How atoms acquire three-dimensional bulk character is one of the fundamental questions in materials science. Before addressing this question, how atomic layers become a bulk crystal might give a hint to the answer. While atomically thin films have been studied in a limited range of materials, a recent discovery showing how to mechanically exfoliate bulk crystals has opened up the field to study the atomic layers of various materials. Here, we show systematic variation in the band structure of high mobility graphene with one to seven layers by measuring the quantum oscillation of magnetoresistance. The Landau fan diagram showed distinct structures that reflected differences in the band structure, as if they were finger prints of multilayer graphene. In particular, an even-odd layer number effect was clearly observed, with the number of bands increasing by one for every two layers and a Dirac cone observed only for an odd number of layers. The electronic structure is significantly influenced by the potential energy arising from carrier screening associated with a gate electric field. Moreover development of techniques to produce high-quality graphene13 with sufficiently long mean free paths has made it possible to study ballistic transport16 and to modify band structures by using moiré structures created by the slight misalignment of bilayer honeycomb lattices of graphene and h-BN17. Since the beginning of research on graphene, the electronic band structure of this material has been expected to show a particular layer number dependence24. Even if we consider only AB-stacking, the band structure should show an even-odd layer number effect24, as shown in Fig. N and 2N + 1  layer graphene show N bilayer-like bands with non-zero band mass, and the 2N + 1 layer additionally shows a monolayer-like band that is massless with a linear dispersion relation. To date, optical measurements of the band structure have been performed on graphene consisting of several numbers of layers28. However, the low-energy band structure, which affects transport properties, has been only studied for samples consisting of a few layers, by measuring the response of the resistance to gate voltage in the absence of a magnetic field31, and from quantum oscillation in magnetic fields34. In this study, we studied the electronic band structure of high-quality samples of graphene consisting of one to seven layers by measuring quantum oscillations and found systematic variations in the band structure.Since the discovery of grapheneh-BN flake12, which has an atomically flat surface, or encapsulated with h-BN flakes13. The number of layers and stacking were determined by examining the G′ peak appearing at approximately 2700 cm−1 in the Raman spectra35, topography obtained using an atomic force microscope, and color intensity analysis of a digitized optical micrograph (see the Supplementary Information). The Raman G′ peak for AB-stacked graphene exhibited a systematic variation with the numbers of layers35, as shown in Fig. 36, except for an offset in the background signal and slight offset in the Raman shift. This enabled us to accurately determine the number of layers and stacking. The carrier density was varied using a back gate built using a conducting Si substrate covered with its oxide1. Typical electrical mobilities ranged between 30,000 and 100,000 cm2/Vs.Figure dRxx/dB) for samples with one to seven layers, as a function of gate voltage and magnetic field. These fan diagrams appear to be the finger-prints of graphene as to its specific layer number and stacking because they reflect specific band structures. In particular, the streaks and bright spots arise from the Landau levels and their crossings. They vary strikingly with the number of layers and directly reflect different electronic structures. Although the results for samples with one to four layers have been already reported in the literature38, we will briefly review their features here to discuss the systematic variation of the electronic structure in multilayer graphene. As shown in Fig. 38 because they have a set of Landau levels for an electron band and a hole band. The fan-shaped diagram for the monolayer originates from the fact that each Landau levels has a degeneracy of 4eB/h. The monolayer and the bilayer are similar to each other but different in terms of the zero-mode Landau levels that appear at the charge neutrality point near Vg ≈ 0 V: the degeneracy for bilayer graphene is twice that for a monolayer1, and it manifests itself as a difference in the widths of the zero-mode Landau levels. Trilayer graphene shows one fan-shaped structure arising from bilayer graphene, as well as a monolayer band whose Landau levels can be seen at low magnetic fields32. The Landau levels of the monolayer bands appearing in the fan diagram are quite different from those found in in monolayer graphene. Since monolayer and bilayer bands in trilayer graphene show different dispersion relations, the carrier density in the monolayer band is much less than that in the bilayer bands. Therefore, the Landau levels of the monolayer band reach the quantum limit at much lower magnetic fields compared to the bilayer band. For four or more layers, i.e., N ≥ 4, Landau fan diagrams become increasingly complicated with increasing number of layers. As can be seen in the figure, bright spots and streaks near the charge neutrality point form complicated patterns, which differ layer to layer. This variation probably originates from the fact that that the Landau level structures are different for different layer numbers. Despite their complexity, we could find monolayer bands at odd layer numbers at low magnetic fields, as indicated by red marks in the figure. In the hole regime,  conspicuous streaks are observed at low magnetic fields. However, in the electron regime, one can discern streaks corresponding to monolayer bands only in trilayer graphene. This electron-hole asymmetry in the band structure arises possibly from the energy offset at the bottoms of the bands. These monolayer bands do not appear for even layers, i.e., two, four and six-layer graphene.Low-temperature magnetoresistance measurements showed Shubnikov-de Haas (S-dH) oscillations arising from Landau quantization. Figure ntot = 0 is possibly due to the formation of semi-metallic band structures because the Landau level crossings originate from electron and hole bands that are offset in energy. Here we will not go into the details of these structures, which will be reported elsewhere. Rather, we will discuss the general features of the band structure by taking the Fourier transform of the S-dH oscillation. The Landau level structure away from the charge neutrality point is rather simple, which enables us to obtain physical insights by estimating the number of bands and their dispersions. S-dH oscillations are periodic against the reciprocal of the magnetic fields and their period Δ(1/B) is relevant to the area A of the energy contour (Fermi surface) of a band, from which the carrier density can be calculated using the formula, nosc = 4 × A/(2π)2 = 4(e/h)/Δ(1/B). We employed a fast Fourier transform (FFT) to observe the periodicity. Figure ntot induced by the back-gate voltage. Here the total carrier density was calculated using calibrated gate capacitances, and the FFT frequency was scaled by a factor of 4(e/h), which directly indicates the carrier density for low energy bands in graphene. In the figure, some noticeable spectra that reflect the carrier density of the Fermi surfaces appear at a given chemical potential tuned by the gate voltage. Other peaks can be assigned to higher-order harmonics whose frequencies can be expressed as multiples of the base frequency or their sums (see the Supplementary Information). These samples show a characteristic layer number dependence. It is clear that FFT spectra for samples with 2N and 2N + 1  layers are somewhat similar to each other in terms of the ntot dependence of nosc except for the presence of bands with a small carrier density in odd layers. This should occur because 2N and 2N + 1 layer graphene have N bilayer bands: for example, two and three layer each have one bilayer band, four and five layer graphene each have two bilayer bands, etc. Reflecting dispersion relations, the density of states of the monolayer band is much less than that of bilayer bands. The relative intensity of the S-dH oscillation arising from the monolayer band decreased as the layer number increased. This is because the carrier density of the monolayer band is reduced because the number of bilayer bands is increased.The pattern that grows in complexity with increasing layer number in the vicinity of nosc of bilayer bands between the samples with 2N and 2N + 1 layers. This difference partly stems from the dependence of the band mass on the layer number; although the number of bilayer bands in 2N layers equals that of 2N + 1 layers, values of the mass in those bands are expected to be slightly different22. Moreover the difference can also result from the fact that the energy offset value of each band is somewhat dependent on the layer number20.In addition to this even-odd layer number effect relevant to the monolayer band, there are small differences in the N < 4. However for N ≥ 4, the disagreement became progressively larger as the layer number increased (see the Supplementary Information). Fine tuning of the SWMcC parameters did not significantly change the situation. Much better agreement between experiment and calculation