The use of carbon based materials on the removal of antibiotics with high concentrations has been well studied, however the effect of this removal method is not clear on the actual concentration of environments, such as the hospital wastewater, sewage treatment plants and aquaculture wastewater. In this study, experimental studies on the adsorption of 7 antibiotics in environmental concentration of aqueous solutions by carbon based materials have been observed. Three kinds of carbon materials have shown very fast adsorption to antibiotics by liquid chromatography–tandem mass spectrometry (LC-MS-MS) detection, and the highest removal efficiency of antibiotics could reach to 100% within the range of detection limit. Surprisedly, the adsorption rate of graphene with small specific surface area was stronger than other two biochar, and adsorption rate of the two biochar which have approximate specific surface and different carbonization degree, was significantly different. The key point to the present observation were the π-π interactions between aromatic rings on adsorbed substance and carbon based materials by confocal laser scanning microscope observation. Moreover, adsorption energy markedly increased with increasing number of the π rings by using the density functional theory (DFT), showing the particular importance of π-π interactions in the adsorption process. Antibiotics have been used for several decades in both human and animals for treatment of microbial infections, and also as feed additives for promotion of growth of livestock animals124811523242526272811Carbon based materials are widely used in biomedicineIn this paper, seven commonly used antibiotics with near actual environmental concentrations were taken as examples, and two kinds of biochar with large surface area and a graphene with small surface area were chosen as sorbents. The objectives of this study were: (1) to determine the adsorption ability of biochar or graphene on antibiotics in environmental concentration, and draw support from fluorescence experiments to directly observe the adsorption process, (2) to reveal the effects of antibiotics with different aromatic rings on adsorption, (3) to explain the adsorption mechanism by experiment and density functional theory. The concentration of antibiotics in the samples were detected by liquid chromatography–tandem mass spectrometry (LC-MS-MS). We found that three sorbents showed rapid adsorption to antibiotics. The graphene with small specific surface area had the best adsorption capacity. For the same sorbents, the number of aromatic rings on antibiotics is a main factor affecting the adsorption rate, which is in agreement with the results of density functional calculations. The main adsorption mechanism is π-π interactions deduced by observing the adsorption process in confocal laser scanning microscope from fluorescence experiments. The cheap carbon materials with more aromatic ring area  can rapidly and efficiently remove the antibiotics from the actual environment. It is also expected to have great applications in removal of polycyclic aromatic hydrocarbons in water.−1), C=C (at approximately 1630 cm−1), C-OH (at approximately 1400 cm−1), and C-O-C groups (at approximately1050 cm−1) were observed on the surface for graphene and biochar37−1, 1630 cm−1, 1400 cm−1 and 1050 cm−1, showing the surface contained carboxyl, phenolic hydroxyl and oxygen containing groups. The broad peak at approximately 3450 cm−1 was assigned as stretching vibration of adsorbed water, and there was not a distinction among three carbon materials. But the absorption peaks of GN and B2 at 1630 cm−1 were stronger than that of B1, which means that the content of aromatic ring on the surface of GN and B2 was higher than on B1. This C-O-C (1050 cm−1) functional group, suggested that oxygen-containing groups were introduced into the graphene.The morphologies of graphene and biochar were visualized by using scanning electron microscope (SEM). As shown in −1, and a D band at 1320–1350 cm−1. The D band relates to disordered sp2-hybridized carbon atoms containing vacancies, impurities, or other symmetry-breaking defects, such as oxygen containing groups, whereas the G band represents the structural integrity of sp2-hybridized carbon atoms. The extent of carbon-containing defects in the biochars and graphene can be estimated by an intensity ratio of D band to G band (ID/IG)D/IG ratios for the B1, B2 and graphene are 1.03, 1.00 and 0.52, respectively. The higher ID/IG ratio in biochars suggests that biochars possessed fewer aromatic rings structures and more carbon-containing defects that led to the formation of oxygen-containing functional groups on the surface of biochars2/g, 881.6748 m2/g and 49.3840 m2/g, respectively. The surface area of biochar