The kink turn is a widespread structure motif that introduces a tight bend into the axis of duplex RNA. This generally functions to mediate tertiary interactions, and to serve as a specific protein binding site. K-turns or closely related structures are found in at least seven different riboswitch structures, where they function as key architectural elements that help generate the ligand binding pocket. This article is part of a Special Issue entitled: Riboswitches. •The K-turn is a widespread structural motif in RNA, generating a tight helical kink.•K-turns fall into two structural classes, depending on a critical H-bond acceptor.•K-turns mediate tertiary interactions in RNA molecules.•K-turns commonly bind specific proteins, exemplified by the L7Ae protein family.•K-turns can be elaborated into more complex forms, including the k-junction. It is found in double-helical RNA, frequently consisting of a three-nucleotide bulge flanked on the 3′ side by a G·A pair, an A·G pair and often one or two further non-Watson–Crick pairs before regular basepairing resumes . This he2http://www.dundee.ac.uk/biocentre/nasg/kturn/K-turns are extremely common in functional RNA molecules. They have been found in a number of riboswitches ; SAM-I 1/2 = 80 μM, and [Na+]1/2 = 30 mM and a Hill coefficient typically ~ 1 First, some  K-turn-forming sequences can be folded by addition of metal ions. This may be studied by the increase in the efficiency of fluorescence resonance energy transfer (FRET) between fluorophores attached to the 5′-termini of the C and NC helices with shortening of the end-to-end distance. For The great majority of K-turns are involved in tertiary interactions, and this provides a second process that can stabilize the folded conformation. The change in axial trajectory at the K-turn is often used to facilitate long-range structure in large RNA molecules. Most of the ribosomal K-turns help mediate such interactions. A rather clear example can be observed in the SAM-I riboswitch, where a long helix (P2) is kinked by a standard, simple K-turn to facilitate the docking of the terminal loop into a receptor in helix P4 The SAM-I riboswitch system was used to demonstrate K-turn structural stabilization by the formation of tertiary interactions. In initial studies, folding was studied indirectly via the ligand binding studied by isothermal calorimetry (ITC) Most K-turns are binding sites for proteins, discussed in greater detail in the following section. A diverse group of proteins bind to K-turns, with varying structure and manner of interaction. The archetypal K-turn-binding proteins are the ribosomal L7Ae and related proteins. Binding of proteins provides a third process that leads to the stabilization of the folded K-turn structure.H. marismortui Kt-7 results in folding into the kinked conformation in the absence of metal ions Kd = 10 pM Binding of L7Ae to We have shown that other ribosomal proteins, L24 and S11, result in the folding of their cognate K-turns . So the stabilization of K-turn structure by protein binding is quite general.7L7Ae and related proteins are a group of RNA-binding proteins that include the eukaryotic and archaeal proteins L7Ae, L30e and S12e H. marismortui Kt-15 in the large ribosomal subunit has been available since the 50S subunit was solved Archaeoglobus fulgidus L7Ae bound to the well-studied H. marismortui Kt-7The structure of L7Ae bound to These proteins bind in the major groove of the K-turn that runs around the outside of the structure. The major groove of normal duplex RNA is deep and narrow and so inaccessible to protein. However the structure of the K-turn splays open the groove, making it very accessible. The binding interface of L7Ae-related proteins comprises two elements. The first is a highly basic β-strand: turn: α-helix element, and the second a short loop of hydrophobic residues (plus a glutamate at one end) that makes a 180° turn.A. fulgidus L7Ae) hydrogen bonds to the 3n/4n phosphate. This interaction is near-universal, proposed in the complexes of the same species L7Ae with box C/D and the human 15.5 kDa protein with U4 snRNA. The lysine at the second turn (K37) is more variable in its interactions, but is bonded to the 4n/5n phosphate in the Kt-7 complex. Two of the basic side chains at the N-terminal end of the β-sheet section are also directed towards the NC helix non-bulged strand. These are not close enough for hydrogen bonding, but should contribute to overall electrostatic stabilization. The N-terminal end of the α-helix is involved in specific recognition of the core of the K-turn. The glutamate side chain from the first turn (E34) is hydrogen bonded to N1 of G1b. This interaction is also observed in the interactions of L7Ae with box C/D and the 15.5 kDa protein with U4 snRNA, and is likely to be universal. The adjacent asparagine residue (N33) is hydrogen bonded to O6 of G2n, also observed in the box C/D interaction. In the L7Ae–Kt-7 complex, as well as the other structures considered here, the O6 atom of G1b is located almost on the axis of the α-helix at the N-terminal end. Guanine O6 has a significant partial negative charge, and this will be electrostatically stabilized by the positive pole of the helix dipole. This appears to be another universal interaction in the L7Ae–K-turn complexes.The three-turn α-helix enters the major groove of the NC helix, directing its N-terminal end towards the major groove edges of the G1b and G2n nucleobases of the G·A pairs B. This r2. The syn conformation of L2 places it maximally in contact with the loop. Isoleucine I88 in particular is located directly over GL2 in the Kt-7 complex. A very similar interaction is observed in the box C/D and 15.5-U4 snRNA complexes. The glutamate of the loop (E89) is hydrogen bonded to N1 of the L1 guanine, thus making a specific contact.The hydrophobic loop covers the nucleobases in the L2 and L1 positions C, makingTaken together, these elements generate a very specific molecular recognition of the structure of the K-turn in double-stranded RNA. By placing the α-helix in the major groove of the NC arm and capping the L2 nucleobase it effectively ‘measures’ the angle between these two elements in the major groove. This then juxtaposes the N-terminus of the α-helix with the major groove side of the guanine bases of the two conserved G·A, where side chains make specific hydrogen bonds, and G1b O6 is placed to maximize the interaction with the positive pole of the helix dipole.The folding of Kt-7 that occurs on the binding of L7Ae might in principle occur by a passive conformational selection process 8B. subtilis lysine riboswitch where the terminal loop of the C-helix probably makes a loop–loop interaction The K-turn motif generates an abrupt change in the trajectory of the axis of duplex RNA. In many cases either the C or NC helix has a terminal loop that is involved in a tertiary interaction, and this is clearly important in facilitating the long-range architecture of RNA molecules. This is true both in relatively small RNA species such as many of the riboswitches and also in large structures like the ribosome. We have seen that in the SAM-I riboswitch the C-helix of the K-turn is involved in a loop–receptor interaction that stabilizes the fold of the functional riboswitch Many of the K-turns also function as the binding sites for specific proteins. The folded, tightly-kinked structure of the K-turn can be stabilized by addition of metal ions for some, but not all sequences, by the binding of specific proteins including the L7Ae family, and by tertiary interactions within a larger RNA structure. There are numerous cases in which the biogenesis of ribonuclear species use one or more of these processes. The first event in the formation of the box C/D snoRNA occurs when an L7Ae-family protein binds to a K-turn H. marismortuiThe majority of ribosomal K-turns mediate short- or longer-range tertiary interactions, and bind specific proteins, so we would expect that interplay between tertiary interactions and protein binding would be very important to the folding path. It could be envisaged that the K-turns are initially relatively flexible in an exchange between folded and unfolded conformations, allowing the local RNA to explore tertiary contacts. Flipping between N3 and N1 conformations might also be important during this process. As the contacts are found the structure could then become fixed by the binding of proteins. An example of this is provided by the 5′-terminal region of the 23S rRNA of


We find that tunneling through a single barrier exhibit a weak dependence on energy. We also show that in double barriers separated by a graphene layer we do not observe resonant tunneling, but a significant increase of the tunneling probability with respect to a single barrier of thickness equal to the sum of the two barriers. This is due to the fact that the graphene layer acts as an effective phase randomizer, suppressing resonant tunneling and effectively letting a double-barrier structure behave as two single-barriers in series. Finally, we use multiscale simulations to reproduce a current-voltage characteristics resembling that of a resonant tunneling diode, that has been experimentally observed in single barrier structure. The peak current is obtained when there is perfect matching between the densities of states of the cathode and anode graphene regions.Research in graphene-based electronics is recently focusing on devices based on vertical heterostructures of two-dimensional materials. Here we use density functional theory and multiscale simulations to investigate the tunneling properties of single- and double-barrier structures with graphene and few-layer hexagonal boron nitride (h-BN) or hexagonal boron carbon nitride (h-BC Vertical heterostructures of two-dimensional materials are the subject of intense investigation for the possibility they offer to engineer and taylor specific electrical and optical characteristicsIn graphene-based electronics, transistors based on vertical heterostructures are studied because they can achieve large current modulation, if large bandgap layers are included that can effectively block current in the off state. Therefore, a few transistor concepts based on vertical transport have been proposed245Vertical heterostructures of two-dimensional materials are qualitatively different from the well known heterostructures based on the III-V and II-VI materials systems. Indeed, adjacent layers can have very different Hamiltonians and transverse energy-dispersion relations. This aspect can lead to the emergence of peculiar transport properties.The choice of the materials stacked with graphene is crucial, since the deposition and growth of graphene on a dielectric substrate can significantly alter its electronic properties and suppress791011Hexagonal boron nitride (h-BN) is considered a promising dielectric131415142N layers, that we have studied by means of density functional theory.In this paper, we report some peculiar properties of vertical transport through single- and double-barrier heterostructures of graphene and h-BN or h-BCIn detail, we will show that tunneling through a single insulating barrier has a small dependence on energy in a large portion of the bandgap (Section II), and that a graphene layer between two tunneling barriers leads to a suppression of resonant tunneling and to an increase of the average transmission (Section III).Such properties are relevant for the operation of vertical electron devices and are not observed in better known heterostructures based on III-V and II-VI materials systems.2N/graphene heterostructures. In this way we are able to reproduce—with a full quantum simulation—experimental resultsFinally, following a multiscale approach, and leveraging the results of ab initio simulations, we have developed a tight-binding model for graphene/h-BN/graphene and graphene/h-BCn ranging from 1 to 5 , denoting the number of insulating layers on the left (l), the number of graphene layers (m) in the central region, and the number of insulating layers on the right (n). Analogously, hereinafter we refer to the single barrier systems with the acronym SB, followed by the number of insulating layer.The systems we consider consist of two identical barriers separated by one, two, or three graphene layers. For the sake of clarity, each double barrier (DB) system is identified by means of a set of three integers  in the current-voltage characteristics can be observed at room temperature. The peak is not due to a resonant-tunneling behavior, but to the perfect matching of the density of states of the cathode and anode graphene regions.Using a multiscale approach, we have then simulated gated graphene/h-BN/graphene and graphene/h-BCFurther study is required to better understand vertical transport in cases in which heterostructures are not lattice-matched, or misalignment and dissipation mechanisms are present.2N atomic layers stacked between semi-infinite graphite leads and arranged according to ref. 2N layers separated by up to three graphene layers and connected to semi-infinite graphite leads. To ensure the continuity of the charge density at the interface between each lead and the scattering region, we include on both sides of the latter at least four atomic layers of graphite.The single barrier structure we consider consist of up to five h-BN or h-BCAb-initio computations have been performed by means of Quantum Espresso28293031It has been shownThe van der Waals interaction anchors the h-BN and graphite layers at the interlayer distance observed in experiments34T as a function of the energy E is evaluated asE and transversal momentum i-th Bloch state is transmitted to the outgoing j-th state of the other contact, and Ti,j over all incoming and outgoing states. Sums run on both spins.We describe the transport properties in the framework of the Landauer formalism25Transmission probabilities have been calculated with the PWCONDHow to cite this article: Bruzzone, S. et al. Vertical transport in graphene-hexagonal boron nitride heterostructure devices. Sci. Rep.5, 14519; doi: 10.1038/srep14519 (2015).


Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62–K70 segment of Rps26 and the 5′ untranslated region of mRNA. The data suggested a specific role of the Y62–K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62–K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5′ untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62–K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast. RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5′ untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62–K70) motif. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62–K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62–K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62–K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast.The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62–K70 segment of Rps26 and the 5′ untranslated region of mRNA. The data suggested a specific role of the Y62–K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62–K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5′ untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62–K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast. Saccharomyces cerevisiae, the ribosome consists of two subunits , which contain 79 ribosomal proteins. Fifty-nine ribosomal proteins are encoded by duplicated genes. Twenty-one duplicated genes are translated into identical polypeptides, while the remainder are translated into very similar proteins  and RPS26b (located on chromosome 5), which are 92% identical. On the protein level, Rps26a differs from Rps26b by only two residues (E106D and D113A) in the C-terminal domain of the protein. Both proteins consist of 119 amino acids and possess a molecular mass of approximately 14 kDa (http://www.yeastgenome.org/). The Δrps26a Δrps26b double deletion is lethal, indicating that Rps26 is essential for the life of yeast . Be. Bein vil P site  (22). Bal P site , 24. In in vivo function of Rps26. Only simultaneous replacement of the highly conserved residues Y62, P65, K66, Y68, and K70 with alanine resulted in moderate growth defects and changes in polysome profiles that were indicative of the accumulation of free 60S subunits. The combined findings of this study point toward an important role for Rps26 in ribosome assembly and subunit joining; however, the findings are inconsistent with the idea of a specific role of the Y62–K70 segment during translation initiation.In order to gain further insight into the mechanism of Rps26 function and, specifically, into the role of the Y62–K70 segment, we employed yeast as a model organism. Based on published data , we expeIt was previously reported that Rps26 contacts the 5′ untranslated region of mRNA , 21 via rps26a Δrps26b deletions was rescued by the expression of RPS26a from a URA3-based plasmid  complemented the growth of the Δrps26a Δrps26b strain on yeast extract-peptone-dextrose (YPD) medium at 30°C or 40°C in the presence of the translational inhibitor paromomycin, high concentrations of dithiothreitol (DTT), or NaCl. Only at low temperature or if the strains were grown at alkaline pH did the Rps26a5A strain display a moderate growth defect  in strain SC288 (see TEF1 promoter (Tef1Promo) in strain SC306. Mutated versions of RPS26a  Wild-type (WT) RPS26a or versions of RPS26a with the alanine substitutions for conserved amino acid residues (shown in boldface) in the 62-YXXPKXYXK-70 motif were expressed under the control of the TEF1 promoter. YPD plates were cultivated at 30°C or 15°C for 2 days or 5 days, respectively. Download Figure S1, PDF file, 0.2 MB.Drop test analysis of growth phenotypes of yeast strains expressing Rps26 variants with single alanine substitutions within the 62-YALPKTYNK-70 peptide. (A) Wild-type Copyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00109-15.3Figure S2 -myc-tagged Rps26. Wild-type (Rps26) or c-myc-tagged Rps26a (Rps26a::c-myc) was expressed under the control of the TEF1 promoter in S. cerevisiae (rps26a::LEU2 rps26b::TRP1) . Download Figure S2, PDF file, 0.1 MB.Drop test analysis of yeast strains expressing wild-type or C-terminally cCopyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the del9) on the functionality of Rps26. The results of the experiment revealed that Rps26del9 failed to rescue the lethality of the Δrps26a Δrps26b mutation , which shares 63% amino acid sequence identity with yeast Rps26  and Rpl24 (large subunit) by Western blotting, the amount of 18S rRNA  and 28S rRNA (large subunit) by agarose gel electrophoresis, and the area below the 40S and 60S peak curves by polysome profiling  Schematic representation of PCRs with the primers used to check marker gene insertions into RPS26a/b. Annealing areas and direction of amplifications are shown by colored arrows. (B) Agarose gels showing results of PCRs with the wild-type yeast (WT) and S. cerevisiae (RPS26a/rps26a::LEU2 RPS26b/rps26b::TRP1) (strain SC222). Lanes (M) contain molecular mass markers; actual sizes are shown on the left and right sides of the gels. The products of reactions with primer pairs #734/#519, #734/#724, #733/#746, and #733/#521 are shown. The appearance of 1.1-kb and 1.25-kb bands with the marker-specific primer pair #521/#519, as well as the synthesis of 2.37-kb and 2.8-kb bands in addition to the 1.7-kb band for LEU2 and TRP1, respectively, demonstrates correct insertion of the LEU2/TRP1 markers into the RPS26 loci. Download Figure S4, PDF file, 0.1 MB.PCR analysis of the Copyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00109-15.6Figure S5 S. cerevisiae (RPS26a/rps26a::LEU2 RPS26b/rps26b::TRP1)  Tetrad analysis of b strain . (B) MarCopyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 5A, or Rps26-Hs were noticeable but not statistically significant .The recently solved crystal structure of the 40S ribosomal subunit revealed a dumbbell-like fold of the yeast protein, in which peripheral, α-helical regions are joined by a handlelike structure consisting of two symmetrical, antiparallel β-sheets  19). Th. Th19). runcated , and RpsThe molecular mechanism by which Rps26 affects the assembly of 80S ribosomes is currently not understood. One possible role of Rps26 in protein synthesis seems to be linked to its interaction with initiation factor eIF3 , which i5A or Rps26-Hs. This was likely due to specific degradation of small subunits containing mutated or heterologous versions of Rps26. Pathways directed to degradation of defective ribosomes and ribosomal subunits, like ribophagy . Genomic DNA from S. cerevisiae strain D273-10B (TRP1 and LEU2 marker genes. S. cerevisiae strain MH272-3fα (ura3 leu2 his3 trp1 ade2) and the diploid strain MH272-3fα/a (ura3/ura3 leu2/leu2 his3/his3 trp1/trp1 ade2/ade2) (Tables S1 to Cloning was performed in D273-10B  was usede2/ade2)  are the e2/ade2) , pYCplace2/ade2) , pYEplace2/ade2) , YEpTef5e2/ade2) , and pES10.1128/mSphere.00109-15.8Table S2 Table S2, DOCX file, 0.1 MB.Plasmids used in the current study. Download Copyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00109-15.9Table S3 Table S3, DOCX file, 0.1 MB.Primers used for gene cloning/mutagenesis in the current study. Download Copyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00109-15.10Table S4 S. cerevisiae deletion mutants. Download Table S4, DOCX file, 0.1 MB.Primers used in PCR analysis of Copyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli strains were grown in LB medium supplemented with the appropriate antibiotic. Yeast strains were grown on rich medium  or on minimal medium containing 0.67% yeast nitrogen base without amino acids  with 2% glucose (SDex) or 2% galactose . SDex and SGal media were supplemented with the appropriate additives .LEU2 and TRP1 marker genes for RPS26 gene disruptions were amplified with their own promoters and terminators from the S. cerevisiae D273-10B genomic DNA. Mutations rps26a::LEU2 (Δrps26a) and rps26b::TRP1 (Δrps26b) were constructed by replacing nucleotides 19 to 324 of RPS26a or RPS26b with the functional LEU2 or TRP1 gene cassette, respectively. Yeast transformations were performed by the lithium acetate method   isolate (SC254) was selected for subsequent experiments. YCplac33-RPS26a was replaced by pRS313-based plasmids encoding different Rps26 variants via the 5-fluoroorotic acid (5-FOA) method  or with the TEF1 promoter (pRS313-based plasmid p896 and YCplac33-based plasmid p887).For expression in yeast, RPS26a was amplified with primers #721 and #725 (a full list of primers used for PCR is shown in 5A) and the Rps26del9 variant lacking 9 amino acid residues (62-YALPKTYNK-70) were generated by the PCR splicing method . Constitutively expressed RPS26a and del9rps26 with a COOH-terminal c-myc tag were constructed in pESC-His, containing the TEF1 promoter instead of the original GAL1/10 promoter, by exchanging the stop codon for a cysteine codon. Subsequently, the fragments were transferred, together with the TEF1 promoter, into the low-copy-number vector pRS313 using EcoRI/ClaI restriction endonuclease sites (plasmids p1687 and p1692).The human gene coding for Rps26-Hs (pET-15-rps26e) was a generous gift from G. Karpova  . The cod595) were spotted onto YPD, SDex, or SGal plates containing the required supplements. The plates were incubated for the times and temperatures indicated in the legends to the figures in which the results of the experiments are shown.Mutant strains were analyzed on agar plates by the drop test. To that end, 5-fold serial dilutions of overnight agar cultures adjusted to the same optical density at 595 nm , and transferred into 2-ml tubes. Extracts were prepared by vortexing yeast cell suspensions with glass beads in the presence of protease inhibitor cocktail  and 1 mM phenylmethylsulfonyl fluoride (PMSF) using a FastPrep-24 device . Extracts were clarified by centrifugation at 8,000 rpm for 10 min, followed by centrifugation at 13,500 rpm for 15 min at 4°C (Yeast strains were grown overnight in liquid medium to an ODn at 4°C .A260 units in 60 µl) were loaded on top of 90 µl of a 25% sucrose cushion in the lysis buffer and were subjected to ultracentrifugation in a CS 150NX micro-ultracentrifuge  equipped with an S100AT3 rotor at 95,000 rpm for 35 min  were loaded on top of an 11-ml linear 15-to-55% sucrose gradient in the lysis buffer and subjected to ultracentrifugation for 2.5 h at 39,000 rpm . Fractions were collected from top to bottom with a density gradient fractionator monitored at A254  and were subsequently analyzed by Western blotting using specific antisera as indicated in the legends to the figures in which the results of the experiments are shown.For polysome profile analysis, clarified supernatants  were mixed with 80% N,N-dimethylformamide and heated at 65°C for 15 min  antibody , yeast anti-Rpl24 antiserum, anti-Sse1 antiserum, and anti-Rps9 antiserum (Yeast extracts were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate buffer  followedntiserum .