was obtained when the potential due to screening by gate induced carriers was considered. Figure h-BN), and gate electrode. A simple capacitor model tells us that the induced charge is located at the surface of the metals and that there are no electric fields inside the gate electrode. However, in the case of an atomic layer material, one must take screening of the induced charge into account, as was discussed in refs41 Hence in the calculation, we assumed screening of the form ∝ exp(−d/λ) where λ is the screening length and d is distance from the surface. The parameter λ was adjusted so that calculations fit the experiments (see the Supplementary Information). The effect of screening becomes increasingly evident as the layer number increases. The screening lengths are equivalent to approximately one to two layers, which approximately agrees with the theoretically predicted value41.We performed a calculation based on the tight-binding model. A simple calculation of the dispersion relation using all the Slonczewski-Weiss-McClure (SWMcC) parameters of graphite could approximately explain the experimental result obtained for 41, and the short screening lengths of a few layers have been regarded as having an effect on the electronic property of multilayer graphene42. Since the screening length has been expected to be equivalent to approximately a few layers in multilayer graphene, it was assumed that only a few layers could be measured if one used gate electrodes to vary the carrier density of the samples42. However, our observations show an even-odd layer number effect and number-of-layers-dependent fan diagrams, which reflects the particular electronic band structures of graphene consisting of four to seven layers. We speculate that these findings indicate that, for at least up to seven layers, coherent quantum states associated with the band structure form along the layer direction, i.e., perpendicular to the sample plane, even in the presence of a gate induced potential variation.Since the beginning of graphene research, there have been investigation into screening of gate induced carriersThe present results answer the question of how graphene becomes graphite as the layer numbers increases. Generalizing this argument, we can conclude that monolayer bands exist even in graphene with larger numbers of odd layers, and presumably even in graphite as long as AB-stacking is preserved in the entire sample and that the number of layers is odd. However, finding a monolayer band would become increasingly difficult because of the large number of bilayer bands.43. Some of these band structures with small layer numbers have been identified from optical measurements. However, transport experiments have only been carried out for ABC-stacked trilayer graphene44. Further studies of graphene with various layer stackings will contribute to a comprehensive understanding of the transition from atomic layers to the bulk.We have limited our concern to band structures in AB-stacked graphene. For a layer number larger than 2, ABC-stacking, which is not equivalent to ABA-stacking, is possible and is expected to show different band structures. The number of possible stacking increases with the number of layers. Each stacking is expected to show different band structures47. Moreover, a similar but different even-even odd layer number effect was recently observed in MoS2 atomic layer devices48; the reported Landau levels showed a valley Zeeman effect in samples with odd layer numbers, and a spin Zeeman effect in samples with even layer numbers. Another example is the topological insulator Bi2Se3. Topologically protected surface states, which appear in the bulk, showed mixing as the number of layers decreases49. Hence, more interesting phenomena might exist that relate atomic layers and bulk materials.A layer number dependence in the electronic band structures was also reported in metal dichalcogenides: the energies of the bottoms of the band were found to vary systematically with the number of layersB-dependence of the magnetoresistance revealed a systematic change in the band structures; in particular, an even-odd layer number effect relevant to the electronic band structure was observed, where a linear band was observed only in odd-layer graphene. The band structure became more sensitive to the gate electric field as the layer number increased.Shubnikov-de Haas oscillations in AB-stacked graphene with one to seven layers were reported. Graphene exhibited characteristic Landau level structures in the fan diagram, which reflected the dependence of the electronic states on the number of layers. FFT analysis of the periodicity in the 1/h-BN crystal. Some samples are encapsulated with h-BN flakes. Graphene samples were patterned by using electron beam lithography. Electric resistance measurements were carried out using a standard lock-in technique. Magnetic fields were applied by using a superconducting solenoid.Graphene samples are mechanically exfoliated from Kish graphite. The graphene is then transferred onto a thin h-BN flake, which was prepared by mechanically exfoliating a high quality Supplementary Information


In this paper, we have to apply the Dirac-Weyl equation to find the analytical energy eigenvalues of the graphene quantum dot interacting in the presence of AB-flux field and external magnetic field. We find that the energy eigenvalue of the graphene quantum dot decreases with both magnetic and AB-flux field but the effect of AB-flux field is more dominant. By ameliorating the intensity of the AB-flux field and keeping the magnetic field constant, the quantum-dot states entangled to produce Landau Levels. We show that besides using the graphene sheet and external magnetic field, the Aharonov-Bohm AB-flux field could as well be used to manipulate the carriers state energies in graphene. It is the thinnest substance ever made. It is the basic structural element of other allotrope, including charcoal, graphite and fullerenes carbon nanotubes. One could also consider it as an indefinitely large aromatic molecule. It has exceptional strength, thermal conductivity, and electric conductivity. The potential applications of graphene is not limited to faster computer chips, it also important in designing hyper-efficient solar cells, flexible touchscreens and desalination membranes Graphene quantum dots are kind of 0D material with characteristics derived from both graphene and carbon dots Manufacturing graphene-based quantum structures seems to be of great challenge for its potential application in electronic devices because of the Klein tunneling effect Magnetic field induced confinement-deconfinement transition in graphene quantum dots was investigated in Ref. It was found in Ref. Beside the graphene sheet and external magnetic field, we suggest that AB-flux field could also be used to manipulate the carriers state energies in graphene. We refer the readers to Ref. Linearity in the behavior of energy spectrum of graphene at the Brillouin zone can be described by the Dirac-Weyl equation which is a characteristic of relativistic massless particles. The study of problems involving Dirac-Weyl Hamiltonian in continuous limit in graphene have been investigated in several literature Motivated by the considerable interest in studying Dirac-Weyl Hamiltonian in continuous limit in graphene, in this paper, we use the Dirac-Weyl equation to find the energy eigenvalue of a circular graphene quantum dot under the influence of magnetic field and AB-flux field. In section 2K. k is related to the energy via the expression In this section, we give theoretical formulation of the problem. In order to achieve our aim of this study, we shall solve the Dirac-Weyl equation in cylindrical coordinate by taking into accounts, the homogeneous magnetic and AB-flux fields perpendicular to the graphene sheet. Thus, the Hamiltonian for this problem becomes3E using the standard root finding methods. We have used Wolfram Mathematica 11 for this and the result has been shown in In this section, we utilize the formula method (FM) 4In this paper, we have described a way of confining Dirac-Weyl quasiparticles in graphene. The energy level of the graphene quantum dot has been obtained in the presence of an external magnetic field and AB-flux field. In The series of crossing and anticrosings that appear in the plot of energy spectrum is as a consequence of the interplay between AB-flux, magnetic field and the quantum dot. We have found that the energy spectrum of the graphene quantum dot decreases with both magnetic and AB-flux field but the effect of AB-flux field is more dominant. By ameliorating the intensity of the AB-flux field and keeping the magnetic field constant, the quantum-dot states integrate to produce Landau Levels. We also found that the energy gap would be closed with increasing AB-flus field if we shift the degeneracy that appear in the energy levels via the magnetic field. The reversal symmetric are broken by the magnetic ordering.The mathematical method utilized in this paper is very efficient and easy to use. We hope that our theoretical work will influence experimental efforts on the effects of AB-flux field and magnetic barriers on Dirac fermions.Fernando Adan Serrano Orozco, Jose Luis Cuevas Figueroa, Hugo Moises Martinez Carrada: Conceived and designed the analysis; Wrote the paper.Juan Gerardo Avalos Ochoa, Xochitl Cabrera Rivas: Analyzed and interpreted the data; Contributed analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.