was obviously higher than that of graphene.Raman spectra of the B1, B2 and graphene can be found in The adsorption processes of various antibiotics on carbons is shown in e and qt (ug/g) is the mass of antibiotics adsorbed on per unit mass of adsorbent at equilibrium and at time t (h), respectively. k1 (1/h) is rate constant of the first-order kinetic model, k2 (g/ug/h) is rate constant of the second-order kinetic model. Adsorption data of biochar and graphene adsorbed antibiotic are modeled by the above two kinetic models, and the results are shown in Table 2S. Comparison of fitting results of Table 2S, the correlation coefficients (R2) of pseudo-first-order kinetic and pseudo-second-order kinetic model are almost the same, however the theoretical values of equilibrium adsorption amount (qe), calculated from the first-order kinetics models were in good agreement with the experimental values. According to the adsorption rate parameter (K1) of first order adsorption kinetics equations, as shown in e and k1 parameters. For SD and SMX, the qe of B2 is larger than that of GN since the BET surface area of B2 is significant higher than that of GN which means larger adsorption capacity of B2. Thus, the smaller BET surface area of GN affect the decreasing of concentration after 2 hours.The adsorption test is the study on the adsorption of trace antibiotics of different carbon based materials, thus some antibiotics reached the removal in the adsorption balance. More concern was given to the adsorption rate of carbon based materials to antibiotics. In order to qualificatory compare the adsorption rate of biochar and graphene with 7 kinds of antibiotics, the adsorption kinetics model was used to simulate the experimental data. Adsorption kinetics is commonly interpreted by first- and second-order kinetic models. When the concentration of the antibiotic was lower than the detection limit, we decided it was 0 ng/ml. According to equations41e for B1, B2, GN were 105.7, 199.0, 190.3 ug/g respectively.The researches of carbon based materials for adsorption of antibiotics confirmed that carbon based materials had a strong adsorption capacity523242526272829Researche can reach 25 mg/gIt is worth noting that the concentration of antibiotics has an important influence on the adsorption process. The adsorption equilibrium time of activated carbon for high concentration AMOX (317 mg/L) is only 35 min, and the values of qIt was observed that graphene had an obvious adsorption effect on FITC in one hour. The fluorescence images at different times  were observed under the same conditions in the same field. The fluorescence results of graphene adsorption of FITC with time were shown in 471) as shown in As previously reported, the adsorption behavior of organic compounds on carbon based material in general follows mechanisms such as π-π interaction4948In order to verify the adsorption mechanism of π rings on graphene and biochar, and the adsorption correlation between graphene and π rings with various number, here, we analyzed the adsorption of a series of increased size π rings on a graphene flake at the ωB97X-D/6-31 + G levels of theory. The optioned structures are showed in Carbon based materials are increasingly recognized as effective, inexpensive, and environmentally friendly sorbents for abating organic contaminants. In this paper, the adsorption of 7 antibiotics by 2 biochar and graphene were studied in actual concentration of environments such as the hospital wastewater, sewage treatment plants and aquaculture wastewater, which pose antibiotics resistance genes, potential human health and ecological risks. In the three cabon based materials, the B1 has large surface area and less aromatic ring area by 800 °C carbonization degree; the B2 has porous, large surface area, high aromatic ring area by 1000 °C carbonization degree; and the GN has high aromatic ring areas, and small specific surface area with few pores and functional groups. The adsorption kinetics model was used to simulate the experimental data. And adsorption processes of FITC, which has similar groups and molecular weight with antibiotics, by graphene particles were visualized by fluorescence microscope.The results show that the three carbon based materials have good adsorption proporties for antibiotics in actual concentration of environments, by which the highest removal efficiency of antibiotics can be up to 100%. The adsorption ability follows this order: GN > B2 > B1. From the view of antibiotics adsorption on same adsorbents, the number of aromatic rings on antibiotics was also an important factor in determining the adsorption rate, the more aromatic ring is, the faster the adsorption rate will be. The adsorption process by graphene lead to fast fluorescence quenching of FITC in 1 hour