The relationship between immunoglobulin A nephropathy and sarcoidosis is uncommon, however a kidney biopsy and appropriate evaluation for diagnosis of sarcoidosis can be applicable.3 deposits (#2+) and a negative C1q deposition was observed. In light microscopic study, a mesangial proliferation with widening of mesangial area which involved more than 50% of all glomeruli was observed  Mesangial proliferation and mesangial area widening in a glomerulus in the kidney biopsy, b) Immune complex aggregation in endocapillary area.HN is the single author of the manuscript.The author declared no competing interests.Ethical issues  have been completely observed by the author.None.


The unusual photocurrent behavior was observed and directly compared with Fowler’s law for photoemission on metals. The Fowler’s law exponent for the graphene-based nanodiode is almost twice that of a thin gold film based diode; the graphene-based nanodiode also has a weak dependence on light intensity—both are significant evidence for CM in graphene. Furthermore, doping in graphene significantly modifies the quantum efficiency by changing the Schottky barrier. The CM phenomenon observed on the graphene/TiO2 nanodiodes can lead to intriguing applications of viable graphene-based light harvesting.Carrier multiplication  in graphene has been extensively studied both theoretically and experimentally, but direct application of hot carrier multiplication in graphene has not been reported. Here, taking advantage of efficient CM in graphene, we fabricated graphene/TiO The conversion efficiency of photon energy harvesting has been enhanced in various ways, such as tandem structures, bandgap engineering, or plasmonics12348910et al. demonstrated that the carrier–carrier interaction is the predominant process in graphene during energy relaxation of energetic photoexcited electrons at femtosecond scaleet al.Recently, CM in graphene has attracted a lot of theoretical and experimental interest because of its sensitive photon response152 Schottky nanodiodes to utilize CM in graphene as an energy harvesting route by which hot carriers are produced and detected through the Schottky barrier. Hot electrons are energetic electrons that are not at thermal equilibrium with the lattice. The energetic electrons lose their energy through inelastic scattering processes to generate secondary electrons within the mean free path of the electrons2 Schottky nanodiodes and observed that hot electron generation can be enhanced by localized surface plasmons, which increases the quantum efficiency of the device2 nanodiode.In this paper, we fabricated graphene/TiO2 Schottky nanodiode, chemical vapor deposition (CVD)–grown graphene was transferred to an n–type TiO2 layer. The number of graphene layers on the TiO2 was controlled: single (SLG), double (DLG), and triple (TLG) layers. 2 nanodiode, which consists of two electrodes (yellow), a TiO2 layer (green), and a graphene layer (black). The actual size and cross–sectional view of the nanodiode are shown in 2 layers builds the Schottky barrier, which can play the role of hot electron collector. The Ohmic contact is formed at the interface between the TiO2 and Ti  lose their energy through impact ionization before being thermalized and producing phonons. The loss of excess energy from the hot electrons leads to multiple secondary electrons (red dots), which may have enough energy to overcome the Schottky barrier, as shown in 2 can be absorbed in the TiO2 layer (band-to-band excitation). Thus, in this case, the total photocurrent is attributed to the excited electrons  from both graphene and TiO2.For fabrication of the graphene/TiO2 and Ti . Figure 2 Schottky nanodiode. 2; an atomic stick–slip image of SLG on the nanodiode is shown in the inset. The ratio of the intensities of the 2D and G peaks decreases as the number of graphene layers increases. The tendency of the intensity ratio of the 2D to G peaks shows an obvious thickness dependence (i.e. 3.1 (SLG) > 1.7 (DLG) > 0.79 (TLG)) and the 2D peak becomes broad and blue shifted, which are typical behaviors of Raman spectra as the number of layers increases22232 diodes (2526272). The photocurrent density increased as the number of layers of graphene increased because of enhanced absorption of incident light on the multilayer graphene.2 diodes . From th2 diodes . Alterna2 diodes , which aTo detect the generation of hot electrons on the Au and graphene layers, the energy conversion efficiency was measured as a function of the photon energy. The IPCE was fitted to Fowler’s law to confirm the generation of hot electrons from the excitation of valence electrons in the graphene and Au film by incident photons. As the photon energy increased above the Schottky barrier energy, there was an increase in the fraction of valence electrons with energy below the Fermi level that could be excited and travel over the Schottky barrier. The conversion efficiency as a function of the photon energy is given byhv is the photon energy (v being the photon frequency), and ESB is the Schottky barrier height. For most metals, α = 2; other exponents apply to semiconductors26where c is a constant, IPCE × hv) vs. ln(hv − ESB)) that is modified from Fowler’s law to determine the exponent, α. As shown in IPCE × hv) can be characterized by two distinct behaviors (i.e. two different slopes), which are separated by the bandgap energy of TiO2 (Eg). The position of the black vertical arrow corresponds to the bandgap  of TiO2 as the boundary between the two distinct behaviors (i.e. photoemission from thin gold/graphene and from direct interband excitation in the TiO2 layer). It is obvious that band–to–band excitation of TiO2 is dominant for generating photocurrent above the photon energy (i.e. higher than ~2.9 eV), which is close to the band gap of TiO2. On the other hand, for photon energy below ~2.8 eV, the IPCE as a function of the photon energy has a different slope because the generation of hot electrons is the main pathway for producing photocurrent.IPCE × hv) increases as a power law in terms of (hv − ESB)α. We found that α is 2.6 ± 0.26 and 4.2 ± 0.35 for gold and SLG, respectively, for hv < Eg  ∝ (hv − ESB)α where α = 2)262 nanodiode, α = 2.6 ± 0.26 is exhibited for hv < Eg, indicating that the photocurrent was induced from photoexcited electrons in Au. However, in the graphene/TiO2 nanodiode, we observed α = 4.2 ± 0.35, which deviates from Fowler’s law. This deviation implies that additional photocurrent mechanisms can exist in the graphene/TiO2 nanodiode. We also found that the exponent α in Fowler’s law with gapless and linear energy dispersion of carriers like graphene is 3, and the exponent cannot be larger than 3 because the exponent for this case is largely determined by the density of states (DOS) of the system. With parabolic dispersion (i.e. with square-root energy-dependent DOS), we have the usual exponent value of 230In the energy range lower than the bandgap, ( hv < Eg . The maiIPCE × hv) ∝ (hv − ESB)ESB. For hot electrons to travel over the surface barrier, the energy component normal to the 2D plane of graphene must be greater than the Schottky barrier. The escape probability is assumed to be unity if this condition is satisfied regardless of the in-plane electron energy in the graphene layer. It is intuitive to consider only carrier generation and escape steps for carrier transport normal to the 2D plane of graphene. By considering the CM process for hot electrons with a normal component of energy greater than the Schottky barrier, we find the total number of hot electrons n after CM,To quantitatively identify this anomalous behavior, we considered the CM process of hot carriers in graphene. By incorporating the CM process of photoexcited electrons, we determined that (nmax = int(hv−Ez), ε is the in-plane energy, D(ε) is the density of state of graphene, and ∆ is the characteristic average energy loss per multiplication step, which is on the order of the Fermi energy2, we found that n ∝ (hv − ESB)IPCE × hv) as a function of (hv − ESB). The graphene/TiO2 nanodiode exhibits α = 4.2 ± 0.35, which represents the effect of CM in graphene on hot electron generation. where et al.2 Schottky nanodiode, doping modifies the Schottky barrier height. As the Schottky barrier increases (i.e. for hole-doped graphene), the number of hot carriers with energy high enough to overcome the Schottky barrier decreases and therefore the diode has low efficiency for internal photoemission. Even though the quantum efficiency depends on the Schottky barrier height (or doping), we show that the exponent α in Fowler’s equation is independent of the barrier height. Thus, we also confirmed the effect of CM on the graphene/TiO2 nanodiode by investigating the doping effects.The transport possibility for hot electrons generated by the CM process is directly related to the Schottky barrier height. It is expected that as the Schottky barrier height decreases, hot electron transport becomes more efficient because energetic electrons can more easily overcome the lower Schottky barrier. Therefore, lowering the Schottky barrier height can induce an increase in photocurrent, improving the quantum yield in a graphene/semiconductor nanodiode. Also, the Schottky barrier height in the photodiode determines the threshold energy above which the photocurrent is observed by photoemission. CM characteristics can be manipulated by doping  of the graphene. Recently, a change in the CM factor dependent on doping was reported by Johannsen 3) were used for n- and p-type doping, respectively33342 Schottky nanodiode to confirm the change in electrical properties  doping conditions, the Schottky barrier decreased to 0.71 ± 0.01 eV (increased to 0.89 ± 0.08 eV), compared with the pristine graphene/TiO2 Schottky nanodiode at 0.75 ± 0.02 eV. The manipulation of the Schottky barrier height ESB in the graphene/TiO2 Schottky diode is illustrated as a function of doping in ESB after effective impact ionization. Upon the decrease of the Schottky barrier in the TETA-doped graphene/TiO2 Schottky diode, the probability of internal photoemission after impact ionization can be enhanced, as indicated in 3-doped graphene/TiO2 Schottky diode, the increase in the Schottky barrier hinders internal photoemission because more energy is needed for the hot electrons to overcome the higher Schottky barrier, as depicted in To dope the graphene, triethylene tetramine (TETA) and nitric acid (HNOoperties  dependinoperties ). The Sc2 Schottky diodes after TETA and HNO3 doping. Based on the IPCE of the graphene/TiO2 Schottky diodes, the power factor α after doping was obtained by fitting the IPCE to a modified Fowler’s law, as shown in nmax changed after doping, but the density of states remains the same. Therefore, the number of hot electrons  depends on the doping because of the change in ∆ and nmax, but we still have the same energy dependence from the equation (i.e. the same exponent α) because the density of states does not change. We note that additional doping may increase the efficiency of Auger recombination; therefore the IPCE does depend on the doping.2 nanodiode by observing the intensity-dependent photocurrent of the incident light. In When the main mechanism for photocurrent is governed by CM, the exponent in Fowler’s law is independent of barrier height, even though the quantum efficiency is determined by the barrier height. We also confirm the CM effects in the graphene/TiO2 is around 2.9 eV. Therefore, the behavior of the photocurrent as a function of light intensity can represent