The assembly of dopants on 2D materials assisted by a polymer matrix, demonstrated by spin-coating wafer-scale substrates in ambient conditions, opens up a scalable technological route toward expanding the functionality of 2D materials.Tuning the charge carrier density of two-dimensional (2D) materials by incorporating dopants into the crystal lattice is a challenging task. An attractive alternative is the surface transfer doping by adsorption of molecules on 2D crystals, which can lead to ordered molecular arrays. However, such systems, demonstrated in ultra-high vacuum conditions (UHV), are often unstable in ambient conditions. Here we show that air-stable doping of epitaxial graphene on SiC—achieved by spin-coating deposition of 2,3,5,6-tetrafluoro-tetracyano-quino-dimethane (F4TCNQ) incorporated in poly(methyl-methacrylate)—proceeds via the spontaneous accumulation of dopants at the graphene-polymer interface and by the formation of a charge-transfer complex that yields low-disorder, charge-neutral, large-area graphene with carrier mobilities ~70 000 cm Incorporating dopants in the graphene lattice to tune its electronic properties is a challenging task. Here, the authors report a strategy to dope epitaxial large-area graphene on SiC by means of spin-coating deposition of F4TCNQ polymers in ambient conditions. Typically microscopic in size, VdW hetero-structures are prone to size-induced mesoscopic effects that obscure novel physical effects expected in graphene at charge neutrality. Large-area graphene, uniformly doped to the Dirac point as in the case of VdW hetero-structures, is required to unveil the intimate details of, for instance, metal-insulator transitions3, interacting Dirac fermions4, and Zitterbewegung of electrons in graphene5. Atomically flat, large-scale epitaxial graphene on SiC (SiC/G) has demonstrated its potential for scalability in applications that demand extremely high-quality graphene over large areas, including quantum metrology7 and high-frequency electronics9. Yet, in practice, carrier density control of SiC/G remains a challenge because of high intrinsic n-doping (n > 1013 cm−2) due to interaction with the substrate, making it difficult to substantially tune its carrier concentration10. While numerous gating techniques have been applied to SiC/G to reduce its carrier density9, such approaches introduce carrier density fluctuations, which prevent further explorations of Dirac physics in this material.Homogeneous doping of graphene down to low carrier densities enables the study of delicate and fascinating physics at the Dirac point. The prime experimental archetypes for the study of Dirac physics in solid-state devices are van der Waals (VdW) hetero-structures of graphene and hexagonal boron nitride (hBN). In these, extremely low carrier densities in graphene are attainable, especially when prepared on atomically flat, electrically conductive substrates that screen long-range potential fluctuations (i.e. disorder) in the graphene layern < 1010 cm−2), low charge fluctuations (at the level of Δn ≈ ± 6 × 109 cm−2), and carrier mobilities up to 70 000 cm2 V−1 s−1 at low temperatures. So far, such electron transport properties have only been attained in microscopic single-crystal graphene flakes encapsulated by hBN11 or in suspended graphene12. The doping method described here was applied to samples over 5 × 5 mm2, resulting in epitaxial graphene devices that display quantum Hall effect at magnetic fields B < 1 T. These measurements indicate, as we elaborate below, a highly homogeneous spatial distribution of charge carriers on graphene. Chemical analysis reveals that air-stable molecular doping of graphene is achieved when organic molecules, embedded in a polymer matrix, diffuse through the matrix and spontaneously accumulate at the graphene surface due to formation of a charge-transfer complex. High-electron-affinity 2,3,5,6-tetrafluoro-tetracyano-quino-dimethane (F4TCNQ) molecules are mixed in a liquid solution with poly(methyl-methacrylate) (PMMA) and spin-coated at ambient conditions on SiC/G, with graphene being used both as the target substrate for molecular assembly and, simultaneously, as a charge sensor. The stability of the samples allows us to study the chemical composition as well as electronic transport properties of the F4TCNQ/graphene system. We expect this method to open up a scalable route toward expanding the properties and functionality of graphene and other two-dimensional (2D) materials well beyond doping control.Here we show a molecular approach to control the carrier density of epitaxial graphene homogeneously over macroscopic areas, yielding graphene with low carrier density (W = 5 mm × L = 5 mm)  epitaxial graphene Hall bars fabricated by conventional electron beam lithography (see Methods section). When PMMA is used as a spacer between graphene and the molecular dopant layer, the carrier density decreases three orders of magnitude from its pristine value, n ≈ 1 × 1013 cm−2 to near charge neutrality, n ≈ 1 × 1010 cm−2  Fig.  and smalm−2 Fig. . Importa1 T Fig. , retainis−1 Fig. . The PMM14 cm2 s−1 and by integrating the areas under the ion current intensity curves, we estimate the density of F4TCNQ molecules near the graphene surface to be ~4.6 × 1014 cm−2  decreases linearly with temperature from its room temperature value, due to suppression of acoustic phonon scattering. Quantum corrections to resistance result in a log(T) dependence below the Bloch-Grüneisen temperature νph = 2 × 104 m s−1 . In contrast, when graphene is gated to the Dirac point, the sheet resistance ρXX monotonically increases as the temperature drops, though remains finite with no indication of a transport gap in the current voltage characteristics down to T = 2 K . Figure ρXX(B), RXY(B) at a fixed gate voltage, corresponds to devices behaving as a system with a single electronic band and spatially homogenous carrier density23. That is, data points in Fig. RXY(B) at low fields22, and fully developed half-integer quantum Hall Effect at high fields23, i.e. ρXX(B) = 0 Ω and strictly quantized RXY plateau over the entire available range of magnetic field  once the magnetic length approaches the average charge puddle size24. In our samples with low carrier density concentration ~1010 cm−2 we have observed no deviation from ρXX(B) = 0 to the largest magnetic field available in our setup (B = 14 T)  we conducted low-temperature magnetotransport measurements on chemically and electrostatically gated devices and found these fluctuations to be on the level of Δ2 . We estimated the molecular density at the graphene surface from the shift in carrier density measured in doped graphene with respect to its pristine concentration (Δn ≈ 1 × 1013 cm−2), and assuming that 0.3 electrons are withdrawn from epitaxial graphene per molecule28 with 1/10 gate efficiency10. The homogeneity in doping is in part enabled by the high degree of F4TCNQ dispersion inside the PMMA matrix, shown by room temperature grazing-incidence wide-angle X-ray scattering (GIWAXs). The diffractogram in Fig. q = 9.6 nm−1 from PMMA  when devices are subjected to thermal excursion from T = 2 to 230 K . The charge homogeneity of the samples is thus determined by the temperature at which the screening charges freeze.It is remarkable that epitaxial graphene displays such low disorder even at extreme dopant coverage, being decorated with a dense layer of molecules of about 3–4 molecules per nm0 K Fig. . This is30  density can lead to suppression of charge scattering in graphene if there is sufficient inter-impurity correlation present in the system35.In summary, we presented a method of guiding the assembly of molecular dopants onto the surface of graphene by using an organic polymer matrix. With the doping stability observed in our samples, the tuneability of molecular coverage, and given the vast catalog of polymers and organic/organo-metallic molecules, we expect this method to open up a scalable route toward expanding the properties and functionality of graphene and other 2D materials well beyond doping control. Moreover, the presented analysis of chemical and electron transport properties of doped graphene sheds light on the complex processes that molecular dopants undergo when embedded in polymers. This is relevant to the understanding of performance, materials, and interfaces in organic electronic devices, especially when combined with 2D materials. This method can be explored in the future to create and study electron transport properties of novel 2D systems of ordered molecular arrays templated by 2D crystals36. Optical microscopy37 revealed a typical surface coverage of >95% monolayer graphene.Monolayer SiC/G was grown on the Si-face of SiC using thermal decomposition of 7 mm × 7 mm 4H(0001)-SiC substrates. The samples were grown in argon atmosphere of 800 mbar, and kept at a temperature of around 1700 °C for 5 min38. The electrical contacts were fabricated using physical vapor deposition of Ti and Au, 5 and 80 nm thick, respectively. After fabrication samples were cleaned using isopropyl alcohol, acetone, and dried using nitrogen gas. For pristine SiC/G  and PMMA . A unit of 25 mg of dry F4TCNQ powder was mixed with 3 ml anisole solvent. A volume of 0.5 ml of this solution was then mixed with 1 ml PMMA A6 (6% PMMA by weight in anisole). The PMMA spacer layer and encapsulation layers used either PMMA A4 (5% by weight in anisole) or PMMA-based copolymer (MMA (8.5) MAA) EL6 (6% by weight in ethyl lactate solvent). All polymer layers were deposited on graphene using spin-coating at 6000 rpm for 1 min. After each spin-coating step a 5 min baking step on a hotplate set at 160 °C follows. The dopant blend is an exception, since