins that associated with ORF1p-FLAG, the bands from the lanes corresponding to the pJM101/L1.3FLAG and pJM101/L1.3 immunoprecipitation experiments were excised from SDS-PAGE gels and submitted for LC-MS/MS see . An LC-MTo confirm the interactions between LC-MS/MS-identified proteins and ORF1p-FLAG, we evaluated 13 of the 39 ORF1p-FLAG interacting proteins for which there were commercially available antibodies and/or cDNA expression clones. Western blot analyses confirmed that these proteins associated with ORF1p-FLAG . The 13 mblastI)  that expresses the blasticidin deaminase gene (We co-transfected HeLa cells with each of the 18 ORF1p-FLAG interacting candidates and pJJ101/L1.3 . An emptThe above data  imply thZAP is a poly (ADP-ribose) polymerase (PARP) family member  initiallPutative ZAP orthologs are present in several species ; thus, wThe ZAP zinc-finger domain binds to RNA and is required for antiviral activity ,92. To ain trans to mediate their retrotransposition [neoTet), and a ZAP-S expression plasmid. Notably, ZAP-S potently reduced Alu retrotransposition to ~25% of control levels  retrotransposons, we tested whether ZAP-S expression affected human Alu retrotransposition. Unlike L1, Alu is a 7SL-derived non-autonomous retrotransposon that does not encode its own proteins . Insteadposition . Brieflyl levels . In contl levels . Thus, ZF21) [F21; ~24% of control levels), zebrafish L2 , and synthetic mouse L1   , a zebraF21) , or a syF21)  that has levels) . The res levels) . NotablymEGFPI) tagged with an EGFP indicator cassette (mEGFPI), which allows retrotransposition activity to be detected by EGFP fluorescence [mEGFPI, which carries two missense mutations that adversely affect ORF1p RNA binding [mEGFPI retrotransposition activity when compared to assays conducted in the presence of a control siRNA  to deplete endogenous ZAP from HeLa cells. Following siRNA treatment, cells were transfected with an L1 plasmid  and ORF2 (ORF2_5804) . Co-tran and ORF2gene10 epitope tag on the carboxyl-terminus of ORF1p and a TAP epitope-tag on the carboxyl-terminus of ORF2p  [mEGFPΔIntron. Thus, EGFP expression is not dependent on retrotransposition. Forty-eight hours post-transfection, flow cytometry was used to isolate EGFP-positive cells    that expΔIntron) . WesternΔIntron) . By compΔIntron) . ControlΔIntron) . Thus, Zgene10 epitope-tag on the carboxyl-terminus of ORF1p [ORF1p, ORF2p, and L1 RNA form RNP complexes that appear as discrete cytoplasmic foci when visualized by fluorescence microscopy ,26,104. of ORF1p ,107) witTo test if the ZAP-S zinc-finger domain is critical for the co-localization of ZAP-S with ORF1p, we co-transfected HeLa cells with pJM101/L1.3Δneo and a tGFP-tagged ZAP-S mutant that expresses the ZAP-S zinc-finger domain , or a ZAP-S mutant that lacks the ZAP-S zinc-finger domain  . In contTo determine if ZAP co-localizes with L1 RNA, we co-transfected HeLa cells with pJM101/L1.3 and either ZAP-S-tGFP, ZAP-S/Δ310-645-tGFP, or ZAP-S/Δ72-372-tGFP. To visualize L1 RNA, transfected cells were probed with fluorescently labeled oligonucleotide probes complementary to sequences within the L1 5' UTR. As a control, cells were co-transfected with pJM101/L1.3 and an empty pCEP4 vector. In pCEP4 control experiments, fluorescence microscopy revealed that ORF1p co-localized with L1 RNA in cytoplasmic foci in ~88% of cells that contained ORF1p cytoplasmic Foci . In HeLaFluorescence microscopy further revealed that in cells co-transfected with pJM101/L1.3 and ZAP-S/Δ310-645-tGFP that L1 RNA was detected in ORF1p and ZAP-S/Δ310-645-tGFP foci in only ~18% of foci-containing cells . In contWe next determined the effect of ZAP-S on ORF1p expression using confocal microscopy. HeLa cells were co-transfected with pJM101/L1.3Δneo and either ZAP-S-tGFP, ZAP-S/Δ310-645-tGFP, or ZAP-S/Δ72-372-tGFP. As a control, cells were co-transfected with pJM101/L1.3Δneo and an empty pCEP4 vector. In pCEP4 control experiments, confocal microscopy revealed that ~10.8% of cells expressed ORF1p after ~ 48 hours . In contL1 cytoplasmic foci also co-localize with an array of RNA binding proteins, including markers of cytoplasmic stress granules (SGs) ,89,104. In this study, we identified 39 cellular proteins that interact with L1 ORF1p and validated 13 of these interactions in biochemical assays. Our data showed that the 13 validated ORF1p-interacting proteins associate with ORF1p via an RNA bridge . NotablyF21), and an engineered zebrafish LINE-2 element (ZfL2-2) (ZAP inhibits the mobility of both human and non-human non-LTR retrotransposons. The overexpression of the human and rat orthologs of ZAP restricts human L1 retrotransposition . Human Z(ZfL2-2) . Althoug(ZfL2-2) . This obThe ZAP CCCH zinc-finger domain is required to both bind and mediate the degradation of viral RNA ,109. OurNotably, the zebrafish ZfL2-2 retrotransposon lacks a homolog to ORF1 and only encodes a single ORF that contains an apurinic/apyrimidinic endonuclease-like (EN) and a reverse transcriptase (RT) domain . The finper se, but instead likely affects the post-transcriptional processing of full-length L1 mRNA. In addition to biochemical data, fluorescence microscopy revealed that L1 RNA was depleted from L1 ORF1p cytoplasmic foci in the presence of ZAP   . Thus, ZIn sum, our data suggest that ZAP restricts L1 retrotransposition by preventing the accumulation of cytoplasmic L1 RNA. Notably, a recent study suggests that ZAP may interfere with translation of viral RNA, and that translation inhibition may precede viral RNA destruction . AlthougIt remains unclear how ZAP might destabilize full-length L1 RNA to restrict retrotransposition. Evidence suggests that ZAP recruits exosome components  along wii.e., a subset of APOBEC3 proteins, TREX1, MOV10, SAMHD1, and RNaseL). L1 elements have been active in mammalian genomes for ~160 million years [ZAP exhibits antiviral activity against a variety of viruses such as MLV , alphavion years –122. Thuon years ,123–125.2 in humidified incubators (Thermo Scientific).HeLa-JVM cells were grown in high-glucose DMEM (Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin-streptomycin (Invitrogen), and 0.29 mg/mL L-glutamine (Gibco) . HeLa-HAOligonucleotide sequences and cloning strategies used in this study are available upon request. All human L1 plasmids contain the indicated fragments of L1.3 (accession no. L19088)  DNA clonThe following cDNA expression plasmids were obtained from OriGene: CDK9 (SC119344); DDX21 (SC108813); GNB2L1 (SC116322); hnRNPDL (SC107613); MOV10 (SC126015); MATR3 (SC113375); ZAP-S (ZC3HAV1 transcript variant 2) (SC101064); ZAP-S-tGFP (GFP-tagged ZC3HAV1 transcript variant 2) (RG208070); hnRNPA2B1 (SC313092); IGF2BP3 (SC111161); PURA (SC127792); UPF1 (SC118343).The following cDNA expression plasmids were obtained from Open Biosystems: hnRNPL (6174088); LARP2 (5164712); LARP4 (5219803); SYNCRIP (5495201).The following cDNA expression plasmids were obtained from Addgene: rZAP   and ZAP-L (pcDNA4 huZAP(L); Addgene plasmid#: 45907, kindly provided by Dr. Harmit Malik) [pJM101/L1.3: is a pCEP4-based plasmid that expresses a human L1 (L1.3) equipped with an mneoI retrotransposition indicator cassette. L1 expression is augmented by a CMV promoter located upstream of the L1 5' UTR and an SV40 polyadenylation signal that is located downstream of the native L1 polyadenylation signal [n signal ,127,128pJM101/L1.3FLAG: was derived from pJM101/L1.3 and contains a single FLAG epitope on the carboxyl-terminus of ORF1p. Dr. Huira Kopera  constructed the