via π-π interaction. We have used density functional theory simulations to find the strong interaction between π rings and graphene, and the adsorption energies increased with number of the π rings. It is concluded that the main adsorption mechanism is the π-π interaction, and it also should be mentioned that other adsorption mechanism such as hydrophobic interaction, H-bonding interaction, electrostatic interaction, pore-filling could not be excluded, which may be auxiliary adsorption mechanism.Comparing the sorption of antibiotics by 3 kind carbon based materials, the biochar with high carbonization degree or graphene, which has rich aromatic ring areas, were more effective in adsorption of antibiotics. From the view of adsorption mechanism, antibiotics with more aromatic rings should be recommended in applications for the easy removal of antibiotics in the environment. At the same time, an efficient, fast and simple sewage treatment process is needed to remove the antibiotics from hospital wastewater, pharmaceutical wastewater and sewage treatment plants.Seven antibiotic standards  including Ofloxacin (OFL), Sulfadiazine (SD), Sulfamethoxazole (SMX), Sulfamethazine (SMZ), Cefalexin (CFX), Amoxicillin (AMOX), Tetracycline (TC) were purchased from Dr. Ehrenstofer GmbH (Germany). Chemical structures are shown in Two types of commercial biochars  and one kind of homemade grapheneIn order to keep the biochar particles size uniformity, eliminate the interference of other substances and microbial interference in biochars, the original samples were treated as follows: the particle size of the biochar was 0.4–0.8 mm after grinding. The biochars and graphene samples were washed by deionized water to an invariable pH value and desiccated by vacuum freeze–drying. The microbial interference was excluded by 30 min UV irradiation. Finally, the carbon materials were characterized by SEM, FTIR spectrum and Raman spectrometer . Surface area of adsorbents (biochar and garphene) were determined by nitrogen adsorption using ASAP 2020 V3.04 H (Micromeritics).0) was chosen as initial concentration.The concentrations of antibiotics in the sewage of hospitals, pharmaceutical plants and sewage treatment plants have been reported in the range of from 64 ng/ml to 540 ng/ml6789Static adsorption method41In order to visually observe the carbon-based material adsorbed antibiotic, the fluorescence properties of the 7 kinds of antibiotics were detected, the results showed that the fluorescence brightness was not suitable for direct observation of the adsorption process. The fluorescent agent fluorescein isothiocyanate (FITC), whose structure is similar to antibiotics  was chosen to directly observe the adsorption process. The molecular weight of FITC is 389.4, which is in the range of antibiotics (251.2-445.2) as listed in The suitable amount of graphene particles was added to the solution of FITC with concentration of 0.1 mg/ml, and then the sample was placed on the stage. By using visual mode, the magnification of the objective was adjusted; the need to test the samples under the fluorescence microscope was found (FLUOVIEW FV1000). By switching to a scanning mode, the double needle and laser intensity parameters, could get clear confocal images. Under the same parameters, we observed the fluorescence change in the same field within one hour.How to cite this article: Peng, B. et al. Adsorption of Antibiotics on Graphene and Biochar in Aqueous Solutions Induced by π-π Interactions. Sci. Rep.6, 31920; doi: 10.1038/srep31920 (2016).


A315T transgenic mice exacerbated the pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and it enhanced the levels of TDP-43 aggregation. These results suggest that inflammation may contribute to development or exacerbation of TDP-43 proteinopathies in neurodegenerative disorders.TAR DNA-binding protein 43 (TDP-43) is a major component in aggregates of ubiquitinated proteins in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here we report that lipopolysaccharide (LPS)-induced inflammation can promote TDP-43 mislocalization and aggregation. In culture, microglia and astrocytes exhibited TDP-43 mislocalization after exposure to LPS. Likewise, treatment of the motoneuron-like NSC-34 cells with TNF-alpha (TNF-α) increased the cytoplasmic levels of TDP-43. In addition, the chronic intraperitoneal injection of LPS at a dose of 1mg/kg in TDP-43 Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the loss of motor neurons in the brain and spinal cord, causing progressive muscle weakness and typically leading to death by paralysis within a few years. Mutations in over twenty genes are known to be associated with familial forms of ALS –2 which Under normal conditions TDP-43 is mostly localized in the nucleus, where it is mainly involved