the origin of the hot electrons, which is associated with the graphene (CM) and the Au film. To distinguish the effect of the graphene (CM) and the Au film, the relationship between photocurrent and light intensity is investigated byIn detail, the intensity dependence of the photocurrent originates from the nature of hot electron generation. The photocurrent from the visible wavelengths is mainly attributed to the hot electrons generated in the graphene and Au film because the bandgap of the TiOn can provide information about each effect of the graphene (CM) and the Au film. The intensity dependence of the photocurrent in the Au/TiO2 nanodiode is much stronger than in the graphene/TiO2 nanodiode, as shown in 2 exhibits very low intensity dependence, whereas the HNO3-doped graphene/TiO2 shows a higher dependence on intensity. The effect of CM is strongly observed at low light intensity2 nanodiode. Our findings indicate that it is possible to detect hot carriers as photocurrent generated by CM in graphene using a Schottky barrier , suggesting that graphene-based photodiodes can work very effectively under a dim light source. A recent report carried out by Kadi F. et al. has theoretically and experimentally dealt with the doping behavior for efficient CM in grapheneThe exponent 2 Schottky nanodiodes and observed CM-induced hot electron generation as photocurrent. The generation of hot electrons by CM in graphene can be observed by directly comparing Fowler’s law for photoemission and the power law behavior of CM. The CM mechanism in the nanodiode is also confirmed from the induced photocurrent in terms of the Schottky barrier height (or doping level of graphene) and the intensity of the incident light. As expected, the exponent in Fowler’s law is independent of barrier height, even though quantum efficiency is strongly related to barrier height. The weak dependence of photocurrent on the intensity of light also indicates strong CM effects in graphene. Thus, based on the light-intensity dependence of the photocurrent, we find that TETA- (HNO3−) doped graphene exhibited strong (weak) CM. We observed that in TETA-doped graphene/TiO2, the photocurrent decreased very slowly as the light intensity decreased, which is different from the behavior of metal-based nanodiodes where the diode current shows a very strong dependence on light intensity. The observed behavior of CM in graphene suggests feasible applications, such as graphene-based ultrasensitive photodetectors, through the detection of hot electrons amplified by CM.In conclusion, we fabricated graphene/TiO2 and graphene/TiO2 Schottky diodes. The details of the device fabrication are described elsewhere41422 is prepared to electrically insulate the silicon wafer and the titanium oxide layer on the Schottky diodes. Then, a 4 × 6 mm, 150 nm-thick film of titanium is deposited onto the silicon oxide through an aluminum shadow mask using electron beam evaporation. To make titanium oxide, the titanium-deposited wafer is then annealed in air at 470 °C for 2 hours, which oxidizes the Ti to TiO2 and produces oxygen vacancies in the TiO2 film, leading to the formation of n-type TiO2. The next step is deposition of a 50 nm film of titanium and then a 150 nm film of gold through a second mask using electron beam evaporation, which constitutes the nanodiode’s two Ohmic electrodes. Finally, for fabrication of the Au/TiO2 nanodiode, a thin gold film (10 ± 2 nm thick) is deposited through a third mask by electron beam evaporation for formation of the Schottky contact between the thin Au film and TiO2. For fabrication of the graphene/TiO2 nanodiode, CVD–grown graphene is transferred onto the TiO2 layer. The SLG is grown by CVD on a 25 um-thick Cu foil . The Cu foil is loaded in a quartz tube furnace, and then heated to 1000 °C under vacuum conditions (8.9 × 10−2 Torr) with a constant flow of H2 (10 sccm) for pre-annealing. SLG is grown by flowing gas mixtures of CH4 (30 sccm) and H2 (10 sccm) under vacuum conditions (3.9 × 10−1 Torr) for 20 minutes. After graphene growth, a rapid cooling step follows under Ar (100 sccm) gas. To transfer the graphene to the TiO2 substrate, the graphene on Cu foil is spin-coated with a PMMA (poly(methyl methacrylate)) supporting layer. After the back of the graphene on Cu foil is etched by O2 plasma treatment , the Cu foil is etched in 0.1 M ammonium persulfate solution. The rinsed PMMA/graphene films are transferred onto the TiO2 substrate and annealed at 70 °C for a few minutes to increase adhesion. The PMMA on the graphene is removed using acetone and isopropyl alcohol. To fabricate the DLG/TiO2 and TLG/TiO2 nanodiodes, this process is repeated two and three times, respectively. The number of graphene layers is confirmed using the ratio of the intensity of the 2D to G peaks in the Raman spectra.To detect hot electron flows, we fabricated Au/TiOI–V) curves were measured by sweeping the voltage between the two electrodes. By fitting the I–V curves of the diodes to the thermionic emission equation, we obtained the Schottky barrier heights, ideality factors, and series resistances of the nanodiodes. The short-circuit photocurrent of the Schottky diodes was measured under illumination by a tungsten–halogen lamp with a normal incidence angle (9 mW/cm2) using a Sourcemeter . The active area for the photocurrent was confirmed by measuring the photocurrent at each position, including graphene/Au, graphene/SiO2, graphene/TiO2, TiO2, and the Au electrode. The effective photocurrent was observed on the graphene/TiO2 interface, as confirmed by For determining the electrical characteristics of the Schottky diodes, current–voltage (−2) corrected by the reference Si photodiode. The IPCE of the nanodiodes was then measured based on the corrected spectra usingThe IPCE data were obtained on the nanodiodes using a PEC–S20. First, the IPCE of the reference Si photodiode was measured to calculate the irradiance of each wavelength. After this, the photocurrent density of the nanodiode was automatically calculated based on the standard solar spectrum (AM 1.5 G–100 mWcmJSC is the short–circuit photocurrent density (Acm−2), Pin is the irradiance intensity at the specific photon energy, and Eph is the incident photon energy.where 3 for n–type and p–type properties, respectively. First, TETA was vaporized on a glass dish at 50 °C for 3 minutes on a hot plate. Finally, the TETA molecules were deposited onto the graphene surface. For HNO3 (63%) doping, the dipping method was used. The graphene/TiO2 nanodiode was dipped in HNO3 (63%) for 5 minutes and dried in air.Graphene was doped with TETA and HNOHow to cite this article: Lee, Y. K. et al. Hot carrier multiplication on graphene/TiO2 Schottky nanodiodes. Sci. Rep.6, 27549; doi: 10.1038/srep27549 (2016).


V are perfectly transmitted even as V → ∞. In this study, theoretical and numerical calculations predict that the transmission probability for an electron wave normally incident to a one-dimensional array of localized scatterers can be significantly less than unity when the electron wavelength is smaller than the spacing between scatterers. In effect, placing periodic openings throughout a potential barrier can, somewhat counterintuitively, decrease transmission in graphene. Our results suggest that electrostatic potentials with spatial variations on the order of the electron wavelength can suppress Klein tunneling and could find applications in developing graphene electronic devices.Graphene's unique physical and chemical properties make it an attractive platform for use in micro- and nanoelectronic devices. However, electrostatically controlling the flow of electrons in graphene can be challenging as a result of Klein tunneling, where electrons normally incident to a one-dimensional potential barrier of height  The wave vectors Since graphene's initial discovery2−28 J-m. In writing One proposed method for controlling and modifying the electronic properties in graphene has been to use superlattice potentials, λ, becomes comparable to the spacing between scatterers, d, non-specular reflection can lead to a dramatic reduction in the transmission through such potentials even at normal incidence. As a result, Klein tunneling in graphene can be suppressed when using a periodic array of localized scatterers.For single or multiple square potential barriers, Klein tunneling is still not suppressed since normally incident waves are only allowed to undergo direct backscattering (specular reflection) from such potentials, which is prohibited due to time-reversal symmetry in graphene. Therefore, to suppress Klein tunneling, electrostatic potentials are required that generate non-specular or diffuse reflection, which is not prohibited by time-reversal symmetry. One such potential that can exhibit diffuse reflection is a one-dimensional periodic array of localized scattering potentials as shown in sr so that the potential for the thn scatterer is given by sr must be greater than the C-C bond length in graphene, i.e., In the absence of an electrostatic potential n where nT is the transmission coefficient for the thn Bragg direction or open scattering channel. The sum in z}+ corresponds to the smallest integer greater than z, and {z}− corresponds to the largest integer less than z.As derived in nR is the reflection coefficient for the thn open scattering channel in nR and nT are given in Likewise, the reflected wave function sr = 20 nm as a function of V0 = 0.8 eV and d = 150 nm, (green) V0 = 0.2 eV and d = 150 nm, and (blue) V0 = −0.9683 eV and d = 156.5 nm. For reference, totT = 1 is represented by a red line, which is the exact transmission probability for a wave normally incident to a constant one-dimensional potential barriertotT = 1 for all V0 since only the n = 0 scattering channel is open, i.e., totT = 1. This can also be understood by the fact that when totT = 1 for n ≠ 0 in totT ≤ 1. Although additional open scattering channels are now available for the incident electron wave to scatter into when totT depends on V0. For example, totT decreased to 0.6277 at V0 = 0.8 eV and d = 150 nm :thl-partial wave t–matrix operator, maxl is the maximum number of partial waves that are included in the calculations, n, and either x > 0] or reflected [x < 0] from the scattering array along with an infinite number of waves nT in nR in nT and nR are given in The theory for scattering of a massless Dirac plane wave spinor from a one-dimensional array of localized cylindrically symmetric scatterers  is derivSupporting Information28293031on Tn in  for  andon Rn in  for  coeV0 and radius sr. For an individual scatterer, the thl partial wave scattering amplitude is given by28lJ(z) and l, respectively. The maximum partial wave used in the calculations, maxl, was chosen to take into account 99.9% of the total scattering amplitude for an individual scatterer, i.e., totT in a 2DEG [In all simulations, each scatterer was modeled as a cylindrically symmetric barrier/well of potential J.D.W. performed theoretical and numerical calculations and wrote manuscript. D.H. worked on initial simulations for the project and helped in manuscript preparation.Supporting Information for


Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by the occurrence of visceral and neurological symptoms. At present, the molecular mechanisms causing neurodegeneration in this disease are unknown. Here we report the altered expression and/or mislocalization of the TAR-DNA binding protein 43 (TDP-43) in both NPC mouse and in a human neuronal model of the disease. We also report the neuropathologic study of a NPC patient’s brain, showing that while TDP-43 is below immunohistochemical detection in nuclei of cerebellar Purkinje cells, it has a predominant localization in the cytoplasm of these cells. From a functional point of view, the TDP-43 mislocalization, that occurs in a human experimental neuronal model system, is associated with specific alterations in TDP-43 controlled genes. Most interestingly, treatment with N-Acetyl-cysteine (NAC) or beta-cyclodextrin (CD) can partially restore TDP-43 nuclear localization. Taken together, the results of these studies extend the role of TDP-43 beyond the Amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD)/Alzheimer disease (AD) spectrum. These findings may open novel research/therapeutic avenues for a better understanding of both NPC disease and the TDP-43 proteinopathy disease mechanism.The online version of this article (doi:10.1186/s40478-016-0325-4) contains supplementary material, which is available to authorized users. NPC1 (95 % of patients) or NPC2 genes, encoding two proteins involved in the intracellular trafficking of cholesterol and other lipids. The deficiency of either protein leads to the accumulation of endocytosed unesterified cholesterol, gangliosides, and other lipids within the lysosome/late endosome compartment  is an autosomal recessive lysosomal storage disorder due to mutations in The clinical presentation of the disease is variable, and the age at onset ranges from the perinatal period to adulthood. The disease is typically characterized by visceral and neurological symptoms. Apart from a small group of patients presenting with a severe perinatal form, leading to death within the first 6 months of life due to liver or respiratory failure, most patients develop a progressive neurological disease. Indeed, NPC has been classically classified on the basis of the age at onset of the neurological symptoms, irrespective of the age of first symptoms .Neuropathological features include meganeurite formation, extensive growth of ectopic dendrites, formation of neurofibrillary tangles, neuroinflammation and neuroaxonal dystrophy . In advaRecently, it has been suggested that synergistic mechanisms, between pathological proteins involved in neurodegeneration , may help explain copathologies and individual diversity that are often detected in neurodegenerative conditions , has emeAt the pathological level, it is now very clear that both gain of toxic function of cytoplasmic aggregates and loss of TDP-43 function in the nucleus may contribute to disease pathogenesis , 14. WhaC9orf72 gene, encoding a protein involved in the regulation of the endosomal trafficking .Brain tissue was obtained from a 61 year-old man who died after a 30-year history of a clinically undiagnosed, familial, neurological disorder. Molecular genetic testing of DNA extracted from frozen brain autopsy tissue  revealedAll procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed, written consent was obtained from the patients (who underwent skin biopsies) included in this report.Fibroblasts were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10 % fetal calf serum (FCS) and penicillin/streptomycin, in a humidified atmosphere containing 5 % CO2 at 37 °C.Multipotent adult stem cells were obtained from fibroblasts at early passages (SKIN-MASCs) from 4 healthy donors and 4 patients affected by NPC disease, as previously described . The sur2 into plates (Corning) or on coverslips. The differentiation protocol was previously described by Bergamin et al. [SKIN-MASCs obtained after 3 passages in selective medium, were seeded at a density of 8000 cells/cmn et al. . Cells wNPC neuronally differentiated cells were treated with vehicle or either 100 μΜ M N-acetyl cysteine  or 400–800 μM 2-hydroxypropyl-b-cyclodextrin  for 24 h.Npc1+/+ and Npc1−/− mice of postnatal days 11, 28 and 75  were deeply anesthetized and then transcardially perfused with 4 % paraformaldehyde (PFA) in PBS. Brains were removed, and processed for immunohistochemical studies, according to a previously published protocol [protocol , TDP-43 For immunofluorescence assays cells were fixed for 15 min in 4 % (w/v) paraformaldehyde in PBS and then permeabilized in 0.3 % Triton X-100 in PBS for 5 min on wet ice. After blocking with 2 % BSA in PBS, cells were incubated overnight at 4 °C with the primary antibodies raised against NeuN (Millipore), tubulin b3 , TDP-43 (Proteintech) Phospho-TDP-43 (Ser409/410 Sigma Aldritch), TIA-1 (Santa Cruz), Lamp-1 (Santa Cruz) and p62 (MBL). Cells were then washed and incubated with Alexa Fluor 555 or 488 labeled secondary antibody for 1 h at 37 °C. Cell nuclei were stained by Vectashield Mountain Medium with DAPI .Images were obtained with a live cell imaging dedicated system consisting of a Leica DMI 6000B microscope connected to a Leica DFC350FX camera .Nuclear and cytoplasmic fractions were prepared as previously described , with feEqual amounts per lane of cytoplasmic and nuclear protein fractions were loaded on a 4-20 % gradient Mini-Protean TGX pre-cast gel for electrophoresis (Bio-Rad Laboratories) while total extracts were electrophoresed on 10 % SDS–PAGE. Fractionated proteins were transferred to poly (vinylidene difluoride) membranes (Roche Diagnostics) or nitrocellulose membrane , respectively. After blocking, membranes were incubated overnight at 4 °C with the primary antibody anti-TDP-43 (Protein Tech), then washed, incubated with the appropriate secondary antibody for 1 h at RT and developed with SuperSignal West Dura reagents . Sp1 and β-III tubulin were used to assess the purity of nuclear and cytoplasmic fractions, respectively, and as reference for TDP-43 expression quantification. Total expression of TDP-43 was normalized by actin levels. Blots were quantified by using a Gel Doc 2000 videodensitometer .Total RNA was extracted from neuronally differentiated cells using the RNeasy Mini RNA extraction kit (Qiagen) according to the manufacturer’s protocols. First strand cDNA synthesis was performed with 1ug RNA using random hexanucleotides (Invitrogen) and SuperScript III reverse transcriptase (Invitrogen). Quantitative RT-PCR was carried out in 15 μl reaction volume containing 1-3ul of obtained cDNA, SsoAdvanced Universal SYBR Green Supermix (BioRad) and gene-specific sense and anti-sense primers. HPRT was used as an internal control for normalization. Primer sequences are listed in Additional file Quantitative RT-PCR was performed in a Roche 480 LightCycler Real-Time PCR System, following the manufacturer’s instructions. LightCycler 480 Basic software (Roche) utilized the second derivative maximum method to identify the crossing point (Cp).Neuropathologic studies of brain and spinal cord were carried out in order to characterize NPC in human brain tissues. Samples of fresh brain tissue were frozen and stored at −80° C. The remainder of the brain and spinal cord was fixed in 10 % buffered formalin. Fixed tissue sections were obtained from multiple brain and spinal cord regions and processed for histology and immunohistochemical studies, according to a previously published protocol . TDP43 ip < 0.05 was considered as statistically significant.Statistical significance was determined by Student’s t-test, using the SPS software. wild-type and Npc1−/− mice during maturation and at adult age were investigated by immunohistochemistry and western blot, respectively  day 11, when neuropathological features of the disease had not yet appeared. Because the development of cerebellar cortex is still in progress at this age, three distinct granule neurons (GN) populations, consisting of proliferating, postmitotic and migrating GNs can be easily identified. Proliferating GNs localize in the outer part of the external granular layer (oEGL) whereas postmitotic GNs stop in the inner part of the EGL (iEGL) before migrating inward through the molecular layer (ML) to reach the internal granular layer (IGL), thus representing the migrating GNs. Despite a reduction of TDP-43 expression level in Npc1−/− mice, EGL-residing GNs of both wild-type and Npc1−/− mice expressed higher levels of this protein compared to IGL-residing GNs. This may indicate a burst of TDP-43 expression in concomitance with GN exit from the cell cycle. A significant reduction of TDP-43 expression in Npc1−/− compared to wild-type mice was also observed in Purkinje cells (PCs)  and confirming the nuclear localization of TDP-43 .The expression and subcellular localization of TDP-43 in the cerebellum of ely Fig. , b. The Cs) Fig. . In bothwild-type and Npc1−/− PN28 mice  of patients affected by NPC. As we have previously shown, this neuronal model has several key features of NPC and is therefore valuable for studying the effects of NPC dysfunction at the cellular level .The levels of both mRNA and protein TDP-43 expression in NPC cells were not significantly different from the levels detected in cells derived from normal controls . Therefore, we first evaluated whether SGs are present in cells by immunofluorescence of differentiated cells derived from healthy controls and NPC patients using the SG marker TIA-1. While in cells derived from healthy controls TIA-1 is completely localized within the nuclei, in about 64 % of NPC cells it was also detected in the cytosol Fig. , b, c. FFAP2A, CNTFR, MAF2D, MADD, TLE1, and TNIK) were differentially expressed in cells derived from NPC patients compared to healthy controls.In order to investigate the possible functional impact of TDP-43 mislocalization on RNA metabolism we then analyzed the mRNA expression of a set of genes known to be regulated by TDP-43 and involved in neuronal survival and differentiation. The expression levels of nine genes were analyzed by real-time Q-PCR comparing normal vs. NPC-derived cells Fig. . ImportaSince SGs are highly dynamic structures that disassemble when the stress stimuli vanish, we also analyzed whether the intracellular localization of TDP-43 could be restored by treating NPC cells with N-Acetyl-cysteine (NAC), a well known anti-oxidant agent, or with 2-hydroxypropyl-b-cyclodextrin (CD), a cholesterol sequestering agent that mobilizes cholesterol from the late endosomal/lysosomal compartment. As shown in Fig. NPC1 gene  and IVS14-2A > G in the Atrophy was prominent in cingulate cortex and cerebellum. Neurons with swollen perikarya contained a pale granular material, a finding consistent with what is observed in storage diseases. Evidence of neuronal storage was noted in the cerebral cortex, hippocampus, amygdala, several deep telencephalic and brain stem nuclei as well as in the spinal cord. Tau-immunoreactive neurons and neurofibrillary tangles were present in hippocampus and entorhinal cortex. Tau-immunopositive threads were also noted in the spinal cord. Amyloid-β deposits were not present.The normal nuclear pattern of TDP-43 immunopositivity was confirmed in multiple brain areas, including the cerebellar dentate nucleus Fig. . HoweverIn recent years, lysosomal storage diseases have become increasingly associated with neurodegeneration and cancer . SeveralMore recently, impaired autophagy and lysosomal dysfunction have also been linked with alterations at the level of RNA metabolism . Indeed,In this work, we have shown aberrant expression and cytoplasmic distribution of TDP-43 in the cerebellum of a mouse model of NPC and in an in vitro human NPC neuronal model system in which the cytoplasmic TDP-43 is sequestered by stress granules. This TDP-43 redistribution and sequestration may represent an important link between neurodegeneration and normal biological processing, as