plasmid.neopAluTet: expresses an Alu element cloned from intron 5 of the human NF1 gene [neoTet reporter gene. The reporter [NF1 gene  that is reporter  was subcreporter .pCEP/GFP: is a pCEP4 based plasmid that expresses the humanized renilla green fluorescent protein (hrGFP) coding sequence from phrGFP-C (Stratagene), which is located downstream of the pCEP4 CMV promoter [promoter .pJJ101/L1.3: is a pCEP4 based plasmid that contains an active human L1 (L1.3) equipped with an mblastI retrotransposition indicator cassette [cassette .pJJ105/L1.3: is similar to pJJ101/L1.3, but contains a D702A missense mutation in the RT active site of L1.3 ORF2 [1.3 ORF2 .pJM101/L1.3Δneo: is a pCEP4 based plasmid that contains an active human L1 (L1.3) [1 (L1.3) .mEGFPΔIntron:pLRE3-EF1- is a pBSKS-II+ based plasmid that expresses an active human L1 (LRE3) that is tagged with an EGFP cassette (mEGFPI) containing an antisense, intronless copy of the EGFP gene. A UbC promoter drives EGFP expression. An EF1α promoter drives L1 expression [pression .pAD2TE1: is similar to pJM101/L1.3 except that it was modified to contain a T7 gene10 epitope-tag on the carboxyl-terminus of ORF1p and a TAP epitope-tag on the carboxyl-terminus of ORF2p. The 3′-UTR contains the mneoI retrotransposition indicator cassette [cassette .pJBM2TE1: is similar to pAD2TE1 except that the pCEP4 backbone was modified to contain the puromycin resistance (PURO) gene in place of the hygromycin resistance gene.mEGFPI:pLRE3- is a pCEP4 based plasmid that contains an active human L1 (LRE3) equipped with an mEGFPI retrotransposition indicator cassette [cassette ,106. Thecassette .mEGFPI:pJM111-LRE3- is identical to pLRE3-mEGFPI except that it contains two missense mutations in ORF1 (RR261-262AA), which render the L1 retrotransposition-defective [efective . Mr. Wilefective .pGF21: contains an 8.8 kb fragment which includes a full length mouse GF21 L1 element that contains the mneoI indicator cassette [cassette .pZfL2-2: is a pCEP4 based plasmid that contains the ZfL2-2 ORF  cloned upstream of the mneoI indicator cassette [cassette .pCEP4smL1: contains a codon optimized full-length mouse element (derived from L1spa) containing the mneoI indicator cassette [cassette .ZAP-S/1-311: encodes the ZAP-S amino acid sequence from 1–311 and the following sequence of non-templated amino acids (IIIYTGFLFCCGFFFFFFFLEGVSLCCPGWS).ZAP-S/Δ72–372: was derived by deleting the SfoI-XhoI fragment from ZC3HAV1 transcript variant 2 , and expresses a ZAP-S mutant protein that lacks amino acid sequence from 72–372.ZAP-S/Δ310-645-tGFP: expresses a ZAP-S mutant protein that lacks amino acid sequence from 310–645 and contains a carboxyl terminus tGFP epitope tag.ZAP-S/Δ72-372-tGFP: expresses a ZAP-S mutant protein that lacks amino acid sequence from 72–372 and contains a carboxyl terminus tGFP epitope tag.LARP5: was derived by cloning LARP5 cDNA  into pcDNA3 (Invitrogen).LARP1: was constructed by cloning the LARP1 cDNA  into pcDNA3 (Invitrogen).pK_A3A: expresses HA-tagged APOBEC3A and was a generous gift from Dr. Brian Cullen [n Cullen .pDCP1α-GFP: expresses a GFP-tagged version of DCP1α and was a generous gift from Dr. Gregory Hannon [y Hannon .pG3BP-GFP: expresses a GFP-tagged version of G3BP and was a generous gift from Dr. Jamal Tazi [mal Tazi .pcDNA6/TR: expresses the blasticidin resistance gene and was obtained from Invitrogen.6 cells/flask and transfected the next day with 20 μg of plasmid DNA using 60 μL of FuGENE HD (Promega). Approximately 48 hours post-transfection, hygromycin B (Gibco) (200 μg/mL) was added to the medium to select for transfected cells. After approximately one week of hygromycin selection, cells were washed 3 times with ice cold PBS and collected with a rubber policeman into 50 mL conical tubes . Cells were then pelleted at 1,000×g and frozen at -80°C. To produce whole cell lysates (WCL), frozen cell pellets were rapidly thawed and then lysed in ~3 mL (1 mL lysis buffer per 100 mg of cell pellet) of lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 0.1% IGEPAL CA-630 (Sigma), 1X complete EDTA-free protease inhibitor cocktail (Roche)) on ice for 30 minutes. WCLs were then centrifuged at 15,000×g for 15 minutes at 4°C. Supernatants were transferred to a clean tube and protein concentration was determined using the Bradford reagent assay (BioRad). For the IP, ~1 mL of the supernatant  was pre-cleared with ~15 μL (packed gel volume) of mouse IgG-agarose beads (Sigma) for 4 hours at 4°C. Pre-cleared supernatants were then mixed with ~15 μL (packed gel volume) of EZview Red ANTI-FLAG M2 Affinity Gel (Sigma) and incubated overnight with rotation at 4°C. The beads then were rinsed 3x with 0.5 mL of lysis buffer, and then washed 3 times with 0.5 mL of lysis buffer for 10 minutes per wash on ice with gentle agitation. Protein complexes were eluted from the beads by adding ~70 μL of 2X NuPAGE LDS Sample Buffer (Novex), supplemented with NuPAGE Sample Reducing Agent (Novex), directly to the washed beads and incubating for 10 minutes at 70°C. Following incubation, the beads were pelleted and the sample was transferred to a fresh tube. For SDS-PAGE analysis, 20 μL of the IP were loaded onto a 4–15% gradient midi-gel (BioRad) and run under reducing conditions. Gels were silver stained using the SilverQuest Silver Staining Kit (Novex) to visualize proteins.HeLa-JVM cells were seeded in T-175 flasks  at ~6–8×10m/z range of 400–1800. For each cycle, the five most abundant ions from each MS scan were selected for MS/MS analysis using 35% normalized collision energy. Selected ions were dynamically excluded for 45 seconds.The Proteomics Facility at the Fred Hutchinson Cancer Research Center  conducted protein identification experiments. Excised silver-stained gel slices were destained and subjected to in-gel proteolytic digestion with trypsin as described . Followie.g. keratin) were filtered from the final L1 RNP candidate list.For data analysis, raw MS/MS data were submitted to the Computational Proteomics Analysis System (CPAS), a web-based system built on the LabKey Server v11.2  and searmblastI, HeLa-JVM cells were seeded at ~1–2×104 cells/well in a 6-well plate . Within 24 hours, each well was transfected with 1 μg of plasmid DNA (0.5 μg L1 plasmid + 0.5 μg cDNA plasmid or pCEP4) using 3 μL of FuGENE 6 transfection reagent (Promega). Four days post-transfection, blasticidin (EMD Millipore) containing medium (10 μg/mL) was added to cells to select for retrotransposition events. Medium was changed every two days. After ~8 days of selection, cells were washed with PBS, fixed, and then stained with crystal violet to visualize colonies. To control for transfection efficiency and off-target effects of cDNA plasmids, in parallel with retrotransposition assays, HeLa-JVM cells were plated in 6-well plates at 500–1,000 cells/well and transfected with 0.5 μg pcDNA6/TR (Invitrogen) plasmid + 0.5 μg cDNA plasmid using 3 μL of FuGENE 6 transfection reagent (Promega). The pcDNA6/TR control assays were treated with blasticidin in the same manner as for retrotransposition assays.The cultured cell retrotransposition assay was carried out essentially as described ,71. For mneoI, HeLa-JVM cells were transfected as described above. Two days after transfection, cells were treated with medium supplemented with G418 (Gibco) (500 μg/mL) for ~10–12 days. As a control, HeLa cells were plated at ~2×104 cells/well in a 6-well plate and transfected with 0.5 μg pcDNA3 (Invitrogen) plasmid + 0.5 μg cDNA plasmid using 3 μL of FuGENE 6 transfection reagent (Promega). The pcDNA3 control assays were treated with G418 in the same manner as for retrotransposition assays.For retrotransposition assays with L1 constructs tagged with 5 HeLa-HA cells were plated per well of a 6-well plate  and transfected with 0.67 μg of pJM101/L1.3Δneo + 0.67 μg of pAluneoTet + 0.67 μg of cDNA plasmid using 6 μL FuGENE HD (Promega). Three days post-transfection, cells were grown in the presence of G418 (500μg/mL) to select for Alu retrotransposition events. As a control, HeLa-HA cells were plated at ~4×105 cells/well in a 6-well plate and transfected with 0.67 μg of pcDNA3 (Invitrogen) + 0.67 μg of pAluneoTet + 0.67 μg of cDNA plasmid using 6 μL of FuGENE HD (Promega). The pcDNA3 control assays were treated with G418 in the same manner as for Alu retrotransposition assays.For Alu retrotransposition assays , ~4×105 5 cells) were plated in 60 mm tissue culture dishes . The next day, the cells were transfected with 50 nM of a control siRNA pool  or siRNA against ZAP  siRNA—SMARTpool, Thermo Scientific) or MOV10  siRNA—SMARTpool, Thermo Scientific) using the DharmaFECT 1 transfection reagent (Thermo Scientific). Twenty-four hours after siRNA treatment, cells were transfected with pLRE3-mEGFPI or pJM111-LRE3-mEGFPI (5 μg), using 15 μL of FuGENE HD transfection reagent (Roche). After 48 hours, cells were trypsinized and an aliquot of the cells (~2×106 cells) was used to monitor endogenous protein levels (72 hours after siRNA treatment) by western blot analysis (see below for list of primary antibodies). Blots were analyzed using an Odyssey CLx (LI-COR) with the following secondary antibodies: IRDye 800CW Donkey anti-Rabbit IgG  (LI-COR) and IRDye 680RD Donkey anti-Mouse IgG  (LI-COR). Knockdown efficiencies were calculated using LI-COR Image Studio Software (v3.1.4) and are the average of three independent experiments. Endogenous tubulin was used as the normalization control. The remaining cells were re-plated at ~2×105 cells/well of a 6-well plate and cultured in medium supplemented with puromycin  to select for cells transfected with pLRE3-mEGFPI. After 4 days of puromycin selection, the percentage of GFP positive cells was determined by flow cytometry using an Accuri C6 flow cytometer (BD Biosciences).In experiments to study the effect of endogenous proteins on L1 retrotransposition, HeLa cells , 10% glycerol, 1mM DTT, 0.1% NP-40, and 1x complete EDTA-free protease inhibitor cocktail (Roche)). Following lysis, whole cell lysates were centrifuged at 12,000xg for 10 minutes at 4°C, and then the cleared lysate was layered onto a sucrose cushion (8.5% and 17% sucrose) and subjected to ultracentrifugation at 4°C for 2 hours at 178,000xg. The supernatant was discarded and the resulting pellet was resuspended in water supplemented with 1x complete EDTA-free protease inhibitor cocktail (Roche). Approximately 20 μg  of the RNP sample or ~30 μg  of the cleared whole cell lysate  were then analyzed by western blot. Blots were analyzed using an Odyssey® CLx (LI-COR) with the following secondary antibodies: IRDye 800CW Donkey anti-Rabbit IgG  (LI-COR) and IRDye 680RD Donkey anti-Mouse IgG  (LI-COR).RNPs were isolated as previously described . Briefly6 cells/dish)  and transfected with 10 μg of plasmid DNA (5.0 μg pLRE3-EF1A-mEGFPΔIntron + 5.0 μg cDNA plasmid or pCEP4) using 30 μL of FuGENE HD. After 48 hours, cells were harvested with trypsin and then subjected to flow cytometry to isolate GFP expressing cells. Approximately 1.2–1.7×106 GFP positive cells were collected for each transfection condition using a MoFlo Astrios cell sorter (Beckman Coulter). The GFP gate was set using untransfected HeLa-JVM cells. The sorted cells were lysed as described in the IP procedure and lysates were then subjected to western blotting using standard procedures. For all other protein expression analyses, HeLa-JVM cells were seeded at ~4×105 cells/well in 6-well plates and transfected with 2 μg of plasmid DNA with 6 μL of FuGENE HD. Cells were collected 48 hours after transfection using a rubber policeman and lysates were prepared as described above. Western blots were visualized using either the SuperSignal West Femto Chemiluminescent Substrate (Pierce) or SuperSignal West Pico Chemiluminescent Substrate (Pierce) and Hyperfilm ECL .To simultaneously analyze the effects of ZAP-S on ORF1p and EGFP protein expression, HeLa-JVM cells were seeded onto 10 cm dishes (~2.7×106 cpm ml-1) overnight at 68°C. For band quantification, northern blot films were analyzed using ImageJ software [HeLa-JVM cells were seeded in T-175 flasks  and transfected with 20 μg of plasmid DNA (10 μg pJM101/L.13Δneo + 10 μg cDNA plasmid) using 60 μL FuGENE HD. Two days after transfection, cell pellets were collected and frozen at -80°C. Frozen cell pellets were then thawed and total RNA was extracted with TRIzol reagent (Ambion), and then poly(A)+ RNA was prepared from total RNA using an Oligotex mRNA kit (Qiagen). Each sample (~1.5 μg of poly(A)+ RNA) was subjected to glyoxal gel electrophoresis and northern blotting using the NorthernMax-Gly Kit (Ambion) according to the manufacturer’s protocol. Following electrophoresis, RNA was transferred to BrightStar Nylon membranes (Invitrogen) and then cross-linked using UV light. For northern blot detection, membranes were prehybridized for ~ 4 hours at 68°C in NorthernMax Prehybridization/Hybridization Buffer (Ambion), and then incubated with a strand specific RNA probe ;(5UTR99: 5'-GGAGCCAAGATGGCCGAATAGGAACAGCT-3' and 5'-AATTAACCCTCACTAAAGGGTGAGTGAGAATATGCGGTGTTT-3').(ORF2_5804: 5'- GACACATGCACACGTATGTTTATT-3' and 5'- Strand-specific RNA probes were generated using the MAXIscript T3 system (Invitrogen). The 5UTR99  probe counderlined) was added to the reverse primer of each primer pair. The pTRI-β-actin-125-Human Antisense Control Template (Applied Biosystems) was used in T3 reactions as a template to generate the β-actin RNA probe. Each northern blot experiment was independently repeated three times with similar results.The T3 promoter sequence  wash buffer  for 5 minutes. To visualize L1 RNA, coverslips were then incubated with 300 nM FISH probes (sequences below) in FISH hybridization buffer  for ~4 hours at 37°C. Following hybridization, cells were incubated for 30 minutes in FISH wash buffer at 37°C and then incubated with FISH wash buffer + 3% BSA for an additional 30 minutes at 37°C. To visualize L1 ORF1p by immunofluorescence, coverslips then were incubated with αORF1p antibodies (1:2000) in 1x PBS + 3% BSA for 1 hour at 37°C. Cells were washed three times with 1x PBS (10 minutes per wash). Cells were incubated with Alexa Fluor 546 conjugated Goat anti-Rabbit IgG (Invitrogen) (1:1000) in 1x PBS + DAPI (50 ng/mL) for 30 minutes at 37°C. Coverslips were mounted on slides with VECTASHIELD mounting media (Vector Laboratories). Combined RNA FISH/immunofluorescence samples were imaged with a Zeiss Axioplan2 microscope . RNA FISH/immunofluorescence images  in a 12-well plate and transfected ~24 hours later with 0.5 μg of plasmid DNA using 1.5 μL of FuGENE 6 transfection reagent. Approximately 48 hours after transfection, cells were fixed with 4% paraformaldehyde for 10 minutes and then permeabilized with 0.2% Triton X-100 in 1x PBS for 7 minutes. Following permeabilization, coverslips were incubated for 5 minutes in FISH  were raised in rabbits and affinity-purified (Open Biosystems). αCDK9 (2316), αUPF1 (9435), and αGFP (2955) were obtained from Cell Signaling Technology. αhnRNPL (NBP1-67852), αILF3 (EPR3627), αLARP1 (NBP1-19128), αMATR3 (NB100-1761), αNCL (NB100-1920SS), and αDHX9 (NB110-40579) were obtained from Novus Biologicals. αFAM120A (ab83909), αPURA (ab79936), and αHA tag (ab9110) were obtained from Abcam. αMOV10 (SAB1100141), αZAP (Anti-ZC3HAV1 (HPA047818)), and αTubulin (T9026) were obtained from Sigma. αZC3HAV1 (16820-1-AP) was obtained from Proteintech. αeIF3 (p110) (sc-28858) was obtained from Santa Cruz Biotechnology. αT7-Tag mouse monoclonal (69522–3) was obtained from Novagen. αTAP rabbit polyclonal (CAB1001) was obtained from Thermo Scientific.S1 Fig(A) The FLAG epitope on ORF1p is compatible with retrotransposition: Constructs were tested in a transient HeLa cell retrotransposition assay [(B) Immunoprecipitation reactions conducted using various wash conditions: Top panel: HeLa cells were transfected with pCEP4, pJM101/L1.3, or pJM101/L1.3FLAG and were subjected to lysis using two different salt concentrations (500 mM NaCl (left gel) or 150 mM NaCl (right gel)). Shown are the images of silver stained gels from immunoprecipitation reactions. The black rectangles indicate the cropped image depicted in (C) Immunoprecipitation reactions under different lysis buffer conditions: Silver stained gels of IP fractions from untransfected HeLa (HeLa UTF) or HeLa cells transfected with pJM101/L1.3FLAG. Lysis buffer contained either 0.1% CHAPS (left gel) or 1.0% Triton X-100 (right gel). Black arrows correspond to the approximate location of ORF1p-FLAG; black bars indicate the approximate location of proteins enriched in the pJM101/L1.3FLAG lane. Molecular weight standards (kDa) are shown on the left side of the gels.on assay ,71. The on assay . Represe(TIF)Click here for additional data file.S2 Fig(A-C) Transfected ZAP is expressed in HeLa cells: Western blots of whole cell lysates demonstrate the expression of ZAP-L (panel A) and ZAP-S and ZAP-S/∆72–372 (panels B and C) 48 hours post-transfection. UTF indicates untransfected HeLa cells. The antibodies are indicated at the right side of the blots. Blue arrows indicate the approximate locations of the ZAP proteins. Tubulin serves as a loading control. Molecular weight standards (kDa) are shown on the left side of the blots. (D) The depletion of endogenous ZAP enhances L1 retrotransposition: Flow cytometry was used to determine the percentage of EGFP-positive, live-gated cells for each siRNA transfection condition (noted above the plots). The X-axis depicts the scattering at 533 nm; the Y-axis depicts the scattering at 585 nm. The EGFP-positive gate was set using the retrotransposition-deficient negative control, pJM111-LRE3-mEGFPI [3-mEGFPI ,69.(TIF)Click here for additional data file.S3 Fig(A) A schematic of the pLRE3-EF1-mEGFPΔIntron: pLRE3-EF1-mEGFPΔIntron expresses a human L1 (LRE3) that is tagged with an mEGFPI expression cassette that lacks an intron. The human elongation factor-1 alpha (EF1α) promoter (arrow) augments L1 transcription. The ubiquitin C (UbC) promoter (upside down arrow) drives EGFP transcription. (B) ZAP-S inhibits ORF1p expression: Western blots were conducted using whole cell lysates derived from cells co-transfected with pLRE3-EF1-mEGFPΔIntron and the ZAP-S expression plasmid or pCEP4 indicated above each lane. UTF indicates whole cell lysates from untransfected HeLa cell. Antibodies are indicated on the right side of each blot. Tubulin is used as a loading control. Western blot images depict a representative experiment that was repeated three times with similar results. Notably, upon extended exposure times ORF1p was able to be visualized in the ZAP-S lane. (C) ZAP-S does not inhibit EFGP expression and/or accumulation: HeLa cells were co-transfected with pLRE3-EF1-mEGFPΔIntron and the indicated expression plasmids (noted above the plots). Flow cytometry was used to determine the percentage of EGFP-positive, live-gated cells for each condition. UTF indicates untransfected HeLa cells. The EGFP-positive gate was set using the UTF sample as a negative control. The X-axis depicts the percentage of EGFP positive cells. The Y-axis indicates the side scattering profile (SSC). Approximately 1.2–1.7 x 106 GFP positive cells were collected and analyzed for each transfection condition.ZAP-S preferentially suppresses the expression of ORF1p. (TIF)Click here for additional data file.S4 Fig(A) ZAP-S-tGFP inhibits retrotransposition in HeLa cells: Top panel: Schematics of tGFP-tagged ZAP constructs. Depicted are the relative positions of the zinc-finger domains (light gray rectangles), cysteine-histidine (CCCH) zinc-fingers , and tGFP tag (green rectangles) ZAP-S expression constructs. Bottom panel: Results of pJJ101/L1.3 retrotransposition assays. The X-axis indicates the cDNA co-transfected with pJJ101/L1.3 or pcDNA6/TR. The Y-axis indicates pJJ101/L1.3 retrotransposition activity (black bars). All values have been normalized to the pCEP4 empty vector control (100%). The numbers above the bar graphs indicate the number of biological replicates performed with each cDNA expression construct. Error bars represent standard deviations. (B-E) ORF1p and ZAP co-localize with stress granules in HeLa cells: HeLa cells were co-transfected with pJM101/L1.3Δneo and ZAP-S-tGFP; proteins were visualized by direct immunofluorescence. ORF1p co-localizes with ectopic ZAP-S-tGFP and eIF3 in cytoplasmic foci (panel B). ORF1p co-localizes with ectopic ZAP-S-tGFP (panels A and B), but not with tubulin (panel C). GFP-tagged G3BP co-localizes with endogenous ZAP in cytoplasmic foci (panel D). GFP-tagged DCP1α forms cytoplasmic punctate structures, which do not appear to co-localize with endogenous ZAP (panel E). The right-most image in each panel represents a merged image. The cell type is indicated at the top left (yellow), the protein name is listed on the bottom left, and the name of the primary antibody used  is annotated at the bottom right. Nuclei were stained with DAPI (blue) and the scale bar represents 25 μM.(TIF)Click here for additional data file.S5 Fig(A) Fluorescence microscopy was used to determine the percentage of cells that contained L1 RNA in cytoplasmic ORF1p foci. The X-axis indicates the plasmid that was co-transfected with pJM101/L1.3. The Y-axis of the graph depicts the percentage of cells where L1 RNA was detected in cytoplasmic ORF1p foci. Experiments were repeated three times. A total of ~60 visual fields (~1600 cells) were examined amongst all three experiments and ~33–41 ORF1p foci containing cells were evaluated for each experimental condition. Error bars represent standard deviations. (B) Confocal microscopy was used to determine the number of ORF1p-expressing cells ~48 hours post transfection. The X-axis indicates the plasmid that was co-transfected with pJM101/L1.3Δneo. The Y-axis of the graph depicts the percentage of cells that express ORF1p. Experiments were repeated twice. Each experiment contained two biological replicates and ~1100–1500 cells were enumerated amongst all experiments for each condition. Error bars indicate standard deviations.(TIF)Click here for additional data file.S1 TableORF1p-interacting proteins were selected based on the criteria that the protein was unique to the pJM101/L1.3FLAG IP and was identified by two or more unique peptides . Column 1 = protein name. Column 2 = protein mass. Column 3 = total number of identified peptides. Column 4 = number of unique peptides. Column 5 = the percentage of amino acid coverage for each of the respective proteins. Columns 6–8 = whether the proteins were identified in the indicated studies ,67,89. G(TIF)Click here for additional data file.