in RNA processing –19. In dPreviously, we reported that levels of messenger RNA (mRNA) and protein for TDP-43 and nuclear factor κ B (NF-κB) p65 were higher in the spinal cord of ALS patients than of control individuals . Surprisin vivo effect of chronic LPS administration in transgenic mice expressing genomic fragment of human TDP-43 A315T gene (hTDP-43A315T) [A315T transgenic mice. These results suggest that chronic brain inflammation may contribute to TDP-43 proteinopathies.Here, we report that LPS exposure induced cytoplasmic redistribution of TDP-43 in cultured microglia and astrocytes. Similarly, NF-κB activation in motor neuron-like cell line NSC-34 by TNF-α enhanced TDP-43 cytoplasmic level. We also tested the 43A315T) –12. InteA315T (hTDP-43A315T) has been generated and characterized by us [The heterozygous transgenic mouse line expressing the human mutant TDP-43ed by us , 23. All2), the tissue was triturated. The cell suspension was washed in glial culture medium  and cultured at 37°C, 5% CO2 in 25 cm2 Falcon tissue culture flasks  coated with 10 mg/ml poly-d-lysine  for overnight and then rinsed thoroughly with sterile distilled water. Four to five days later medium was changed and supplemented with 5ng/ml of mouse recombinant macrophage colony stimulating factor  and every second day thereafter, for a total culture time of 10–14 days. At the moment of tissue dissection the genotype of each pup was unknown. Each brain was then dissected and cultured separately (one brain/25 cm2 flask). The transgenic pups were identified by DNA extraction from tails and PCR amplification of the human TARDBP gene. After 10–14 days, when the genotype of each pup was known, cultures with the same genotype were pulled together during the separation of astrocytes from microglia.Primary astroglial cultures from brain tissues of neonatal (P2-P3) mice were prepared as described previously . In brie2 and astrocytes at 40,000 cell/cm2 in 16-well chamber slides (Thermo scientific) for immunocytochemical analysis and 6-well-plates for protein or RNA extraction. Astrocytes and microglia cultures were treated with LPS (Sigma) at different concentration and for different periods as indicated in figures and results section.The procedure followed to separate astrocytes from microglia is based in previous published protocols . In brieA315T mice and their non-transgenic (wild-type) littermates received intraperitoneal (i.p.) injection of LPS  diluted in 100 μl of vehicle (Veh) solution . Mice were i.p. injected once a week for duration of two months. Mice were not exhibiting overt phenotypes due to LPS injection. Control mice were given same volume of saline.To trigger a systemic innate immune response in the CNS, presymptomatic 6-month-old hTDP-43After two months of systemic injections, animals were deeply anesthetized by i.p. injection of pentobarbital (50mg/kg) and then rapidly perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M borax buffer, pH 9.5, at 4°C. Spinal cords were rapidly removed from the animals. Dissected spinal cord tissues were postfixed for 24 h in 4% paraformaldehyde and equilibrated in a solution of PBS-sucrose (20%) for 48 h. Spinal cord tissues were cut in 25 μm thick sections with a Leica frozen microtome and kept in a cryoprotective solution at -20°C. Immunohistochemistry was performed on 25μm-thick sections. TDP-43 was probed (1:200) with Rabbit polyclonal (ProteinTech) or (1:200) mouse monoclonal (Abnova) antibodies. Neu N was immunostained with (1: 500) monoclonal (Invitrogen) antibody.Real-time RT-PCR was performed with a LightCycler 480 (Roche) sequence detection system using Light-Cycler SYBR green I at the Quebec Genomics Centre. Total RNA was extracted from cell cultures using TRIZOL reagent (Life Technologies). Total RNA was treated with DNase (QIAGEN) to get rid of genomic DNA contaminations. Total RNA was then quantified using Nanodrop, and its purity was verified by Bioanalyzer 2100 (Agilent Technologies). Gene-specific primers were constructed using the GeneTools software (Biotools Inc.). Three genes, Atp5, Hprt1, and GAPDH, were used as internal control genes. The mRNA copy number of each analyzed gene was divided by the mRNA copy number of the house-keeping gene Atp5 in the same sample, in order to take in account variations in samples size. Then, in order to get the fold changes in the expression of each gene with the LPS-treatment, levels of mRNA in the LPS-treated sample were divided by the levels of mRNA in the respective untreated control. Fold change equal to 1 indicate no effect of LPS in the gene expression, while fold changes higher than 1 indicate that the LPS treatment led to a higher expression of the analyzed gene.For sub-cellular fractionation, cells were lysed by a hypotonic buffer  and protease