affected by mutations, other disease processes, or prolonged environmental stress –41. In aIn contrast to previous studies showing that p62 co-localizes with TDP-43-positive cytoplasmic inclusions in patients with FTLD-MND, we did not observe co-localization of TDP-43 and p62 in NPC cells. This finding may represent a feature specific to NPC pathology. A previous study showed that even though p62 co-localizes and physiologically interacts with TDP-43, the p62 in the cerebral cortex of FTLD-TDP patients co-immunoprecipitated a smaller amount of TDP-43 as compared with normal brains . This obBesides accumulating cholesterol within the lysosomal compartment, NPC cells are characterized by oxidative stress, which may contribute to disease pathology , 44. A rBased on the reported data, it is possible to hypothesize that in NPC, chronic stress conditions (including oxidative stress) may trigger the translocation of TDP-43 from the nucleus to the cytosol where it is phosphorylated and recruited within stress granules. As a consequence, its function in the regulation of RNA processing may be impaired.The presence of TDP-43 pathology has also been observed in brain tissue obtained from a patient affected by NPC. This result is in line with the findings observed and in the human neuronal model of the disease. However, it must be interpreted with caution and confirmed by analyzing brain tissue from additional NPC patients.NPC−/− mouse and in NPC human cells/tissue, the results obtained in mice do not exactly reflect the findings in human samples, suggesting that NPC−/− mouse would not be the best model of TDP-43 pathology in NPC. The obtained results are not surprising considering that some characteristic features of NPC human neurons, such us the high degree of ectopic dendritogenesis and the presence of neurofibrillar tangles [Although TDP-43 pathology was observed in both  tangles , 46, areFinally, it is worth noting that in NPC cells, TDP-43 showed a diffuse cytoplasmic distribution, but no well-formed aggregates. Recently, a similar pattern of TDP-staining was described by Van Deerlin et al., in anterior horn cells and other neurons in brains from ALS patients. It was suggested that this TDP43-diffuse, often stippled or granular distribution represents a “pre-inclusion” state .The reported findings are significant for several reasons. First, they potentially expand the presence of TDP-43 proteinopathies  beyond tIn conclusion, these results extend the importance of the role of TDP-43 in neurodegenerative disease and further highlight the need to prioritize the targeting of this protein to develop novel therapeutic strategies. Furthermore, our results suggest that metabolic diseases which involve neuropathological consequences could represent a novel field of study to search for alterations in RNA binding proteins, especially those that are already described to play a role in motorneuron diseases. The reason is that both the differences and similarities observed in the expression or regulation of these proteins in very different cellular disease contexts, might help us to pinpoint the basic functional alterations that trigger neurodegeneration. In addition, confirming their eventual presence in a particular metabolic disease will also represent an advantage with regards to future therapeutic strategies. In particular, it is to be expected that novel therapeutic strategies that show promise in one type of disease might also prove to be very beneficial also in other disease contexts.


Delia radicum and its three major parasitoids: the two staphylinid beetles Aleochara bilineata and A. bipustulata and the hymenopteran parasitoid Trybliographa rapae. For each species, two populations from Western France were sampled and microbial communities were described through culture independent methods (454 pyrosequencing). Each sample harbored at least 59 to 261 different bacterial phylotypes but was strongly dominated by one or two. Microbial communities differed markedly in terms of composition and abundance, being mainly influenced by phylogenetic proximity but also geography to a minor extent. Surprisingly, despite their strong trophic interaction, parasitoids shared a very low proportion of microbial partners with their insect host. Three vertically transmitted symbionts from the genus Wolbachia, Rickettsia, and Spiroplasma were found in this study. Among them, Wolbachia and Spiroplasma were found in both the cabbage fly and at least one of its parasitoids, which could result from horizontal transfers through trophic interactions. Phylogenetic analysis showed that this hypothesis may explain some but not all cases. More work is needed to understand the dynamics of symbiotic associations within trophic network and the effect of these bacterial communities on the fitness of their hosts.All animals are infected by microbial partners that can be passengers or residents and influence many biological traits of their hosts. Even if important factors that structure the composition and abundance of microbial communities within and among host individuals have been recently described, such as diet, developmental stage or phylogeny, few studies have conducted cross-taxonomic comparisons, especially on host species related by trophic relationships. Here, we describe and compare the microbial communities associated with the cabbage root fly  T. T34]. Tematodes . Dependinfection . In manyd others .i.e. an insect and its three main natural enemies. The cabbage root fly Delia radicum (Diptera: Anthomyiidae) develops in the roots of brassicaceous crops. It is mainly attacked by three solitary parasitoids: two coleopteran staphylinids (Aleochara bilineata and A. bipustulata) and the parasitoid wasp Trybliographa rapae  or or11] orha rapae . Both Als adults , 52]. W. Wi.e. aus flies . The parasitoids .Since the bacterial communities present in the food and the environment are supposed to influence insect microbiota we expected that the bacterial communities of parasitoids exploiting the same host might be especially alike as a result of sharing the same kind of food and microenvironment during development. On the other hand, phylogeny, the level of host specificity or geography could also influence which bacterial partners are found.Wolbachia, Rickettsia and Spiroplasma are a very different case. For them, host/parasitoid trophic interactions represent a gate to colonize more species during exceptional horizontal transfers. For example, laboratory experiments have shown that parasitoids are a possible transmission route for facultative endosymbionts in aphids . F. F64]. FRepresentative sequences of OTU corresponding to bacteria known to be vertically transmitted and shared by at least two species of our sample were then aligned with mafft 6.822 using default parameters  and theiMaximum Likelihood (ML) trees were built for Rickettsia spp., Spiroplasma spp. and Wolbachia spp. First, we used the Akaike information criteria corrected for small sample sizes  to selecDelia radicum, Aleochara bilineata and A. bipustulata were more precisely characterized as follows. For each DNA pool (two pools per species i.e. one per population), two independent PCR took place using the same PCR reaction mixtures and PCR parameters. In the first one, a 415bp fragment of the bacterial 16S rDNA gene was amplified using primers W-Specf—CATACCTATTCGAAGGGATAG—3’ and W-Specr: ACGTTCGAGTGAAACCAATTC—5’ , r, r76], rid wasps  even thooperties . This dioperties . On the d genera . Some rad genera . Howeverd genera . Some cod genera . In thisronments .Wolbachia sp. dominated D. radicum bacterial communities and was also detected in all but one sample (eastern T. rapae) in the three other species. This Alphaproteobacterium is the most frequent symbiont in insects and it also infects a large number of arthropod species, often manipulating the reproduction of its host , . T. TWolbacc fungus . These bect guts . Another tracts; , plant h tracts;  and ants tracts; . The rolymbionts .A. bilineata (8/37), 25% for D. radicum (5/20), 33% for A. bipustulata (11/33) and 38% for T. rapae (29/76) were shared by the two populations sampled, giving an idea of the core microbiota of each species. In the same way, only a few OTU were shared between at least two species and the comparison of bacterial communities based on their UniFrac distance clustered as follows: (i) the two replicates of each sample, demonstrating the reproducibility of the sequencing method used, then (ii) samples from the same species and finally, (iii) Aleochara sp. samples, showing that the main environmental filter influencing the microbial communities in our samples is probably phylogenetic proximity. Host phylogeny has already been shown to explain much of the variation found in insect bacterial communities , b, bA. bilelopment . It coulAleochara species is interesting because A. bilineata samples were directly sampled in fields and were killed as soon as they emerged from their host while A. bipustulata individuals were adults sampled from a rearing cage and came from a stock reared in the laboratory for more than ten generations using eastern D. radicum as a host. Two opposite hypotheses could explain such a similarity between two bacterial communities: (i) the two Aleochara species share a similar core microbiota in the wild and rearing them in the laboratory does not modify it significantly, or (ii) the generalist A. bipustulata has a microbiota markedly different from that of the specialist A. bilineata in the wild, but this community was modified by rearing it on D. radicum pupae only, leading to a "specialist diet". The lack of freshly wild-captured A. bipustulata samples in our study does not allow us to settle this case, but the potential of laboratory rearing to modify microbiota is known. For example Lehman et al. [Poecillus chalcites. Temperatures in the laboratory are also more regular and most of the year higher than those experienced in the fields sampled, which can raise the densities of several facultative or obligate endosymbionts like Wolbachia [Buchnera [The proximity between communities found in the two n et al.  found tholbachia  or BuchnBuchnera . AccordiWolbachia, the combining of 16SrDNA (W-Spec) and fbpA sequences allows to describe with confidence at least three Wolbachia variants, two from supergroup A and another from a distinct supergroup, possibly B. One variant from supergroup A is found in D. radicum while the other A-group Wolbachia is found in its specialist parasitoid A. bilineata. The two variants are very closely related but not identical. Accordingly, horizontal transfer might have occurred between host and parasitoid, but if so it was not a recent one. On the other hand, the variant found in A. bipustulata appears related to supergroup B , Trybliographa rapae (B), Aleochara bilineata (C) and Aleochara bipustulata (D).Rarefaction curves showing the number of detected MOTUs as a function of the number of high quality reads in  Coding of populations sampled and homology thresholds : see legend included in the figure.