Long interspersed element-1 (L1) is a transposable element that can move within the genome, potentially leading to genome diversity and modified gene function. Although L1 activity in somatic cells is normally suppressed through DNA methylation, some L1s are activated in tumors including colorectal carcinoma. However, how L1-retrotransposition (L1-RTP) is involved in gastrointestinal disorders remains to be elucidated. We hypothesized that L1-RTP in somatic cells might contribute to colitis-associated cancer (CAC). To address this, we employed an experimental model of CAC using transgenic L1-reporter mice carrying a human L1-EGFP reporter gene. Mice were subjected to repeated cycles of colitis induced by administration of dextran sodium sulfate (DSS) in drinking water with injection of carcinogen azoxymethane (AOM). L1-RTP levels were measured by a quantitative polymerase chain reaction targeting the newly inserted reporter EGFP in various tissues and cell types, including samples obtained by laser microdissection and cell sorting with flow cytometry. DNA methylation levels of the human L1 promoter were analyzed by bisulfite pyrosequencing. AOM+DSS-treated mice exhibited significantly higher levels of L1-RTP in whole colon tissue during the acute phase of colitis when compared with control naïve mice. L1-RTP levels in whole colon tissue were positively correlated with the histological severity of colitis and degree of neutrophil infiltration into the lamina propria (LP), but not with tumor development in the colon. L1-RTP was enriched in LP mesenchymal cells rather than epithelial cells (ECs), myeloid, or lymphoid cells. DNA methylation levels of the human L1 promoter region showed a negative correlation with L1-RTP levels. L1-RTP was absent from most tumors found in 22-week-old mice. In conclusion, we demonstrated that L1-RTP was induced in the mouse CAC mucosa in accordance with the acute inflammatory response; however, retrotransposition appears not to have direct relevance to colitis-induced cancer initiation. Long interspersed element -1 (L1) is a transposable element that can move within the genome to induce gene deletions, inversions, and insertions, potentially leading to genome diversity as well as altering gene function . L1 is pIn this study, we found that L1-RTP was positively associated with the severity of colitis. Activation of L1 was correlated with hypomethylation of CpG islands at the L1 5’-UTR. However, L1-RTP was not increased in the tumors in this model suggesting that L1-RTP is not significantly involved in tumor initiation in this CAC model.All experiments using mice were performed according to the Institutional Guidelines for the Care and Use of Laboratory Animals in Research with the approval of the Animal Care and Use Committee of the Research Institute, National Center for Global Health and Medicine . Animals were euthanized with ketamine and xylazine and every effort was made to minimize suffering.hL1-EGFP), named Line 4, was developed in our facility [A line of transgenic mice harboring a human L1-EGFP reporter gene . The method for detection of the frequency of L1-RTP with a PCR targeting retrotranspositioned EGFP was described previously . For anaWhole colon tissues obtained during the acute inflammatory phase were rolled up and snap-frozen in liquid nitrogen. Frozen sections were fixed with cold acetone and stained with hematoxylin and eosin for histological analysis. Histological scores for the degree of crypt loss and cell infiltration were blindly assigned to each colon. Crypt loss was evaluated as the relative decrease in epithelial cell area in comparison with the naïve colon using a captured image analyzed by image J software : 0—no change or less than 10% decrease from naïve colon, 1—10–30% of epithelial cells were lost, 2—30–50%, 3—50–70%, 4—70–90%, 5—more than 90%. Cell infiltration scores: 0—no change from naïve colon, 1—focal infiltration limited to the mucosal layer, 2—focal infiltration in both mucosal and submucosal layers, 3—diffuse mucosal and submucosal infiltration. A score for each mouse was determined as the sum of the crypt score and the cell infiltration score. Neutrophil infiltration was determined by the nuclear morphology. In some experiments genomic DNA was obtained from frozen sections using a laser microdissection system  according to the manufacturer’s instructions.-CD3+CD11b-CD11c-; B cells including B220-low plasma cells, EpCAM-B220+CD11b-CD11c-; dendritic cells/macrophages, a mixture of EpCAM-CD3-CD11b+ cells, EpCAM-CD3-CD11c+ cells, and EpCAM-CD3-F4/80+ cells; granulocytes, EpCAM-Gr-1high cells; non-epithelial non-hematopoietic cells, EpCAM-CD3-B220-CD11b-CD11c-F4/80-Gr-1- cells.Epithelial cells (ECs) were isolated from the colon and treated with 10 mM EDTA in Hank’s Balanced Salt Solution . Lamina propria (LP) cells (LPCs) were isolated using a Gentle MACS Dissociator  with the Lamina Propria Dissociation Kit for the mouse (Milteny Biotech). LPCs were stained with fluorescein isothiocyanate (FITC)-, R-phycoerythrin (PE)-, or allophycocyanin (APC)-labeled anti-CD3, anti-CD11b, anti-CD11c, anti-B220, anti-Gr-1, or anti-EpCAM antibodies , and 7-aminoactinomycin D to identify viable cells, and the cells were sorted with a flow cytometer . Cell type sorting was performed with the following surface markers: T cells, EpCAMTo assess the methylation status of L1, bisulfite-pyrosequencing was performed with a PyroMark Q24 pyrosequencing machine  using pyro LINE kit .Statistical analysis was performed using the Prism 6 statistical program  with the methods indicated in the figure legends. For the analyses of difference unpaired one- or two-tailed Student’s t tests were used. Correlations were tested with two-tailed Pearson calculations. Values of P < 0.05 were considered significant.hL1-EGFP) [The experimental protocol for the CAC model is illustrated in L1-EGFP)  were useL1-EGFP) . As a reAlthough L1-RTP was significantly induced during the acute phase of the CAC model, the levels of induction showed considerable individual differences . Therefo+ T cells, B220+ B cells/plasma cells, CD11b+/CD11c+/F4-80+ macrophage/dendritic cells, or Gr1+ granulocytes -treated mice (a positive control for the human L1-RTP reporter ), and mein vivo, induced by administration of a carcinogen with induction of colitis. In this study of CAC, we show that L1-RTP is not induced in epithelial cells or hematopoietic cells, but most likely in a mesenchymal cell type. Furthermore, we successfully measured DNA methylation levels of the human L1 promoter in this mouse model, and found a negative correlation between L1-RTP and DNA methylation of its promoter. This result supports the notion that hypomethylation of L1 is associated with L1-activation. On the other hand, we did not find L1-RTP in colon tumors or their background mucosa in this murine colon cancer model, although a correlation has been established between L1-RTP and cancer in humans as described in a recent review [in vivo system does not detect the endogenous mouse L1-RTP, although detection of the RTP of the human L1 transgene was clear. Levels of human L1-RTP in the colon were generally low, and this detection sensitivity may not exactly represent the endogenous retrotransposition in the mice, which might affect the frequency of CAC. However, the fact that we detected increased L1-RTP in acute colitis but not in tumors indicates that the impact of L1-RTP on tumorigenesis is at least indirect. Second, previous study reported that L1 retrotransposition was present in preneoplastic liver but not in the subsequent liver adenocarcinoma [This is the first report in which L1-RTP was directly demonstrated t review . Insteadarcinoma . The autRecently, the role of mesenchymal stromal cells in various inflammatory conditions including colitis has been reported. The reparative and anti-inflammatory properties of mesenchymal stromal cells have been tested in a variety of animal models and have been applied in specific clinical settings . Particu