the baking time dictates the final doping level.The electrostatic top gate was fabricated using a shadow mask, and physical vapor deposition of Au in order to avoid disruption of the chemically doped sample. In order to preserve the integrity of the doping strength of graphene, additional layers of dopant blend and PMMA encapsulation layers were deposited before fabrication of the gate. The samples with the full stack of five polymer layers are the most resilient to drift in carrier concentrations in ambient conditions.4He gas flow cryostat, which allowed for temperatures down to 2 K and magnetic fields up to 14 T. All reported values of charge carrier mobility and charge carrier concentration were extracted from four-probe Hall and quantum Hall measurements. The standard measurement setup used current biased samples at maximum of 100 nA DC , in this range samples showed linear IV characteristics down to cryogenic temperatures. The electrostatic top gate was DC biased with voltages ranging from −100 to +200 V, where leakage current was <0.05 nA.Standard electrical characterization was performed in a liquid 3+ cluster ion gun as the primary ion source and a 10 keV C60+ ion source for sputtering and etching. The samples were analyzed using a pulsed primary ion beam  with a focus of approximately 2 μm at a field of view of 150 µm × 150 µm. The mass resolution using this setup was at least M/ΔM = 5000 full width at half maximum at m/z 500. All spectra were acquired and processed with the Surface Lab software . Depth profile analysis was performed using a C60++ beam at 20 keV with a current of 0.2 nA in a non-interlaced mode with 1 s of analysis, 1 s of sputtering, and a pause of 1 s for each sputter cycle at 350 µm × 350 µm. The maximum ion dose density of Bi3++ was kept between 4 × 1012 and 7 × 1012 cm−2 over the whole depth profiling experiment, while the ion dose for C60++ ranged from 2 × 1014 to 4 × 1014 ions per cm2. Low-energy electrons were used for charge compensation during analysis.ToF-SIMS analysis was performed using a TOF.SIMS 5 instrument . The instrument is equipped with a 25 keV BiData were obtained at the D1-beam line at the Cornell High Energy Synchrotron Source at Cornell University. The measurements used a positron beam with synchrotron radiation of a wavelength of 1.162 Å, Pilatus 200K detector with pixel size of 172 μm × 172 μm and sample to detector distance of 177.2 mm. Measurements were done in ambient conditions at room temperature. The exposure time for each individual measurement was 20 s.Supplementary InformationPeer Review File


We developed a multi-functional graphene composite with electromagnetic interference (EMI) shielding and de-icing properties. Two-dimensional graphene fillers were homogeneously dispersed in a polymer by three-roll milling. The electrical properties and percolation threshold of the graphene composites were measured with various graphene contents. The variation in the EMI shielding properties of the graphene composites with respect to the filler content was measured. The shielding efficiency improved with increasing graphene filler content. Furthermore, we conducted electrical heating tests on the graphene composites. The composites could be heated rapidly to 200 °C by electrical Joule heating with low electric power because of the high electrical conductivity of the composite. Moreover, the composite film was suitable for application in a de-icing unit because of its rapid and homogenous heating performance.  Because of their excellent mechanical strength and good electrical and thermal properties ,2,3, carHowever, carbon-based nanomaterials are easily aggregated because of the strong Van der Waals forces that bind them. To fabricate the ideal graphene composite, a uniform dispersion method is required to distribute the filler homogenously in the polymer matrix. The conventional method used is the ultra-sonication technique ,16,17,18Conductive polymer composites offer some merits as EMI shielding materials. These include the low weight of the materials, corrosion resistance, and flexibility ,25,26,27In this paper, we describe the fabrication of graphene polydimethylsiloxane (G-PDMS) composite films using a three-roll mill process. We measured the electrical properties of the composites while varying the filler content. Furthermore, EMI shielding and electrical heating performances were evaluated. We confirmed the possibility of application of the composites for EMI shielding and de-icing units, based on these properties. The composite shows outstanding EMI shielding and de-icing performance as a result of the homogeneous distribution of the filler. Thus, we show that graphene composite films can be applied in electric vehicles and in the aviation industry, where both EMI shielding and de-icing properties are simultaneously required.We fabricated a G-PDMS composite film for EMI shielding and de-icing applications. Fillers were purchased from Angstron Materials Inc. (graphene powder N006-P). Graphene powder comprised flakes that were 10–20 nm in thickness and measured 5.0 μm in the x-y directions. PDMS (Sylgard 184) was used as the base polymer of the composite film. First, for preparing the composite paste, Sylgard 184 (10:1) and graphene were premixed using a paste mixer . After the premixing step, we used a three-roll milling machine  to homogeneously disperse the fillers by applying mechanical shear forces. The G-PDMS paste was dispersed with the help of a three-roll milling machine at 100 rpm for 6 min. Graphene-dispersed G-PDMS paste was used to form a film of 500 μm thickness using hot press-machine; this technique yielded a film with uniform thickness. The paste was cured at 150 °C for 40 min to achieve stable curing. The pressure of the machine was set at 15 MPa for film fabrication.We measured the EMI SE of the G-PDMS film using a network analyzer  with an S-parameter. A circular sample with 1 mm thickness and a diameter of 10 cm was fabricated; a reference sample with the same dimensions was also prepared. The SE value of the composite was measured (based on the ASTM D4935-99 method) in the 1.0–3.0 GHz frequency range.The electrical heating properties of G-PDMS were characterized for de-icing unit application. Silver and copper electrodes were formed on the film. The silver electrode was made using silver paste; after curing the silver paste at 175 °C for 1 h, copper was attached to the silver electrode of the composite film. To heat the film, we used a power supply  to input electrical power to the G-PDMS. An IR camera was used to measure the surface temperature of the film. In de-icing tests, a cooling spray was used to control the low temperature of −50 °C.We fabricated G-PDMS composites for application in EMI shielding and in a de-icing unit. The composite film was produced using a three-roll mill. Nano-sized fillers usually aggregate because of the Van der Waals forces; hence, special methods are required obtain a homogeneous dispersion of graphene so that its excellent material properties can be exploited. Therefore, we used a solvent-less dispersion method that makes use of mechanical shear forces. The fabrication process of the graphene dispersion is shown in We observed the inside morphology of the graphene composite using scanning electron microscopy (SEM). In addition, the morphology of G-PDMS indicates a homogeneously dispersed state without aggregation of fillers. For HG-PDMS, a sufficiently dense distribution of the fillers in the interior of the composite can be seen. The electrical properties of G-PDMS composites were measured for various filler contents. Pure PDMS is a non-conducting polymer; the addition of a small amount of graphene can improve the electrical properties of the composite because of the superior electrical conductivity and high aspect ratio of the filler. In c is the volume fraction (P) of the electrical percolation threshold, and β is a critical exponent. From the best fit of log-log plots of the power low (inset c (0.48 vol%) and β (3.9) are obtained. The Pc and β values are quite similar to those of a previously reported graphene composite (Pc and β are 0.5 vol% and 1.31) [This equation suggests that the conductivity increases when the filler volume fraction increases. Furthermore, the results of the equation are known as the electrical percolation threshold tendency, where σ is the composite conductivity, Pow inset , Pc (0.4nd 1.31) .In i and Pt data measured by the network analyzer (Equation (2)), where Pi and Pt are the magnitudes of the incident and transmitted power densities, respectively. The EMI SE of the graphene composite is measured over the frequency range of 1.0–3.0 GHz. Generally, pure PDMS does not show EMI shielding properties. We measured the EMI SE values of graphene composites. When graphene filler is added to the polymer, the EMI SE increases. For the LG-PDMS composite, an EMI SE value of 15 dB is measured. Furthermore, the EMI SE value of the MG-PDMS graphene film improved to 21 dB. Finally, for the composite with the highest graphene content , the EMI SE value increased to ~25 dB in the frequency range 1.0–3.0 GHz. Because of the sufficiently high values of the EMI SE at low graphene contents, G-PDMS can be applied for EMI shielding in electric vehicles and aircraft. EMI SE is calculated with PFor application of the composite in a de-icing unit, we performed de-icing experiments by electrical heating. Graphene composites are used as heating units because of their rapid heating performance resulting from the presence of carbon-based fillers that have superior electrical and thermal properties. When the graphene fillers are well-dispersed, the surface temperature is increased homogeneously upon application of electric power. These properties of the graphene composite enable rapid and uniform heating units to be fabricated. 2 S/m and the EMI SE was improved to levels required for application in EMI shielding systems. The SE of the flexible composite was 25 dB in the 1.0–3.0 GHz frequency range; this EMI SE value indicates possible application of the composites in electromagnetic wave shielding. In addition, the G-PDMS unit can be heated rapidly (from room temperature to 200 °C in 50 s) to enable fast de-icing. Thus, in view of its superior EMI SE and rapid heating performance, the composite material is suitable for EMI and icing prevention units and can be applied in vehicles and the aviation industry.Graphene-based composites have been investigated for various applications, such as EMI shielding and de-icing. We fabricated G-PDMS composites by three-roll milling. The fillers were dispersed homogeneously inside the composite, by which the electrical conductivity of the graphene composite improved to 10