inhibitor (Sigma)) for 30 min on ice. Cells were further broken down by passing 30 times though a 22.5/27 G needle. Membranes were separated from the remaining cellular components by centrifugation at 3000 rpm, 10 min., 4°C. Pellet was then resuspended in extraction buffer  and protease inhibitor (Sigma)) and incubated for 60 min at 4°C with rotation. Nuclear fraction was then separated from the cytosolic fraction by ultra centrifugation at 30,000g for 30 min at 4°C. Pellet containing the cytosolic fraction was resuspended in RIPA buffer. Protein concentration was estimated using the Bradford method.It was done as previously described by Hart et al, with some modifications as described below . Frozen Total protein was extracted from cells using the RIPA buffer  and protease inhibitor (Sigma)). Protein concentration was estimated using the Bradford method. After denaturation, protein samples were resolved by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membrane . Membranes were incubated in blocking solution  solution) for 1h at room temperature. Membranes were then incubated with a primary antibody diluted in blocking solution at 4°C overnight. After rinsing 3 times with 0.1% Tween-20 in TBS solution, membranes were incubated in a horseradish peroxidase-conjugated secondary antibody made in goat  diluted 1:5000 in blocking solution. After rinsing 3 times with 0.1% Tween-20 in TBS solution, immunoreactive proteins were visualized by chemiluminescence with the Renaissance kit . Primary antibodies used were the following: (1) Anti human TDP-43 monoclonal antibody  (2) anti-TDP43 rabbit polyclonal antibody , which reacts with both human (transgenic) and mouse (endogenous) proteins and (3) anti-actin mouse monoclonal . Bands intensity was measured using Image J 1.45p . The intensity of all TDP-43 bands was divided by the intensity of the respective actin band to take in account the differences in the protein loading. Fold change is the ratio of the band intensity in the LPS-treated cultures over the band intensity in the respective untreated controls, after normalized by the intensity of the correspondent actin bands.Paraformaldehyde (4%) fixed cell cultures and 20-um-thick spinal cord sections were incubated in blocking solution  for 1h at room temperature. Cells were then incubated in primary antibodies diluted in blocking solution at room temperature overnight. After rinsing 3 times with PBS, cells were incubated with Alexa 488 or Alexa 594 conjugated secondary antibody made in goat (Life Technologies), diluted 1:200 in blocking solution for 2h at room temperature. Nucleus was stained with 4', 6-diamidino-2-phenylindole  and then rinsing 3 times with PBS. Cover slips with stained cells were mounted on glass slides with mounting medium. The primary antibodies used were the following: anti-glial fibrillary acidic protein (GFAP) mouse ; anti-Iba1 rabbit ; anti-CD11b rat ; anti-TDP43 rabbit polyclonal antibody  and anti-TDP-43 mouse monoclonal . Cells were viewed using a 40X objective on a DM5000B microscope (Leica). Statistical analyses were performed with GraphPad Prism 5.A315T transgenic mice went from a ‘resting’ form with long branching processes and small cellular body, in the absence of LPS, to a large ameboid form when treated with LPS    or CD11b marker) .A315T transgenic astrocytes resulted in redistribution of TDP-43 from nucleus to cytoplasm. As observed in A315T mice.As shown in Subcellular fractionation of astrocyte cultures was performed. Nuclear and cytoplasmic fractions were analyzed by Western blot . TDP-43 As shown in The cellular/molecular events that may lead to abnormal neuronal distribution of TDP-43 in neuronal cells are largely unknown. We investigated whether inflammatory stimuli could promote mislocalization of TDP-43 from nucleus to cytoplasm in motor neuron-like NSC-34 cell lines. Non-transfected NSC-34 cells and NSC-34 cells stably transfected with hTDP-43WT-HA were treated with recombinant mouse TNF-α at a concentration of 10 ng/mL for 6h. Non-TNF treated cells served as controls. Following treatment, the subcellular distribution of TDP-43 was determined by Western blot analysis complemented by immunocytochemistry. As expected, the majority of TDP-43 was located in nucleus. However, TNF-α treatment induced cytoplasmic mislocalization of TDP43 in non-transfected cells as well as in cells stably transfected with hTDP-43WT-HA . Thus, tin vivo, 6 months old non-transgenic mice and hTDP-43A315T transgenic mice were chronically i.p. injected once a week with LPS (1 mg/kg) or vehicle solution starting at 6 months of age for a duration of 2 months. After two months of LPS treatment, the spinal cords were collected and examined by immunostaining or western blotting for TDP-43 immunodetection. Spinal cord