The EB process is found to depend on both the graphene type and on the ambient conditions. For the mechanically exfoliated graphene, performing EB under vacuum leads to a higher yield of nanometer-gap formation than working in air. Conversely, for graphene on SiC the EB process is not successful under vacuum. Finally, the EB is possible with turbostratic graphene discs only after the creation of a constriction in the sample using lithographic patterning.Graphene-based electrodes are very promising for molecular electronics and spintronics. Here we report a systematic characterization of the electroburning (EB) process, leading to the formation of nanometer-spaced gaps, on different types of few-layer graphene (namely mechanically exfoliated graphene on SiO The vision of molecular electronics is the exploitation of single molecules as the active units in complex devices offering novel functionalities beyond the present technology –3. To ac2 carbon bonds assure a high mechanical stability even above room temperature. Finally, the possibility to exploit specific functionalizations to attach the molecular units to the graphene electrodes through carbon bonds and/or π-stacking seems to be a promising route to develop well-defined and robust junction configurations. In addition to these advantages, several theoretical papers have investigated the possibility to use graphene as an electrode to contact individual molecules [Recently, the use of graphene as electrode material for molecular electronics has been proposed ,10. Witholecules –16, predolecules , edge efolecules , and supolecules .2. Therefore it is important to test it also on other types of graphene. In particular, since many envisaged applications require the use of a gate electrode to tune the molecular junctions properties, it seems appealing the use of few-layer graphene, which is still thin but much less gate-dependent than the single layer [Recent works have successfully made use of graphene for the realization of electrodes in molecular devices ,17. Specle layer ,20.2 substrate and we show that the yield of nanometer-gap formation can be increased significantly when working at a reduced pressure. In addition, we report, for the first time, the EB process (in air and under vacuum) also on other types of few-layer graphene, which are better suited for large scale integration, namely multilayer graphene grown on the C-face of SiC [In this work we compare the EB process in air and in vacuum in few-layer graphene flakes exfoliated on SiOe of SiC  and thine of SiC –25.−4 mbar) so that the final comparison between the results in air and under vacuum is made independent on the geometrical factors. In total, we measured 23 graphene flakes of thickness of 1–20 layers, corresponding to 115 junctions.We first consider the case of few-layer graphene flakes obtained by the mechanical exfoliation technique . A typical flake is shown in the inset of I–V curves become strongly non-linear, probably related to the removal of contaminants induced by the current annealing [21To open a nanometer-size gap in the graphene devices, we polarized the junctions with a single voltage ramp with a fast feedback loop in order to stop the current immediately after the opening of the junction. We used the same method previously employed for the electromigration of gold nanowires . A typicnnealing ,20–2127.ealing 21. When thI–V measurement keeping the bias voltage below ±1.5 V to avoid any modification of the gap [After the EB process, the device is characterized by an  the gap ,28. An eI–V curves according to the Simmons model [d, the junction area A and the height of the tunnel barrier Φ as the fitting parameters. For the fit shown in d = 1.83 nm, A = 5 nm2, Φ = 0.6 eV. It is known that the value of d is a quite robust fitting parameter, while the other two are not, since good fits are still possible even with very different values. Indeed, we can fit our data with almost the same accuracy by using a very broad range of values for A and Φ  the values for d are in the range from 0.5 nm to 3 nm. This narrow distribution, which is expected since in the model the current depends exponentially on the width of the gap, is a strong indication that we can obtain electrodes with distances in the true nanometer range.In order to characterize the gap size in more details, we fit the ns model  (see SupI–V characterization after the EB process. The first class (A) is composed by the devices showing a sizeable tunneling current (I > 10−13 A) within the range of ±1.5 V. In these devices, the EB process created a gap with sizes of a few nanometers. In the second group (B) we consider junctions that underwent an EB process but no tunneling current is observed in our measurement range. These are “open” devices, with a gap larger than about 10 nm, which can be still useful for contacting long molecules or nanoparticles. Finally, the last group (C) comprises those devices that did not break under the maximum voltage/current we applied. The results of these studies are summarized in the upper panel of In the following, we discuss the yield of this EB process, with particular attention on the differences encountered between air and vacuum working conditions. In total, 58 junctions were processed in air and 57 under vacuum and they have been divided in three categories depending on the Considering the junctions processed in air, 25 of them (ca. 43%) exhibited a tunneling current after the EB, in 20 (ca. 35%) no detectable current was measured and finally 13 (ca. 22%) did not break during the process. Such a yield is comparable with what has been found by Burzurì et al. [V = 0.5 V in different devices. We stress that the I–V tests after the EB process were repeated also under different conditions (air or vacuum) and we did not find a significant dependence on the measurement conditions (only the conditions of the EB process are relevant). Since the tunneling current is inversely proportional to the size of the gap, we conclude that under vacuum conditions the EB is more controllable, leading to higher yield of success and generally smaller gap sizes.Interestingly, the tunneling currents measured after the EB process generally display a much higher value when the process is carried out under vacuum than in air, as shown in the bottom panel of It is also worthy of note that the number of devices that were not burned is almost the same for air and vacuum conditions. We noted that very thick or very large flakes led to devices displaying very small two-probes resistance (≤200 Ω), which did not undergo the EB process for our maximum applied voltage (10 V) and/or led to the saturation of our current-meter (30 mA). Indeed, the largest part of the devices falling in the category C of If we consider only the flakes that were effectively electroburned, i.e., neglecting devices falling in category C, the difference in yield between the EB process in air and in vacuum is even more evident: the rate of success (junctions displaying tunneling current after EB) is 55% in air and 80% in vacuum. In addition, the gaps obtained by the vacuum process generally display much larger currents implying a smaller size of the gap itself. Similarly, an improved yield in the fabrication of nanometer-sized gaps in graphene junctions by changing the environment conditions from air to vacuum was also reported by Nef et al.  who, howIbreak (upper panel) and the voltage Vbreak (lower panel) at which the EB process occurs as a function of the initial resistance of the junctions for