Due to the exceptional properties of graphene, numerous possibilities for real applications in various fields have been provided. However, it is a challenge to fabricate bulk graphene materials with properties arising from the nature of individual graphene sheets, and which assemble into monolithic three-dimensional structures. If 3D structured graphene foam were made instead of 2D structured graphene, it is expected that it would be a facile fabrication, with relatively low cost with the possibility of scale-up, and would maintain the intrinsic properties of graphene. To solve the weaknesses of 2D structured graphene, this study aimed to fabricate a 3D graphene-carbon nanotubes (CNT) hybrid foam. In this study, CNT was used to reinforce the graphene foams. In addition, two different surfactants, known as sodium dodecylbenzene sulphonate (SDBS) and cetyltrimethylammonium bromide (CTAB), were applied to help CNT dispersion. The π–π interaction was induced by SDBS/CNT, while ionic interaction was derived from CTAB/CNT. To confirm the charge effect with different surfactants, SEM, Zeta-potential, FT-IR, Raman spectroscopy, and compression tests were performed. When using a cationic surfactant, CTAB, compressive modulus, and strength increased due to the formation of relatively strong ionic bonding. Graphene has come into the spotlight owing to its extraordinary properties ,2,3,4,5.Recent studies have revealed that 3D graphene architectures have arisen as a keyword to resolve the aforementioned negative points of fabrication of graphene . OtherwiHerein, a strong graphene foam produced by introducing carbon nanotubes (CNTs) as reinforcement is suggested. CNTs have been attracting significant interest as reinforcement in composites as well as in graphene due to their various unique properties ,18,19,20Hence, to achieve the facile fabrication, a modified Hummers’ method was applied to synthesize of GO. Ethylenediamine (EDA) acts as a reducing agent to functionalize and connect the GO sheets. These foams are fabricated by subjecting a wet-gel precursor to freeze-drying to remove the ice crystals without collapsing the cell structure. SEM was measured to identify structural and morphological changes with low and high magnification. In addition, Raman, Zeta-potential, and FT-IR were conducted to identify a good connection with each different surfactant on CNTs. Finally, compression tests under compressive stress were performed, and the modulus and strength were increased by 157% and 54.3%, respectively.2SO4, SAMCHUN, Seoul, Korea, 95%), potassium permanganate , sodium nitrite , hydrogen chloride , hydrogen peroxide , single-walled carbon nanotubes , Houston, TX, USA), SDBS (Sigma Aldrich), and CTAB (Sigma Aldrich) were used to synthesize and fabricate the graphene oxide and 3D graphene-CNT hybrid foams.Graphite, sulfuric acid . Finally, the resulting sediment was dried in the freeze-dryer.Graphite oxide was prepared via a modified Hummers’ method ,22. 0.6 2O for 5 min. Then, 0.1 g of SWNT was added with this solution under ultrasonic agitation for 3 h at room temperature with the aim of making a good dispersion. Finally, the dispersed SWNT solution was centrifuged for 30 min to collect the supernatants. The chemical interaction between CNT and surfactants (SDBS and CTAB) is illustrated in Before the preparation of CNT dispersion, 1 g of each surfactant, such as SDBS and CTAB, was ultra-sonicated with 100 mL of HThe synthesis process of the reduced graphene foam is illustrated in −1 and Raman spectroscopy  within a range of 400–4000 cm−1 were used, respectively. In addition, the surface charges  of the samples were measured by Malvern instrument . Finally, compression tests were conducted to evaluate mechanical properties under compression . The specimens were cylindrical in shape and had dimensions of 5 mm of diameter and 2.5 mm in height. Tests were conducted along with ASTM 1621-10, while strain rate is 0.3 mm/min.The cell structures of the graphene foam and graphene-CNT hybrid foam were measured by scanning electron microscopy . To investigate the interaction for the dispersion of GO and GO-CNT, and the chemical bonding between the CNT with each surfactant and GO, the Fourier transform infrared spectroscopy  within a range of 500–4000 cm3D graphene architecture, called reduced graphene oxide, was prepared via self-assembly . GO has As shown in Additionally, the surfaces of GO sheets are highly negatively charged, apparently as a result of the existence of the carboxylic and hydroxyl groups on these sheets. Although CNT is a neutral, SDBS/SWNT and CTAB/SWNT are negatively and positively charged, respectively. For this reason, there could be a tight connection between each surfactant and CNT. In cases of GO-SDBS/SWNT and GO-CTAB/SWNT, they are also negatively and positively charged, respectively. However, the magnitude of these hybrid samples decreased. From this, they have a good connection between CNT with each surfactant and GO.−1 due to the stretching vibration of O–H, while the peak at 1717 cm−1 is attributed to C=O bond stretching. The deformation band at 1387 cm−1 indicated tertiary C–OH, while the peaks at 1042 cm−1 and 1220 cm−1 belong to C–O of alkoxy and epoxy bond, respectively. The other bands at 2975 cm−1 and 1617 cm−1 indicate the C–H stretching of aliphatic groups and C=C bonds.As shown in −1, 2870–2950 cm−1, and 1470–1520 cm−1 correspond to slightly shifted O–H, aromatic with =C–H and ring C=C stretching vibration, and with –CH3 and –CH2 stretching vibration, respectively. These peaks are due to the attachment of surfactant molecules. Specifically, it was determined that CNT exist in GO-SDBS/SWNT and GO-CTAB/SWNT hybrid foams when compared with FT-IR spectra of CNT [On the other hand, for both of GO-SDBS/SWNT and GO-CTAB/SWNT, the characteristic peaks at 3720 cma of CNT .In addition to SEM images shown in −1 and ~1580 cm−1 corresponding with D and G bands due to C–C stretching of the graphitic lattice. In comparison, the Raman spectra of GO-SWNT hybrid foams indicated the distinctive D, G, 2D, and radial breathing mode (RBM) bands of CNTs. In addition to the presence of the D band, the G band of the GO-SWNT hybrid split into two bands, called G+ and G− due to the effect of curvature on the nanotube G peak [−1 can be used to determine the amount of disorder. It implies that the disorder caused by oxidation and reduction of GO is partially recovered by adding SWNTs. In addition, the RBM is representative of SWNTs due to an isotropic radial vibration in a range from 120 cm−1 to 350 cm−1.In case of GO foam, it had two prominent peaks at ~1350 cme G peak . The newD/IG) is denoted as a structural disorder; an increase in the ID/IG ratio is attributed to an increase in the disorder [D/IG ratio of GO, GO-SDBS/SWNT, and GO-CTAB/SWNT. The ID/IG ratio of two kinds of the GO-CNT hybrid foams is much smaller than that of GO foams. It also means that the crystallinity is recovered.Besides the 2D band, the integrated intensity ratio between the D and G bands  and strength than GO-SDBS/SWNT because the interaction between GO and CTAB/SWNT is ionic bonding and π–π interaction, but GO and SDBS/SWNT is only π–π interaction. Therefore, CTAB/SWNT is more effective reinforcement for GO foams than SDBS/SWNT , Table 1Although graphene has superior properties, such as chemical and mechanical properties, it has not been commercialized or translated into practical outcomes, yet. Recently, 3D graphene architectures have been attracting significant interests as a key solution to solve problems, such as low productivity, high cost, difficulty in controlling high-quality materials, defects and dispersion problem. However, recent researches on 3D graphene architectures struggle with strength. To solve this obstacle, we synthesized Graphene-CNT hybrid foams by introducing CNT as a reinforcement. In addition, when CNT dispersions were processed, a strategy of interaction was applied by using anionic and cationic surfactants.This study confirmed that GO-CNT hybrid foams had good connections between CNT with each surfactant and GO. The charge effect of surfactants, covered CNT, was experimentally investigated. Furthermore, by applying CNT as a reinforcement, the compressive modulus and strength at 10% strain dramatically increased. Therefore, it can be concluded that the CNT with different ionic surfactants as reinforcement can act as a good reinforcement. Moreover, it was experimentally investigated that the CTAB/SWNT is a more effective reinforcement for GO foams because the formation of ionic bonding between GO and CTAB/SWNT is stronger than the π–π interaction induced from GO and SDBS/SWNT.