sections double immunostained with polyclonal antibody for TDP-43 and neuronal marker (Neu N) showed increased cytoplasmic immunostaining in motor neurons of transgenic mice treated with LPS  (A315T transgenic mice (Fold change > 1)  . In contnge > 1) . The effnic mice .A315T transgenic mice exhibited more cytoplasmic TDP-43 immunostaining when injected with LPS rather than vehicle. LPS treatment increased the number of neuronal cells with nuclear depletion of TDP-43 and with cytoplasmic TDP-43 aggregates (in vivo results are in agreement with our findings with in vitro cell cultures that activation of NF-κB pathway by inflammatory stimuli (LPS or TNF-α) can promote cytoplasmic mislocalization and aggregation of TDP-43.To further confirm the effect of LPS treatment on abnormal distribution of TDP-43, spinal cord sections from LPS and vehicle-treated mice were immunostained with monoclonal antibody specific for human TDP-43. As revealed by immunostaining, spinal neurons from hTDP-43gregates . These iThe results presented here demonstrate that inflammatory stimuli such as TNF-α or LPS can promote cytoplasmic mislocalization and aggregation of TDP-43 in glial and neuronal cells, a proteinopathy similar to what has been observed in ALS cases . Some faA315T linked to familial ALS [A315T mice overexpress at moderate levels the TDP-43 transgene ubiquitously under its own promoter. Thus, it was possible to derive from the hTDP-43A315T transgenic mice primary cultures of astrocyte and microglia that expressed TDP-43A315T mutant. The LPS treatment increased the total amount of TDP-43 protein in both microglia and astrocyte cultures  Primary astroglia and microglia cultures were prepared from brain tissues of neonatal mice and then subjected to LPS treatment as described in Materials and Methods. (B) To trigger a systemic innate immune response in the CNS, presymptomatic 6-month-old hTDP-43A315T mice and their non-transgenic (wild-type) littermates received intraperitoneal i.p. injection of LPS (1 mg/kg of body weight) diluted in 100 μl of saline. Mice were i.p. injected once a week for duration of two months.(PDF)Click here for additional data file.


Nanoelectromechanical systems constitute a class of devices lying at the interface between fundamental research and technological applications. Realizing nanoelectromechanical devices based on novel materials such as graphene allows studying their mechanical and electromechanical characteristics at the nanoscale and addressing fundamental questions such as electron–phonon interaction and bandgap engineering. In this work, we realize electromechanical devices using single and bilayer graphene and probe the interplay between their mechanical and electrical properties. We show that the deflection of monolayer graphene nanoribbons results in a linear increase in their electrical resistance. Surprisingly, we observe oscillations in the electromechanical response of bilayer graphene. The proposed theoretical model suggests that these oscillations arise from quantum mechanical interference in the transition region induced by sliding of individual graphene layers with respect to each other. Our work shows that bilayer graphene conceals unexpectedly rich and novel physics with promising potential in applications based on nanoelectromechanical systems. Graphene nanoelectromechanical systems enable the study of the interplay between electrical conductivity and mechanical properties. Here, the authors observe oscillations in the electromechanical response of bilayer graphene due to wrinkling, rather than the linear response seen in single layers. Separating graphene3581011121310On the other hand, bilayer graphene has been reported to show appealing physical properties such as the possibility of opening and tuning an electronic bandgap17Here we integrate mono- and bilayer graphene into NEMS to study the effect of strain on their transport properties. Mono- and bilayer GNRs with widths between 60 and 300 nm are investigated using nanoindentation techniques based on atomic force microscopy (AFM) for high-resolution imaging and controlled deformation of the GNRs. Electrical conductance of the suspended GNR is measured simultaneously with mechanical deformation using a low-noise lock-in amplifier. While monolayer graphene displays increasing resistance with strain, we observe oscillations in the electromechanical response of bilayer graphene. This behaviour is explained from the point of view of quantum interference in the transition region induced by sliding of individual graphene layers with respect to each other.Vs to the source electrodes and a back-gate voltage Vg to the degenerately doped silicon substrate. The drain current is measured using a current–voltage converter connected to a lock-in amplifier. The