both the working conditions (air and vacuum).In order to analyze further the dependence of the EB process on the environment conditions, in Ri while it is clearly higher when the process is performed under vacuum. The dependence of Ibreak on Ri, which is primarily determined by the number of layers of the flakes , suggests that the EB occurs at a constant current density (which only depends on the geometry of the device), regardless of the environment conditions, and in agreement with previous works [We found that the current is generally similar in the two cases (air or vacuum) and it always scales with the initial resistance. On the other hand, the voltage at which the device rupture is found does not show a clear dependence on us works . On the us works . We willI–V curve is shown in Vbreak > 40 V for vacuum processed devices, while it is in the range 10–15 V when working in air) and no detectable currents were ever measured after the EB process. Interestingly, the SEM images revealed that the rupture is always at the metal contacts and not in the graphene devices  in large quantities (hundreds of discs with a diameter of about 1–2 µm in a single deposition).We also tested the EB process in multilayered graphene microstructures, employing thin discs of turbostratically stacked graphene . These discs are comprise up to 100 graphene layers exhibiting a rather large charge carrier mobility in the range of 10ed discs . They caI–V-characteristics of such an electroburned device can verify the actual presence of a gap. Typically a bias voltage of 1 V results in a current of 80 pA, as exemplified in In a first attempt, simple pairs of source–drain electrodes have been defined on the discs by electron beam lithography. More than 10 discs have been contacted and underwent the EB process in air. In all of these experiments the EB took place at the contact areas as exemplary shown in 2 and achieve a better control on the gap formation.We now compare the different behaviors encountered in the three types of graphene analyzed. Concerning the exfoliated few-layer graphene flakes, we found that performing the EB in vacuum leads to a higher yield of nanometer-gap fabrication. At the same time the EB process happens at higher voltages under vacuum than in air, the current being almost identical under the two conditions. This means that the dissipated electrical power and the local temperature of the devices are similar or even slightly higher in the case of vacuum. Since a higher local temperature is usually considered to be detrimental for a controlled gap formation, we conclude that the environment pressure must be the key parameter to optimize the EB process of few-layer graphene on SiO2 vs graphene/SiC).Graphene grown on the C-face of SiC exhibits a similar behavior under ambient conditions, while under vacuum the oxygen–carbon reaction does not even initiate. This difference must be related to the different stacking feature (turbostratic vs graphite-like) of the graphene itself and/or to the different characteristics of the graphene/substrate systems (exfoliated graphene/SiO2 (which is known to be more reactive than SiC). Such impurities may be working as catalysts, initiating the EB process of graphene even under a reduced oxygen atmosphere.Focusing on these parameters, it is interesting to consider the case of turbostratic microdiscs, which have a morphology similar to that of graphene on SiC but are deposited on the same substrate as the exfoliated graphene. Here, we found that the EB process leads to a breaking of the graphene devices only after the patterning of a constriction. This hardness with respect to exfoliated graphene suggests that the different morphology of the edges also plays a role to initiate the burning. Indeed, the presence of nonsaturated carbon bonds makes the edges the most reactive part of the device. Edges cleaved during the exfoliation (exfoliated graphene), edges created during the oxygen plasma (graphene on SiC and turbostratic discs after the patterning), and edges of the untreated microdiscs  do have We presented a systematic study of the electroburning (EB) process in few-layer graphene devices for different graphene types. We focused our attention on exfoliated graphene, graphene epitaxially grown on the C-face of SiC and turbostratic graphene microdiscs comparing the results obtained when working in air and under vacuum. We showed how the process strongly depends on the specific type of graphene and on the environment pressure in the chamber. For mechanically exfoliated graphene, the vacuum process leads to the formation of nanometer-sized gaps with a higher yield and generally smaller sizes. On the other hand, for graphene on the C-face of SiC, the EB process creates a gap in the graphene devices when it is performed in air, while under vacuum conditions it simply leads to the blow-up of the metal contacts. As-deposited turbostratic graphene discs are found to be extremely resilient against the EB process: The opening of a gap in the device is possible only after creating a hot spot in the discs, as demonstrated in our work after the patterning of a constriction.Our work suggests that the oxygen pressure is a key factor in the EB process but also other factors such as the type of graphene stacking, the morphology of the edges and the specific graphene/substrate system play an important role. Further studies will focus on tuning the partial oxygen pressure during the electroburning to determine the optimal working conditions for the different types of device. We believe that these results will contribute to the realization of reliable graphene based electrodes for molecular electronics and spintronics.Few-layer graphene flakes were deposited by the standard “scotch tape” mechanical exfoliation method from natural graphite pieces on top of a p-doped silicon wafer coated with 300 nm of oxide. Flakes of suitable thickness (1 to approx. 20 layers) were located with an optical microscope on the basis of their contrast with the substrate. In some cases, the effective number of layers is also checked by micro-Raman spectroscopy, see Turbostratic graphene was obtained on on-axis SiC(000−1) semi-insulating wafer dice. First, the SiC dice were hydrogen-etched in order to obtain atomically flat surfaces . This prTo assure good ohmic contacts, the first fabrication step was the thermal deposition of 3 nm Cr/30 nm Au as the initial metal contacts. Successively, graphene was patterned in the desired device geometry  by electron beam lithography and oxygen plasma etching (30 s in a Diener Femto plasma system at maximum power). Finally, the remaining metal parts (the bonding pads and the connections from the contacts to the pads) were obtained by the evaporation of 10 nm Cr/100 nm Au and lift-off.−4 mbar). After the electroburning process, I–V measurements were taken with an AdWin-Pro system (16 bit output and input ) using a FEMTO pre-amplifier.For mechanically exfoliated and epitaxial graphene the electroburning process was performed by applying an increasing voltage ramp (20 mV/s) while continuously measuring the conductance of a junction. The burning of carbon atoms initiated the formation of a gap and increased the resistance. Once the resistance overcome the chosen value of 200,000 Ω, the voltage was immediately reset to zero (<100 µs). The process was performed at room temperature either in air or under vacuum ; the AFM characterization of the graphene grown on SiC; additional details about the fitting procedure of the non linear I–V curves according to the Simmons model.See Supporting Information for the Raman spectra of the different graphene materials (exfoliated, graphene on the C-face of SiC, turbostratic micro-discs deposited on SiOFile 1Titel: Material characterization.