Incidental findings of non-caseating granulomas and metastasis in sentinel lymph nodes are rare but cause clinical challenges. We report a case of coinciding unexpected asymptomatic lymphoid sarcoidosis and a micrometastasis in a sentinel node of a patient, who was newly diagnosed with 2.0 mm thick melanoma on the left calf. Sarcoidosis is a relatively uncommon systemic autoimmune disease characterised by the development of non-caseating granulomas in multiple organ systems, mostly the lungs and the lymph nodes. Clinical symptoms are fatigue, weight loss, and pulmonary symptoms. Sarcoidosis predispose individuals to cancer  and melaSarcoidosis is caused by an aberrant immune response towards an unknown agent, yielding repeated cycles of Tumour Necrosis Factor-α production and infiltrating T-helper-1 cells and macrophage. The interaction between these pleomorphic manifestations in the lymph nodes and predisposition to metastatic melanoma is unknown.A 43-year old healthy male with no familiar disposition of or previous history of melanoma or symptoms of pulmonary, systemic, or cutaneous sarcoidosis was referred with a 2.0 mm thick, intermittently bleeding superficially spreading malignant melanoma on the left calf. AJCC (7th edition) stage was pT2a. No lymhadenopathy was found by clinical examination. A 2.0 cm margin of excision to fascia was performed. The sentinel node was located by preoperative lymphoscintigraphy . Blue dy18 Fluorodeoxyglucose positron emission tomography and Computed Tomography (18FDG-PET-CT) revealed multiple metabolically active mediastinal lymph nodes  or reviews (3 articles) on the topic. One article was omitted due to foreign language. A total of fifteen case reports describing coincidence of sarcoidosis and metastatic melanoma were found . In a sy articlesThe patient gave written informed consent prior to publication of this case report.


Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for hematological malignancies (i.e. leukemia and lymphoma), because graft-versus-leukemia (GVL) activity mediated by alloreactive T cells can eliminate residual malignant cells and prevent relapse. However, the same alloreactive T cells also mediate a severe side effect, graft-versus-host disease (GVHD), and prevention of GVHD while preserving GVL activity remains an elusive goal. The immune checkpoint molecule PD-L1 and its interaction with PD-1 receptor in regulating cancer immunity is under intensive and wide-spread study, but knowledge about this interaction in regulating GVHD and GVL activity is very limited. In this review, we summarize the literature exploring how PD-L1 interaction with its receptors PD-1 and CD80 regulate GVHD and GVL activities, how PD-L1 signaling regulates T cell metabolic profiles, and how a differential role of PD-L1 interaction with PD-1, CD80 or both may provide a novel avenue to prevent GVHD while preserving strong GVL effects. Hematopoietic cell transplantation (HCT) provides curative therapy for hematological malignancies, such as lymphoma and leukemia, owing to graft-vs.-leukemia/lymphoma (GVL) effects mediated by alloreactive T cells –5. HowevAlloreactive T cells are activated by host antigen presenting cells (APCs) in lymphoid tissues early after HCT, and they then migrate to GVHD target tissues that were initially damaged by conditioning regimen to cause tissue inflammation and GVHD , 7. Acut+ cells (Treg), donor APCs, B regulatory cells (Breg), myeloid-derived suppressor cells (MDSC), and immune checkpoints including interactions of programmed death ligand 1 (PD-L1) with programmed death-1 (PD-1) and CD80 , is a member of the B7 family of immuno-coinhibitory and costimulatory molecules and functions as an immune checkpoint via its interaction with its receptors PD-1 and CD80 . PD-L1 is an Ig-V like transmembrane protein constitutively expressed by hematopoietic cells such as T, B and dendritic cells and by parenchymal cells in response to cytokine  induction. PD-L1 was co-discovered in 2000 by two groups who reported it to have conflicting functions in the regulation of T cell activation, proliferation and apoptosis , 29. Thein vivo studies on the role of PD-L1 were also conflicting. Tissue-specific transgenic expression of PD-L1 under the insulin promoter in islet beta cells augmented the rejection of islet grafts, which was associated with increased proliferation and reduced apoptosis of infiltrating CD8+ T cells . Under these conditions, costimulation with CD80-Ig decreased the proliferation of double-deficient T cells, indicating that CD80 can signal through PD-L1 expressed by T cells to inhibit proliferation . CD80, a member of the B7-super family, is a dimeric transmembrane protein, is constitutively expressed by T cells and is further upregulated upon T cell activation . Generalferation . Furtherferation . Taken tin vivo studies using an anti-PD-L1 mAb that specifically blocks PD-L1/CD80 interaction while preserving PD-L1/PD-1 interaction have established PD-L1/CD80 “reverse signaling” into T cells as being pro-tolerogenic. In a murine model of immunization, blockade of PD-L1/CD80 interaction led to increased expansion and reduced induction of T cell anergy during the contraction phase following immunization . Phosphorylated T-bet localized to the Ifng locus and subsequently increased IFN-γ expression  after HCT in a minor MHC-mismatched model of GVHD (DBA/2 → BALB/c)  . Surviva of GVHD . Moreove of GVHD . Finally of GVHD .+ T cell pathogenesis in GVHD target tissues after HCT  on day 5 after HCT led to a reduction in proliferation and apoptosis, leading to an augmentation of donor T cell survival and an augmentation of GVHD  and reduced expression of gut homing and chemokine receptors  that blocks both PD-L1/CD80 and PD-L1/PD-1 interactions augmented GVHD and led to an increase in Glut1 expression and glycolysis with a subsequent reduction in mitochondrial respiration (OXPHOS) , oxygen consumption [indicative of increased utilization of O2 in the electron transport chain (ETC)], mitochondrial membrane potential and ROS production   . In contxpansion . Specifiiration) . In contiration) .−/− recipients suggest that in the absence of host PD-L1, PD-L1 interactions with PD-1 and CD80 among donor cells may modulate donor T cell metabolism. In addition, PD-L1/CD80 interaction may play an important role in augmenting donor cell expansion in lymphoid tissues. We recently demonstrated that PD-L1 expressed by alloactivated donor T cells augment donor T cell expansion in lymphoid tissue through T cell-T cell interaction to augment GVL , inhibit IL-2 activity or augment PD-L1/PD1 interactions in GVHD target tissues could tolerize infiltrating T cells while maintaining the function of T cells in lymphoid tissues, such that GVHD could be effectively prevented while strong GVL activity is preserved.KC wrote the manuscript. PM and DZ revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We report a graphene-based energy harvesting system powered by enzymatic biowaste reaction through two-dimensional (2D) nanofluidic channels; the integrated 2D nanofluidic generator shows distinct advantages such as flexibility, low cost, and high output in ionic currents. Effective recycling of biowaste energy in a compact system remains a great challenge. Here, we report a graphene-based energy harvesting system powered by enzymatic biowaste reaction through two-dimensional (2D) nanofluidic channels. The integrated 2D nanofluidic generator shows distinct advantages such as flexibility, low cost, and high output in ionic currents. The NH4+ ions migrate across the membrane under the force of chemical gradients from the reaction chamber to the receive chamber, resulting in a net ionic current (Ic) and charge imbalance across the membrane . When the ionic flow goes through the membrane in vertical direction driven by the enzymatic biowaste reaction, energy conversion is generated as a net transmembrane ionic current. Additionally, this design strategy of the graphene-based 2D nanofluidic system can be generally extended to other 2D and polyelectrolyte materials for smart nanofluidic electrogenic devices and clean energy.Here we demonstrate a graphene-based nanofluidic energy harvesting system powered by an enzymatic biowaste reaction . Polyacr–1, assigned to the C–H stretching frequencies of PAA. In comparison, GO shows very weak absorption bands at the same region. The obvious signal difference indicates the successful functionalization of PAA onto the surface of graphene. Futhermore, it is known that chemically reduced graphene obtained through the hydrazine method precipitates as agglomerates owing to their hydrophobic nature. Our study shows that, with the assistance of negatively charged polymer, the graphene–PAA mixture is well-dispersed even after chemical reduction  was prepared by vacuum filtration of dispersed graphene–PAA complex colloid solution through a poly(vinylidene fluoride) (PVDF) filter film–4 M, and then reached equilibrium below 10–4 M , which was connected with the test chamber through an agar-saturated KCl salt bridge (Scheme S1Ic) is solely generated from the enzyme-triggered ion transport through the graphene nanochannels. The transmembrane potential (Em) and the diffusion current (Ic) were recorded as a function of time elapsed  and charge imbalance on both sides of the film (Em). Peak values of 851 nA and 9.72 mV can be reached within 8 min, suggesting the complete degradation of urea, and the electric power density reaches 2.7 mW m–2. Based on our experimental data and a previous report,ηe) were calculated as 177 mV and 0.3%, respectively . After adding urea, the ionic current gradually reaches the maximum value within 8 min. The amplitude of the resulting ionic current increases with the urea concentration from 0.2 mM to 10 mM at a constant rate of 82 nA mM–1 (The time dependency of ionic current with respect to the urea concentration is shown in  nA mM–1 . However nA mM–1 . More urvia harvesting of both ionic current and generation of membrane potential. The values of current, potential and electric power density are 600 nA, 7 mV and 1.3 mW m–2, respectively. The above results reveal practical application for energy conversion using human biowaste. At the present stage, the output voltage is generally less than 10 mV. There is still a long way to go for this technology to be available for most real-world applications. From our point of view, there are two possible strategies to improve the performance of the 2D membrane. First, as key influencing factors, the channel structure and charge density inside the 2D nanochannels could be further tuned through the optimization of film preparation steps. Second, enzymatic catalytic reaction could also be optimized to increase the charge selectivity of the membrane. Further efforts are needed toward a better performance of energy conversion through 2D nanofluidic networks.To examine the possibility of the GPM for practical biowaste conversion, urine from human metabolism was added into the reaction chamber. In conclusion, we demonstrate an energy harvesting device powered by enzymatic biowaste reaction through polyelectrolyte functionalized 2D graphene nanofluidic channels. PAA-functionalized graphene forms a layered structure, and constructs interconnected 2D nanofluidic networks inside the membrane. Negatively charged GPM governs the surface-charge-governed ion transport, preferentially permeating counter-ions while excluding co-ions. Under the force of the enzymatic biowaste reaction, energy conversion is observed in the generation of ionic current and electric potential across the membrane. Our results suggest that the as-proposed 2D graphene–PAA nanosystem shows a sensitive and rapid response to human urine. We believe that the strategy is generally applicable to other types of nanofluidic devices for energy harvesting.Supplementary informationClick here for additional data file.