transfer and output characteristics of a monolayer- and a bilayer-suspended device are presented in Is–Vs characteristic typical of ohmic contacts. The gating dependence Is–Vg shows ambipolar dependence with the charge neutrality point VCN lying at the conductivity minimum and corresponding to the Dirac point.Our samples consist of field-effect transistors based on suspended GNRs . The devDcantilever, (lower graph) as a function of the position of the stage, Zpiezo. We extract the deformation of the GNR ribbon at the point where load is applied by the AFM tip (DGNR) from the expression Zpiezo=Dcantilever+DGNR  and chemical vapour deposition (CVD) grapheneIn the case of monolayer graphene, we have performed electromechanical measurements on five devices with widths varying between 60 and 300 nm , resistaR/R0 as a function of nanoribbon deflection DGNR  defined as GF=(ΔR/R0)/ɛ. This observed strain-induced resistance increase is in agreement with the previous reports on monolayer graphene142122To compare the electromechanical response of the various GNRs, we represent the relative variation of the resistance Δion DGNR . In almo2  of the individual graphene layers with respect to each other. This lateral displacement is expected due to the weak van der Waals force between the two graphene layers25272829303132333435383926293335362732342830W1 and W2, is different  in one of the layers of bilayer graphene, as illustrated in W of the charge carriers in the decoupled graphene layers. Increasing the strain leads to higher amount of local corrugation and changes ΔW, which would cause constructive or destructive interference.We note, however, that the effective width of the transition region within the two individual graphene layers measured on the graphene lattice, E and momentum parallel to the transition region, k||, for various carrier path differences a=0.246 nm—the lattice constant of graphene). The trivial case of ΔW=0  reveals the massive character of Dirac fermions in bilayer graphene. Finite path differences ΔW result in significant amount of backscattering developing a clear sub-band sequence resulting from the quantum confinement of massless Dirac fermions in the transition region (indicated by the dashed line in the last panel of k=4π/(3a). Therefore, one period of oscillations corresponds to the deformation-induced lateral displacement of 3a=0.74 nm.To verify this hypothesis, we perform numerical simulations of electronic transport in a model bilayer graphene device . The simRC, act as a ‘bottleneck' in a realistic device and are thus responsible for most of its total resistance. We obtained RC=41 kΩ by fitting both the average value of the calculated resistance and the magnitude of oscillations to the experimental data reported in a. The direct comparison of W=0 corresponds to the minimum of resistance (no backscattering) while in experiments resistance oscillates reaching both higher and lower values compared with the zero-displacement point. This implies that the stacking domain boundary was already present before indentation. In other words, the origin in experimental resistance curves corresponds to a finite value of ΔW in To gain further insight, we compare the calculated resistance with actual experimental observation. A quantitative comparison requires accounting for the role of contacts as well as for the diffusive transport in the rest of device. Both factors, below collected in a single value et al.In summary, we have investigated the electromechanical response of mono- and bilayer GNRs. Monolayer graphene devices show an increase in resistance under strain, which is related to a change in the Fermi velocity under strain. Within our experimental conditions at room temperature, we observe neither a spectral nor a transport bandgap larger than 4 meV for strain under 5%, in agreement with theoretical predictions by Pereira 2 layer2 plasma, followed by mask removal in acetone. The electron beam lithography step was used to define contacts using a standard bilayer technique (methyl methacrylate (MMA)/PMMA) followed by metallization and lift-off in acetone. The graphene channel is released and suspended using hydrofluoric acid wet etching and critical point drying.Single- and bilayer sheets of graphene have been exfoliated from commercially available crystals of graphite (NGS Naturgraphite GmbH) using the scotch-tape micromechanical cleavage techniqueElectrical characterization of the devices is carried out using Agilent E5270B parameter analyser and a home-built shielded probe station with micromanipulated probes. AFM imaging and electromechanical nanoindentation experiments are performed using a home-built set-up combining the Asylum Research Cypher AFM with the low-noise lock-in amplifier (SRS-830). We use nonconductive Si AFM tips, model NSC36/AlBS from MikroMash .How to cite this article: Benameur, M. M. et al. Electromechanical oscillations in bilayer graphene. Nat. Commun. 6:8582 doi: 10.1038/ncomms9582 (2015).Supplementary Figures 1-11, Supplementary Table 1, Supplementary Notes 1-8 and Supplementary References