Through careful control of the subtle balance between the linear periodic potential of rippled graphene and the C60 surface mobility, we demonstrate that C60 molecules can be arranged into a quasi-1D C60 chain structure with widths of two to three molecules. At a higher annealing temperature, the quasi-1D chain structure transitions to a more compact hexagonal close packed quasi-1D stripe structure. This first experimental realization of quasi-1D C60 structures on graphene may pave a way for fabricating new C60/graphene hybrid structures for future applications in electronics, spintronics and quantum information.We report the preparation of novel quasi-one-dimensional (quasi-1D) C Due to its many exciting chemical and physical properties, numerous applications have been demonstrated in single-electron transistors, superconductivity, and photovoltaics660 often serves as a model system for understanding molecule-surface interactions. C60 on epitaxial graphene, a system exclusively formed of elemental carbon, provides an ideal model system to study interactions at the molecular level, electromigration, molecular electronics and spintronics. On most metal or graphite surfaces, C60 molecules self-assemble into isotropic thermodynamically favorable hexagonal close-packed monolayers8910Buckminsterfullerene, C60 molecules is quasi-one-dimensional (quasi-1D) structures. Highly ordered quasi-1D molecular configurations are excellent model systems and prototypes of 1D quantum confinement of electronic states, and thus have potential importance in electronic nanodevices, spintronics and solid-state quantum computation12131460 nanostructures have been rarely realized experimentally due to their highly anisotropic configuration. A few previously reported experimental results include C60 peapods by embedding C60 molecules in carbon nanotubes, 1D C60 structures aligned along step edges on vicinal surfaces of metal single crystals, and C60 chains on self-assembled molecular layers161718192021222324252760 nanostructures on rippled graphene by utilizing the linear periodic potential in graphene as a template. Through carefully controlling the subtle balance between the weak periodic potential and the surface mobility of C60, C60 molecules on rippled graphene are arranged into a novel quasi-1D C60 chain structure with widths ranging from two to three molecules wide. At a higher annealing temperature, the quasi-1D chain structure transitions to a more compact hexagonal close packed quasi-1D stripe structure. To the best of our knowledge, this is the first experimental realization of quasi-1D C60 structures on graphene. Our experimental results may pave a way for fabricating new C60/graphene hybrid structures for future applications in electronics, spintronics , and quantum information.A particularly interesting arrangement of C60 molecules were deposited onto graphene grown by the CVD method on Cu foil at the background pressure below 2.0 × 10−8 mbar (more details are in the Method section). After post-deposition annealing at 423 K, we observed small atomically flat facets with typical sizes ranging from 50 nm to 200 nm on the surface. These facets can be classified into two predominant types based on the distinguishable surface characteristics, labeled as type A and type B  plus a trimolecular cell is 5.08 ± 0.02 nm. From these models, we can clearly see the large spacing (1.23 nm) between adjacent chains 60 on graphite based on the thermal desorption spectroscopy study60 chains and stripes are grown, show a well-defined linear periodic graphene rippled superstructure with an orientation close to the chain and stripe orientation. 60 chains and stripes, as indicated by the blue arrow in 30272827283334353637To gain detailed insight into the growth mechanisms of the quasi-1D C60 chain structure. Based on the rippled graphene structure, it is possible for us to explain the formation of the chain structure from the point of view of mechanical stability60 on flat graphene is the hexagonal close-packed structure due to the weak diffusion barrier on flat graphene and the much stronger C60-C60 interaction4160 to arrange into more stable quasi-1D configurations. The linear periodicity of rippled graphene, 0.75 ± 0.01 nm, is smaller than the typical inter-row distance of 0.87 nm in the C60 closed packed arrangement. C60 molecules randomly occupy the valley sites of the linear periodic potential at the initial stage of growth. It is possible for C60 molecules to settle down in the adjacent ripples at a less stable state because C60 molecules are forced to stay slightly off the valley sites. In this way, two or three adjacent linear graphene ripples are filled up. This is consistent with the observation that almost all C60 chains consist of two or three rows. Based on a geometrical and energetic argument, it is easy to see that chains with 4 or more rows are less likely to form. Compared with the formation of the bimolecular chains, the possibility to form trimolecular chains (both sides need a C60 molecule sitting down around the middle C60 molecule) is about half the probability of the formation of the bimolecular chains, which only need one side to be occupied with C60 molecules. From a Monte Carlo statistical approach, C60 molecules adsorbed on such a linear graphene sheet will be stabilized in one of the valleys60 molecules with less thermal energy can form monomolecular chains. That is one of the possible directions to further tune and control the formation of these quasi-1D structures.The orientation of the graphene ripples is fully consistent with that of the C60 stripe structure after annealing at 487 K, we will start by considering the diffusion of Cu atoms underneath graphene and the arrangement of C60 molecules on graphene. When annealing at a higher temperature of 487 K, C60 molecules have a higher probability to overcome the potential energy difference between the hilltop and the valley sites of rippled graphene and rearrange into chains with larger width due to the higher thermal excitation. The junction structure in the bimolecular and trimolecular chains probably provides diffusion channels for C60 molecules to transfer between adjacent chains resulting in emergent chains with larger widths. Considering that the arrangement of bimolecular and trimolecular chains is usually 2-3-2 or 3-2-2 as described above, it is reasonable to assume that the emergent chains with width of five C60 rows are the configuration with highest probability to form, compared with chains of other widths. The distribution of chain widths and stripe widths may imply that at the annealing temperature of 487 K, the thermal excitation can only drive C60 molecules to cross over one or two energy barriers, but not more. When adjacent bimolecular and trimolecular chains merge into a 5-row chain, the interchain spacing between the merged 5-row chain to its neighboring chains can be generated from the initial interchain spacing in the chain configuration (1.23 nm), which is 1.59 nm. This spacing is slightly smaller than the interchain spacing of 1.73 nm required for a new C60 row to fill in the empty space. It has been previously reported that Cu atoms can easily diffuse underneath a graphene sheet even at room temperature. These detailed observations suggest that copper atoms can diffuse along smooth step edges and kinked sites, and even dissociate from step edges, indicating a weak interaction between the graphene sheet and the underlying copper atoms60 row to form along the straight step edges and results in hexagonal close packed quasi-1D C60 stripes with the typical width of six rows. The origin of C60 stripes with other number of rows can be attributed to the emergence of different combinations of C60 chains, such as two bimolecular chains emerging into a stripe with five C60 rows.In order to understand how the bimolecular and trimolecular chains transform into the C60 chain and stripe structures  was loaded into the homemade Knudsen cell mounted on the load lock of the LT-STM system. The C60 source was degassed at a pressure of low 10−8 mbar before deposition. C60 molecules were then sublimated at a deposition rate of ~0.9 ML/min with the background pressure below 2.0 × 10−8 mbar. During the deposition process, the substrate was kept at room temperature. The samples were then subsequently annealed at 423 K, 487 K, and 578 K respectively in the preparation chamber of the STM system with the base pressure at low 10−10 mbar. All the STM measurements were performed at room temperature in the STM characterization chamber with a base pressure of about 3.0 × 10−11 mbar.All experiments were carried out in an ultra-high vacuum (UHV) scanning tunneling microscope system (Omicron LT-STM). Before CHow to cite this article: Chen, C. et al. Temperature Evolution of Quasi-one-dimensional C60 Nanostructures on Rippled Graphene. Sci. Rep.5, 14336; doi: 10.1038/srep14336 (2015).


There are over 0.8 million people living with dementia in the UK. The needs of people with dementia are increasingly complex as the illness progresses. Eating and drinking difficulties are a major source of ill health and stress across the stages of dementia in multiple settings.The evidence on what interventions support people with dementia in continuing to eat and drink well needs to be updated, and the full set of interventions assessed. To ensure people with dementia and their carers have access to the best current evidence, which address the questions that are important to them, we consulted with stakeholder groups and formulated specific questions to be addressed by a systematic review. The evidence will be summarised in light of these questions.http://www.crd.york.ac.uk/ PROSPERO/ DisplayPDF.php?ID= CRD42014007611) was shared with members of two patient and public involvement groups. The members were asked to comment on the protocol and suggest questions they would like the review to address. Compiled questions were added to the protocol to inform our search strategies.The review protocol (We have conducted a comprehensive search of 13 databases for studies that assess the effectiveness of interventions to improve, maintain or facilitate oral food and drink intake, nutrition and/or hydration status, in people with dementia. Screening studies for eligibility, assessing risk of bias and extracting relevant data is underway and duplicated independently. Studies included in the review will be used to address the specific questions asked.The stakeholders’ questions, that were not originally specifically addressed by the review protocol included issues around personalisation of interventions, the relationship with the carer, meaningful activity around food as well as specific issues around oral hygiene and swallowing difficulties. These questions are guiding the way that the review is conducted and will help focus the way that information from the review is provided to people with dementia and their carers.This systematic review aims to review the effectiveness of interventions to improve, maintain or facilitate oral food and/or drink intake in people with dementia and address specific questions raised by stakeholders. By soliciting and then addressing questions within the systematic review that are important to our stakeholders we will improve the usability of our findings - the questions and their summary answers will be used to inform best practice.