Ghrelin is a 28-amino-acid peptide that regulates energy hemostasis, glucose and lipid metabolism. We aimed to explore the effects of ghrelin on the proliferation and differentiation of 3T3-L1 and human primary preadipocytes.3- 2,5-diphenyl tetrazolium bromide (MTT) spectrophotometry, Oil Red O staining, intracellular glycerol-3-phosphate dehydrogenase (G-3-PDH) assays and semiquantitative reverse transcription polymerase chain reaction were used to investigate the action of ghrelin.p < 0.05). Ghrelin also promoted the proliferation of human primary preadipocytes from 24 h (p < 0.05) to 48 h (p < 0.05) at a concentration of 1000 ng/ml. Further investigation showed that IGF-1 levels were notably increased in ghrelin-treated 3T3-L1 and human preadipocytes, and IGF-1 antibody was capable to attenuate this stimulatory action of ghrelin . Additionally, ghrelin significantly suppressed the differentiation of 3T3-L1 and human primary preadipocytes; 10 ng/ml ghrelin notably downregulated G-3-PDH activities in 3T3-L1 cells on day 3 and in human cells from days 4 to 12 following differentiation , and the intracellular lipoprotein lipase mRNA levels were lower than that of the controls (p < 0.05). Further investigation showed that the mRNA levels of peroxisome proliferator-activated receptor γ2 (PPARγ2) and CCAAT/enhancer binding protein α (C/EBPs) were also suppressed in ghrelin-treated human differentiating adipocytes.Ghrelin (0.01–1000 ng/ml) significantly increased the numbers of 3T3-L1 cells, and the maximum stimulatory effect was observed with the 10 ng/ml ghrelin treatment for 24 h (Ghrelin promotes the proliferation of 3T3-L1 and human primary preadipocytes by increasing the expression of IGF-1. Ghrelin inhibits murine and human adipocyte differentiation by downregulating PPARγ2 and C/EBPα levels, consequently leading to decreased lipid accumulation and lipogenic enzymes expression. Obesity and its related disorders, such as type 2 diabetes, dyslipidemia, and cardiovascular disease has increased in prevalence in recent years and has become a vital health concern worldwide . ObesityPreadipocytes are recruited from multipotent stem cells and regularly undergo mitotic clonal expansion, thereby upregulating adipocyte number. When treated with appropriate cytokines or hormones, preadipocytes are growth-arrested and induced to differentiate . In the Factors influencing adipogenesis and lipid retention have been receiving substantial attention from researchers, and several hormones, such as insulin, glucocorticoid and thyroxine, have been found to affect proliferation and differentiation of preadipocytes . GhrelinSeveral studies have shown that ghrelin stimulates the adipogenesis of 3T3-L1 preadipocytes , but the3T3-L1 preadipocytes were received as a kind gift from the Department of Genetics, Institute of Life Sciences, Peking University, Beijing. Human primary preadipocytes were obtained from the abdominal subcutaneous adipose tissues (SATs) of three patients undergoing surgery . The clinical details of these three patients are listed in g for 5 min. The supernatant was discarded, and the cell pellet was re-suspended in DMEM/F12 medium. Unfiltered tissues were digested once more according to the steps above. Cells were collected and counted before being seeded in DMEM/F12  with the addition of 10% FBS , then cultured in a 5% CO2 95% air atmosphere incubator. The inverted phase contrast microscope  was used to observe the growth and differentiation state. The cells were induced to differentiate in AIM-V  medium supplemented with 0.5 μM insulin, 0.25 μM dexamethasone, 0.2 nM thyroxine, and 0.5 nM 3-isobutyl-1-methylxanthine (IBMX) . The differentiation medium was refreshed every 2 days.Human subcutaneous adipose tissues were prepared in 5–10 g samples, then rinsed by 1 × phosphate buffered saline (PBS) three times. The remaining connective tissue and blood vessels were cleared. The adipose tissues were digested with 5 ml collagenase (2 mg/ml) for 40 min at 37°C. Samples were filtered through a 25 μm mesh and centrifuged at 600 3/well in 10% FBS/DMEM medium for 12 h, then transferred to AIM-V medium. After 4 days, 3T3-L1 preadipocytes were added with 0.01, 0.1, 1, 10, 100, 1000 ng/ml ghrelin, and human cells were added with 0.1, 10, 1000 ng/ml ghrelin. After 4, 8, 24, 48 and 72 h, the preadipocytes were rinsed twice using 1 × phosphate buffered saline (PBS). 3- 2,5-diphenyl tetrazolium bromide  solution was added to each well with a final concentration of 1 mg/mL, and the cells were incubated for 4 h. Then, 100 μL dimethylsulfoxide solution was added, and the cells were shaken for 10 min. Afterward, optical density (OD) value was recorded using an ELISA reader  at a wavelength of 492 nm. IGF-1 antibody was added after treatment with ghrelin . Experiments were repeated six times with individual octuplicate samples in each experiment.Preadipocytes were seeded in a 96-well plate with a density of 5 × 103/well in 10% FBS/DMEM medium for 12 h. The cells were rinsed twice using 1 × phosphate buffered saline (PBS), then transferred to X-Vivo 15 medium  medium for 4 days. After treatment with 1, 10, and 100 ng/ml ghrelin for 24 and 48 h, 10 μL of the Cell Counting Kit-8  solution was added to each well of the plate. The cells were then incubated for 2 h in the incubator, and the absorbance was measured at 450 and 620 nm using a microplate reader .3T3-L1 preadipocytes were seeded in a 96-well plate with a density of 8 × 103/well as mentioned above. After 4 days, 3T3-L1 preadipocytes were added with 0.25, 1, 2.5, 5, 10, 25, and 100 ng/ml IGF-1, and human primary preadipocytes were added with 1, 10, 100 ng/ml IGF-1 for. After 24 h, MTT assays were conducted to determine the effect of IGF-1 on proliferation.3T3-L1 and human preadipocytes were seeded at a density of 5 × 103/well in 96-well plate and the differentiation was performed based on the protocol mentioned above. After 3 days in AIM-V medium, cells were treated with 0.1, 10, 1000 ng/ml ghrelin and 1, 10, 100 ng/ml IGF-1 in AIM-V differential medium for 4, 8, 12 days for human primary preadipocytes and 1, 3, 6 days for 3T3-L1 cells due to a faster differentiation rate in murine preadipocytes. Oil Red O  staining was conducted as described previously  was measured to observe the triglyceride synthesis action of cells . Murine The differentiating cells were treated with 10 ng/ml ghrelin and IGF-1 for 8 days. Total RNA was extracted using a Trizol RNA kit , and 2 μg RNA was reverse transcribed with an RT-PCR system including M-Mul reverse transcriptase , RNasin , Oligo (dT) primer  and deoxyribonucleoside Triphosphate . 