An approximate growth model was employed to predict the time required to grow a graphene film by chemical vapor deposition (CVD). Monolayer graphene films were synthesized on Cu foil at various hydrogen flow rates from 10 to 50 sccm. The sheet resistance of the graphene film was 310Ω/□ and the optical transmittance was 97.7%. The Raman intensity ratio of the G-peak to the 2D peak of the graphene film was as high as ~4 when the hydrogen flow rate was 30 sccm. The fitting curve obtained by the deviation equation of growth model closely matches the data. We believe that under the same conditions and with the same setup, the presented growth model can help manufacturers and academics to predict graphene growth time more accurately. Graphene as a TCE has a wide range of applications, including in solar cells, solid-state lighting, and detectors, owing to not only its higher optical transmittance but also its more favorable conductance[Graphene, comprising two-dimensional monolayer of spductance-4 than tductance. (Additiductance. Howeverductance. Neverthductance. Four-laductance. Additioductance-14. This3PO4 in deionized water of 100 mL) was utilized to smooth out the foil, and a voltage from 2 to 4 V was applied until the Cu foil glowed. Thereafter, the Cu foil was rinsed in a large amount of deionized water with sonication and then blow-dried with nitrogen gas. The Cu foil was placed in the reaction chamber, the Ar and H2  gases were introduced into the chamber during temperature ramp-up. The Cu foil was annealed at 1,070°C for an hour. Then, 0.3 sccm of methane  was used as a source of carbon to grow the graphene. The H2 flow rate was varied from 10 to 50 sccm prior to observation of the morphology of the graphene domains and the nucleation density. Following the growth process, the as-grown graphene/Cu foil was removed from the heating zone for rapid cooling. Polymethyl methacrylate (PMMA) was spin-coated on the as-grown graphene/Cu foil as a supporting layer to prevent any cracking during the transfer process. The graphene grew on both sides of the Cu foil. The graphene at the back of the foil was removed by floating on nitride acid solution (30% in deionized water) for 10 s. The Cu foil was etched away overnight using an ammonium persulfate solution (0.1 M) and then rinsed three times in deionized water. The PMMA/graphene was placed on the substrate, and the PMMA was then dissolved in hot acetone bath for 24 h. The residual PMMA was removed by annealing in air at 200°C for an hour and reduced to pristine graphene using an H2/Ar (7/20 sccm) mixture. The morphology and the nucleation density of the graphene domain were measured by scanning electron microscopy (SEM); The surface profile of Cu foils were measured by atomic force microscopy (AFM); the sheet resistance was measured using a four-probe stage; the Raman shift of the graphene was measured by Raman spectroscopy using a laser with a wavelength of 532 nm, the laser power at the focused spot was 2 mW, and the numerical aperture value was 0.75 on the sample with an area of 1 μm2.Graphene films were grown by chemical vapor deposition (APCVD) on 25-μm-thick Cu foils  in a 3-in. quartz tube furnace under atmospheric pressure. Beforehand, electrochemical polishing  and the red spots represent the growth times of graphene that yield the specified area, based on Equation 7. A red spot at the dashed line indicates that the graphene fully covered the Cu foil. Figure -1, verifying the presence of a single-layer graphene[The graphene.An approximate growth model of the synthesis of graphene by APCVD was developed. When the hydrogen flow rate was 30 sccm, the transmittance as a function of wavelength for single-layer graphene reached its maximum of 97.7% at λ =550 nm. The 2D/G ratio and the FWHM indicated that the graphene comprised a single layer of high quality. The lowest obtained sheet resistance of a single layer of graphene was about 310Ω/□. The results of the experiments closely matched the fitting curve. We believe that, under the same conditions and with the same experimental setup, the proposed growth model can help manufacturers and academics predict the growth time of graphene more accurately.The authors declare that they have no competing interests.SHC (Chan) designed the study and wrote the paper. JWC, HSW, MCL, and HBC analyzed the data. SHC (Chen), CCL, and CCK are advisors. All authors read and approved the final manuscript.


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.