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Flower-like palladium nanoclusters (FPNCs) are electrodeposited onto graphene electrode that are prepared by chemical vapor deposition (CVD). The CVD graphene layer is transferred onto a poly (PEN) film to provide a mechanical stability and flexibility. The surface of the CVD graphene is functionalized with diaminonaphthalene (DAN) to form flower shapes. Palladium nanoparticles act as templates to mediate the formation of FPNCs, which increase in size with reaction time. The population of FPNCs can be controlled by adjusting the DAN concentration as functionalization solution. These FPNCs_CG electrodes are sensitive to hydrogen gas at room temperature. The sensitivity and response time as a function of the FPNCs population are investigated, resulted in improved performance with increasing population. Furthermore, the minimum detectable level (MDL) of hydrogen is 0.1 ppm, which is at least 2 orders of magnitude lower than that of chemical sensors based on other Pd-based hybrid materials. Therefore, hydrogen sensors that have high sensitivity, rapid response, and reversibility are required to detect and/or monitor minute hydrogen leakages in industrial applications32) films meet these demand but require an operating temperature of over 200 °C, which increases the overall power consumption of the sensing device56Hydrogen H gas is u2 molecules are selectively adsorbed onto the surface of Pd by dissociation into hydrogen atoms (H2→2H), and diffused into the interstitial sites of Pd structure. As a result, the phase of Pd transfer a solid solution of Pd/H (α-phase) and a palladium hydride (β-phase), resulted in resistance changes at room temperature. However, materials based on Pd is susceptible to structural changes (such as vacancy and dislocation), which are increased during the phase transition of Pd (α to β) that occurs at hydrogen concentration higher than 2%, causing have been known to collapse during the sensing reaction due to an irreversible phase change8910111213Palladium (Pd) is an attractive candidate to replace metal oxides, because HThe shape control of metal nanostructure is important factor to enhance the activity and stability172-hybridized carbon atoms, has attracted as a substrate materials. Among these graphene substrate, CVD graphene is one of promising substrate owing to its unique physical properties , and excellent mechanical strength2324et al. fabricated flexible hydrogen gas sensors using CVD graphene decorated with Pd nanoparticles via electrodepositionet al. synthesized CVD graphene on SiO2/Si decorated with thin Pd film using electron beam evaporationGraphene, a two-dimensional materials with honeycomb structure composed of single-layer sheet of sp2 gas is both sensitive and reversible, and is attributed to the more active site from flower-like shape of the Pd nanostructures and the high carrier mobility of the underlying graphene. The minimum detectable level (MDL) of H2 is as low as 0.1 ppm, which is considerably lower than that of other chemical sensors based on carbon-based palladium composites. To our knowledge, no previous reports have described the electrodeposition of flower-like metal nanoclusters on CVD graphene.Herein, this study demonstrates a simple strategy for fabricating flower-like palladium nanoclusters on CVD graphene (FPNCs_CG) electrode through the electrodepostion. The flower shapes are formed by modifying the graphene surface with 1,5-diaminonaphthalene (DAN). The population of FPNCs is well-controlled by adjusting the DAN concentration in the functionalization solution. Furthermore, flexibility of electrode can be obtained through transfer of CVD graphene onto PEN film. The resulting FPNCs_CG electrode films are used as signal transducer for the detection of hydrogen gas. The response of these electrode toward H4) as carbon source and hydrogen (H2) as a catalyst28−1) generates after the DAN treatment, owing to the disorder of the graphene basal plane. Furthermore, The I2D/IG intensity ratio of surfaces that is untreated with DAN (ca. 3.7) is higher than that of DAN-treated surfaces (ca. 1.8). This change suggested that charge impurity or rippling on the surface cause an inhomogeneous charge distribution and electron-donating containing aromatic molecules induced a ratio decrease due to the doping effect. Additionally, the upshift of 2D and downshift of G band wavenumber can be observed by electrical gating, owing to the dynamic effect of carrier population3032−1, corresponding to C-N stretching vibrations of primary amino groups, and at 3414 and 3322–3227 cm−1, corresponding to asymmetric and symmetric N-H stretching of aromatic primary amino groups, respectively342+ ions react with numerous functional group on the graphene surface induced by moisture, oxygen, and fabrication process, resulted in evenly spread hexagonal pyramid structure  are then deposited on the graphene surface. With increasing electrodeposition time, flower-like palladium nanoclusters (FPNCs) are formed from these Pd NPs as nucleation sites (3742−) in sulfuric acid theoretically prohibit the growth of Pd at the adsorption sites. Furthermore, this effect is maximized on the Pd (111) plane, which is preferred adsorption plane for SO42− ions. To confirm the influence of sulfate ions, sulfuric acid as electrolyte is prepared with various concentration (0.01 and 0.1 M) as shown in 2SO4, due to the uniformly hindrance of the growth of (111) plane on the Pd surface. Adversely, low concentration induce to the partial hindrance, resulted in more sharp structure. This result indicate that Pd is predominantly composed of (111) plane and the growth of Pd can be controlled by sulfate ions concentration.tructure 36. Conveon sites . FPNCs fca. 300 nm in size and compose of numerous needles of hexagonal pyramids  face-centered-cubic (fcc) Pd1617181920FE-SEM micrographs of FPNC_CG surfaces with various populations of FPNCs are shown in pyramids  with ca.n length . The HR-I–V) curves of the FPNCs_CG films and indicates that the FPNCs are in ohmic contact with graphene surface. Contact resistance increases slightly with increasing of the FPNCs population, implying that the FPNCs acts as impurity for decrease of quality. However, this slight change in contact resistance may be ignored in practical applications due to the overall high conductivity of the FPNCs_CG films.θ = 24°, suggesting that the single layer of CVD graphene is highly crystalline , (200), (220), and (311) reflections of the fcc lattice. Energy Dispersive X-ray (EDX) spectra revealed that the FPNCs are composed of Pd and carbon elements, implying that the FPNCs are deposited on the graphene surface (2+ ions are completely reduced to Pd0 during the electrodeposition process (Powder X-Ray Diffraction (XRD) of the FPNCs_CG films contains an intense and sharp peak corresponding to graphitic carbon (002) at about 2stalline . XRD spe surface . The che process .2 atmosphere at room temperature. The resistance change, ΔR, of the electrode is monitored during sequential or periodic exposures to H2. ΔR/R0 (sensitivity) is defined as the percent resistance change upon exposure to a gas with a fixed concentration of hydrogen and is calculated as follows:R0 is the resistance of the sensor exposed to dry air and R is the maximum resistance after exposure to a gas containing hydrogen. When the FPNCs_CG is exposed to hydrogen gas, the phase of the FPNCs is transfered from palladium (Pd) to palladium hydride (PdHx). Consequently, decreasing of the work function is beneficial to the flow of more electrons, resulted in decreasing of the resistance  is lower than that of FPNCs_CG_L (10 ppm). This is likely because higher populations of FPNCs provide more reactive sites for hydrogen atoms. Furthermore, the detection limit of FPNCs_CG_H is three orders of magnitude lower than that of noble metal/graphene based electrodes  as a function of hydrogen concentration is shown in 2 flow; humidity: at below ca. 10%), however, the humidity was important factor for sensitivity and response/recovery time. The sensing performance was measured at wet air  to investigate the effect of humidity. The resistance of FPNCs_CG electrodes decreased at both water drop and bubbled water, implying that water obviously influenced the resistance (4445The electrodes were operated in dry air (using only Nsistance . When thsistance . This is2 gas compared to nitrogen dioxide (NO2) and ammonia (NH3) gas 4748492 and NH3 were lower than that of hydrogen gas, due to the different sensing mechanism of hydrogen (phase transition), indicating that FPNCs_CG based sensor show the high selectivityThe FPNCs_CG based sensors also exhibited high selectivity for HNH3) gas . When thecreased . On the tendency . This isca. 10 mm. 2 exposure  of 0.1 ppm H2. This report describes an effective method for the fabrication of flower-like metal-graphene composites with population control for various flexible electrochemical applications.In conclusion, flower-like palladium nanoclusters (FPNCs) are electrodeposited on CVD graphene (FPNCs_CG) electrode. The shape and population of FPNCs can be controlled by modifying the graphene surface with DAN. The phenyl groups of DAN interact with the graphene surface v2), and sulfuric acid (H2SO4) were purchased from Aldrich Chemical Co. and used as received.1,5-diaminonaphthalene (DAN), palladium (II) chloride , respectively. Finally, the graphene was transferred to a flexible polyethylene naphthalate (PEN) film and dipped into the acetone solution at 60 °C for 2 hr to remove the PMMA.Graphene was synthesized by chemical vapor deposition (CVD) on copper (Cu) foil in a CH2 into 100 mL of 0.05 M H2SO4. Prior to electrodeposition, as-prepared CVD graphene piece (1.5 cm × 1.5 cm) was immersed in a methanolic solution containing a certain concentration  of DAN  for 30 min. The concentration of DAN was used as the variable in controlling the population of deposited FPNCs. The electrodeposition process was conducted with a three-electrode system: the DAN-treated CVD graphene was used as the working electrode with a Ag/AgCl reference electrode and a Pt foil counter electrode. Pd was deposited by applying a constant potential of −0.1 V for 10 min. Then, the gold electrode was deposited on the FPNCs_CG by thermal evaporation.Electrolyte for the electrodeposition of FPNCs was prepared by dissolving 0.1 M PdClField Emission Scanning Electron Microscope (FE-SEM) images were obtained using a JEOL 6700 instrument. JEOL JEM-200CX and JEOL JEM-3010 instruments were used for Transmission Electron Microscopy (TEM) and High-Resolution Transmission Electron Microscopy (HR-TEM), respectively. Samples were dispersed in ethanol and cast onto perforated carbon grids. X-ray Photoemission Spectroscopy (XPS) and X-Ray Diffraction (XRD) experiments performed on a JPS-9000MS  and M18XHF-SRA  instruments, respectively. RAMAN spectra were recorded on an FRA 1106/S FT-Raman (Bruker) spectrometer and excited with a 514-nm Ar laser. Samples for RAMAN spectroscopy were prepared on silicon-oxide-treated silicon wafers. The electrical properties and sensing performance of the FPNC_CG films were investigated using a current-source meter (Keithley 2400). I–V characteristics were determined using a WBCS 3000 potentiostat (WonA Tech). Fourier-Transform InfraRed (FT-IR) spectra were acquired using a Frontier FT-IR spectrometer (Perkin Elmer Inc.).2) gas at various concentrations (0.1–100 ppm) was introduced into the chamber using a mass flow controller . The real-time resistance was measured at a constant applied current of 10−4 A. After exposure, the H2 gas was removed by blowing across the FPNC_CG film with compressed air (N2). This process was repeated several times. H2 gas/N2 air mixtures were supplied at various flow rates of 1–5 sccm and 2–8 slm using a MFC controller.Resistance changes in the FPNC_CG films were monitored with a source meter connected to a computer. The FPNC_CG sensors were placed in a vacuum chamber within a vapor inlet/outlet pressure of 100 Torr. Hydrogen .


We theoretically study the effective third-order nonlinear response and optical bistability of the 3D graphene based composite consisting of graphene wrapped dielectric nanoparticles embedded in dielectric host at terahertz frequencies. Taking into account the nonlinear conductivity of graphene, we derive the analytical expressions for the effective third-order nonlinear coefficient  Graphene is a two-dimensional hexagonal crystal carbon sheet with only single layer of atoms, which has recently attracted enormous interest for its outstanding optical properties123567891011121516181920212223242627One remarkable feature of graphene is its nonlinear properties which have already been considered both in theory and experiments30313233343536384132Although graphene is employed not only for planar geometries, it is possible now to create graphene wrapped objects experimentally44a are randomly embedded in the linear dielectric host as shown in 4445482234ε and εh, respectively. Assuming the electric displacement vector Dn inside (n = c) and outside (n = h) the sphere have linear relation with the electric fields En, so that Dn = εnEn. In the case when a is far less than the wavelength of the incident light, we can adopt the so-called the quasi-static approximation. Hence the electric potentials both inside and outside the spherical particles would satisfy the Laplace equation: We consider a random composite in which graphene wrapped spherical dielectric nanoparticles with radii ϕh, we have Es is the scattering field in the host. E0 is the amplitude of the external applied field and A, B are the unknown coefficients to be determined with the appropriate boundary conditions. For this monolayer graphene coated dielectric sphere in which the graphene layer is considered as an extremely thin conducting shell with conductivity σ, we employ the non source-free boundary conditionsHere, the electric potential inside the particles includes the incident part and induced one, i.e., ρ is the surface density of charge which has the relation ω. Here, the operator ∇s stands for surface divergence and σ.where Solving the above equations, one yields the unknown coefficients,B is dipole polarizability of the coated sphere. According to the Clausius-Mossotti relation, we can get the effective dielectric coefficientwhere N is the numbers of spherical particles per volume. Substituting B into the in which where In the weakly nonlinear limit, to derive the third-order coefficient of the composite, it is convenient to introduce the macroscopic effective linear dielectric constant Next, we rewrite σ0 = σintra + σinter, σintra and σinter are the intraband and interband terms which have the following formsin which the linear term n2D on the gate voltage, τ is the electron-phonon relaxation time, and T is the temperature in K. e, kB and σ3 can be expressed as32In the above equation, In THz frequencies, the nonlinear term of the surface conductivity of graphene is much weaker, i.e., where Ec and outside the sphere Eh there is a relationOn the other hand, in the composite system, for the local electric field inside the sphere E0 light upon the composite and Ec, one may introduce an average field theory for the composite,To derive the relationship between the external field Therefore, we can finally derive the following relationσ3, we obtainIn the weakly nonlinear limit, substituting Ec in It is sufficient to only retain the first term in By comparing andEc inside the particles as the function of E0. Taking the square of modulus of In what follows, we would like to check the strong field case. Generally, T = 300 k), hence the linear part of the surface conductivity in For frequencies εh and ε are pure dielectric and have no dissipation, we can alternatively rewrite In the case that by defining new dimensionless variables In order for Thus the positions of the local minimum (maximum) a and permittivity ε of the particles could further reduce the magnitude of the resonant peak. However, in contrast to EF, increasing the radius a and permittivity ε of the particles will result in the peak of a and ε, or decrease the graphene related variable EF. Note that, for larger a, ε and EF at a specific λ. This conclusion may be derived directly from analysis of a and ε will go a further step to verify this validity.In The effective third order nonlinear coefficient Criterion > 0, this composite could exhibits OB. Viewing ε and λ within our interested parameters regions. To achieve low switching threshold for OB, we choose ε = 2.25 and λ=19.3 μm as our primary parameters in λ = 20 μm and λ = 22 μm) are plotted in the same figure. The longer the λ is, the lower value of Only for the condition that λ, a, ε and EF should be the same as that of λ, a, ε and EF), which are shown in λ and higher EF would lead to higher switching thresholds. On the contrary, larger ε and a give rise to lower switching thresholds. Below, we will further demonstrate that the switching thresholds always vary monotonically with these variables.Considering the relation between τ = 0.1 ps in our calculations. It is interesting to see how the relaxation time τ influence the third-order nonlinear coefficients and optical bistability. As an example, we replot τ. By comparing these two figures, we found that the magnitude of the optical nonlinearity peaks in our interesting spectra is generally increased with the increase of τ, especially for high ε. And the peak positions of τ becomes larger, and the critical boundary (Criterion = 0) goes closer to the maxima line of EF with different τ in τ, and it decreases dramatically with longer τ especially for high EF region. In contrast, τ and shows slight decreasing. EF. In other words, if we further reduce the Fermi energy EF, the switching thresholds will be even lower. It should be noted that the parameters for the solid lines in EF = 0.3 eV. That is why these two solid (red and black) lines finally cover each other at the leftmost. In this case, further reducing EF will go beyond the condition for OB. But in the dashed line case, there still exists the region for decreasing EF, hence achieving lower switching thresholds.In the discussion above, we employ the relaxation time of carriers as τ, on one hand, will reduce the switching thresholds especially for Increasing τ−1 term in the linear part of the surface conductivity. Therefore Next, we want to discuss about one specific case in which P0 in the non-dissipation case. As a consequence, λ into a bit higher value so that the OB criterion can be satisfied. Once the τ−1 is neglected hence σ0 is pure imaginary, the switching thresholds It is interesting to note that τ should be included and any small τ−1 will lead to τ−1. In addition, any amount of τ−1 will lead to the criterion boundary deviating from the maxima line of We are surprised to see the switching-down threshold is theoretically zero with any parameters only if the OB criterion is satisfied. After zero see . That mea and permittivity ε of the particle, or reduce the external wavelength λ and Fermi energy EF. The introduction of graphene provides us a new freedom to operate the OB in the composite. Once the structure parameters, i.e., a and ε are fixed, we can still change the OB profile by varying EF as well as the conventional variable λ. In principle, we can achieve very low switching threshold OB at both low and high THz frequencies. Unlike the graphene-dielectric multilayer structures38EF, as well as low switching thresholds.Up to now, we demonstrate that, in order to reduce the switching thresholds one can either increase the particle radius εe similar as In order to verify the properties of this composite material for more practical purpose, we next study the transmittance and reflectance of the subwavelength slab consisting of this kind of composite. Without loss of generality, we employ the field-dependent effective permittivity ε1 and slab with field-dependent permittivity εe respectively. θ and d are the incident angle and thickness of the composite slab [see λ = 17.4 μm in the spectrum, and high transmittance dominates in the spectrum for the non-resonant incident wavelengths. Actually, this surface plasmon resonant wavelength can be well determined by our effective permittivity of the composite system λ=17.4 μm. Therefore although the composite slab is only 5 μm thin the light can not go through the slab, not only at normal incidence but also at all angles [see where slab see . First, gles see . On the gles see .EF = 0.3 eV at the resonant wavelength λ = 17.4 μm [see the red line in 5 V/m. With the present parameters, the red line does not show a bistable curve. Once we adjust the Fermi energy EF = 0.35 eV, the transmittance curve indicate OB. In contrast to the red line, the transmittance of OB (black line) is high at low T ≈ 0) but finally jumps to a dramatically high transmittance (T = 0.96) at low incident intensity. And we find its switching thresholds are not very high, only on the level of ∼105 V/m. This novel properties of transmittance can be used as tunable optical switches and sensors. For comparison, we plot the R/T curves off the resonant wavelength λ = 17.8 μm in The enhancement of local fields within the nonlinear graphene thin shell at the surface plasmon resonant wavelength represent an ideal condition to boost the nonlinear effects in the graphene thin shell. Next we would like to check the transmittance spectra when the high field is applied. We find that it still has the opportunity to go through the composite slab and has a very high transmittance for tion see . Again, In the end, some comments are in order. At first, we assume that the model is within the quasi-static approximation since the radius of particle is far less than the incident wavelength. Note that the radius is less than100 nm, while the incident wavelength is around 10 μm, hence the quasi-static approximation is safely satisfied. Secondly, to describe the surface conductivity of graphene, we introduce the random-phase approximation, in which the surface conductivity has a simplified form. And this conductivity expression further reduces to a simpler one see  when we λ and higher EF would lead to higher switching thresholds. And larger ε and a give rise to lower switching thresholds. Furthermore, the influence of the relaxation time of graphene is discussed. It is interesting to note that when the relaxation time is infinitely long, the switching-down threshold is down to zero which indicate the state that extremely small input could lead to large enhanced local field inside the composite. Finally, we study the optical reflectance and transmittance of the subwavelength slab consisting of such graphene-based nonlinear composite. Complete zero transmittance is found at all incident angles, and the transmittance also show giant OB with the variation of incident field intensity which can be tuned by the Fermi energy of the graphene. Graphene optical bistable devices appear to be particularly promising and could open a new possibility of all-optical switching, optical transistor, optical logic and memory.To conclude, we propose a new nonlinear composite composed of graphene-wrapped nanoparticles embedded in dielectric host. In the quasi-static limit, we derive the effective third-order nonlinear coefficient and study the optical bistability (OB) of the composite at terahertz frequencies. We found that by decreasing the Fermi energy of the graphene layer, as well as the radius and permittivity of the inclusions, one could effectively increase the third-order nonlinear response. With rigorous derivation, the conditions for achieving optical bistability is presented in addition with the switching thresholds of OB. It is shown that the switching thresholds are highly dependent on the Fermi energy of graphene, therefore, it provides a new degree of freedom to control the inside local field with input one. Moreover, tunable conventional variables (radius or permittivity of the inclusions) of the nonlinear composite could be used to reduce the switching thresholds in the meantime keeping a low Fermi energy level, which might be practicable for experiments. In general, longer How to cite this article: Huang, Y. et al. Low-threshold optical bistability of graphene-wrapped dielectric composite. Sci. Rep.6, 23354; doi: 10.1038/srep23354 (2016).