2 μl RT products (cDNA) were amplified with human LPL, PPARγ2, C/EBPα, β-actin primers and mouse LPL, IGF-1 primers as shown in TM-ACF Plus Medium  based on the protocol mentioned above. Concentrations of 1, 10, and 100 ng/ml ghrelin  were administrated for 24 h to preadipocytes and 8 days to differentiating cells. RT-qPCR analysis was performed using SYBR Premix Ex Taq  and an ABI7500 PCR system  as previously described . RT-qPCR was conducted in 3T3-L1 preadipocytes and adipocytes to assess the expression of proliferation and differentiation-related genes. The primer sequences of target genes are listed in Ct–ΔΔ. RT-qPCR was duplicated in 2 wells in a final volume of 20 μL.3T3-L1 preadipocytes were cultured in X-Vivo 15 medium and MesenCultescribed . The tott-test was used to analyze for two groups, and one-way ANOVA was used for three or more groups. The Mann–Whitney U or Kruskal–Wallis test was used if data were still not normally distributed. p < 0.05 was considered significant for all analyses.All statistical analyses were performed using SPSS 10.0 for Windows, and all values were expressed as the mean ± standard deviation (SD). A th day of differentiation, large lipid droplets could be notably observed, and 95 to 98% of the cells differentiated to mature adipocytes , and this effect was further enhanced with the increase in ghrelin concentrations. The maximum promotion effect was observed at 10 ng/ml, which was up to 114.9% of the control group (p < 0.05). When the concentration of ghrelin was increased to 100 and 1000 ng/ml, its promotional effect was slightly weakened but was still higher than that of the control groups (p < 0.05).3T3-L1 preadipocytes were treated with 0.01–1000 ng/ml ghrelin for 24 h. As presented in p < 0.05).To investigate the time curve of ghrelin on 3T3-L1 preadipocyte proliferation, 3T3-L1 preadipocytes were treated with 10 ng/ml ghrelin for 4–48 h. As shown in p < 0.05). This stimulatory effect was not observed at lower concentrations (0.1 and 10 ng/ml). p < 0.05) to 48 h , but this stimulatory effect disappeared at 72 h.We observed that ghrelin promoted the proliferation of 3T3-L1 preadipocytes, and a similar result was also noted in human primary preadipocytes. Human primary preadipocytes were treated with 0.1–1000 ng/ml ghrelin for 24 h. As shown in p < 0.05), but the concentrations showed no significant difference at 48 h compared with the control group , and this effect was concentration-dependent. The maximum promotion effect was observed at 100 ng/ml, which was up to 134% compared with the control group (p < 0.05). IGF-1 also presented a similar concentration-dependent stimulatory effect in human cells , and 1.32-fold of that at both 8 h (p < 0.05) and 24 h (p < 0.05), suggesting that IGF-1 may be involved in the stimulatory proliferation of ghrelin in 3T3-L1 cells. To further confirm the hypothesis, the various dilutions of IGF-1 antibodies  were added together with 10 ng/ml ghrelin. As presented in p < 0.05), whereas no significant changes were observed at the lower dilutions of 1:1000 (p > 0.05) and 1:5000.IGF-1 is a growth factor known to promote cell proliferation. To determine whether IGF-1 is involved in the proliferation of 3T3-L1 preadipocytes, the cells were treated with 10 ng/ml ghrelin for 4, 8, and 24 h, and the levels of IGF-1 mRNA were assessed using RT-PCR. As presented in p = 0.0175) in human cells  of IGF-1 antibody was capable to attenuate the stimulatory action of 1000 ng/ml ghrelin . The inhibitory effect was observed at 10 and 1000 ng/ml ghrelin on days 1, 3 and 6 as well, but this effect was not further enhanced with the increase of doses and the extension of action time.Oil Red O staining can indirectly assess triglycerides in the cytoplasm of differentiated adipocytes. To investigate the effect of ghrelin on the differentiation of 3T3-L1 preadipocytes, these cells were treated with 0.1, 10, and 1000 ng/ml ghrelin for 1, 3, and 6 days. In this study as depicted in rd day after differentiation (p < 0.05).As the enzyme G-3-PDH is specifically expressed in differentiating adipocytes, we further investigated the effect of ghrelin on G-3-PDH activity. 10 ng/ml ghrelin was added to 3T3-L1 preadipocytes in a serum-free differential medium for 1, 3 and 6 days, and G-3-PDH activity was measured. As shown in p < 0.05).The experiments discussed above suggested that ghrelin inhibited the differentiation of 3T3-L1 preadipocytes based on triglyceride content and G-3-PDH activity. Consistent with the results of the mouse 3T3-L1 cells, the OD values of the human differentiating adipocytes also decreased with administration of 0.1 to 1000 ng/ml ghrelin for 4, 8, and 12 days , but thep < 0.05), 10.5%  and 9.3%  on days 4, 8, and 12, respectively.3T3-L1 preadipocytes were treated with 1, 10, 100 ng/ml IGF-1 for 1, 3, and 6 days. In contrast to the observation of ghrelin, as shown in p < 0.05) in 3T3-L1 adipocytes (p < 0.05) in human adipocytes  and 30.1%  compared with the controls, suggesting that PPARγ2 and C/EBPα were involved in the inhibitory differentiation of ghrelin in human differentiating adipocytes.PPARγ2 and C/EBPα are two important transcription factors that can activate and mediate synergistically in the differentiation of preadipocytes. To clarify the effect of ghrelin on these adipogenic markers, the expression of PPARγ2  and C/EBp < 0.05).Acetyl-CoA carboxylase is an essential enzyme for fatty acid synthesis. 3T3-L1 preadipocytes were treated with 1, 10, and 100 ng/ml ghrelin for 8 days after inducing to differentiation. As depicted in Ghrelin is a 28-amino-acid peptide that has been widely found to regulate energy homeostasis, glucose and lipid metabolism, and its receptor, GHS-R1a has been expressed in rodent and human adipocytes . Our stu–7 to 10–15mol/L , which is consistent with our results. The study performed by –7, 10–11 to 10–15mol/L) promoted 3T3-L1 preadipocyte proliferation, and 10–9 and 10–11mol/L ghrelin also presented a stimulatory effect at 6 h. In our study, we found that the 10 ng/ml ghrelin stimulated 3T3-L1 cell growth at 24 h. A similar effect on human primary preadipocyte proliferation was also observed, but it was at a higher concentration (1000 ng/ml) compared with 3T3-L1 cells. Because the expression of ghrelin receptor-GHSR has been found in both human and rodent adipocytes, an explanation may be the difference in the receptor number and affinity of these two species. Rat ghrelin was used in our present study, and it may have higher affinity with the receptors of murine 3T3-L1 preadipocytes than human preadipocytes (3 ng/ml) in cardiomyocytes /Akt and ERK1/2 pathways . In our –11 to 10–13 mol/L  for 6 or 9 days. The stimulatory effect of ghrelin was also observed in human visceral adipocytes at concentrations of 10 to 1000 pmol/L   inhibited preadipocytes differentiation. The stimulatory effect of ghrelin was observed at concentrations of 10–11 to 10–13 mol/L in other studies, whereas 0.1 to 1000 ng/ml ghrelin inhibited differentiation in our study. A possible explanation may be that ghrelin is able to promote differentiation at low doses but markedly suppresses adipogenesis at the high doses. Interestingly, we found that ghrelin modulated proliferation by increasing IGF-1 levels  . In cont1 levels , whereas1 levels , and ano1 levels . GhrelinIn the present study, we demonstrated that ghrelin downregulated the expression of lipogenesis enzymes, thereby reducing the lipid content in adipocytes. Additionally, ghrelin has been reported to have other effects on adipocytes, such as inhibition of lipolysis and anti-inflammation. In this regard, ghrelin suppresses lipolysis by activating of PI3Kγ/Akt and phosphodiesterase (PDE)  in mouseGhrelin enhanced the proliferation of 3T3-L1 and human primary preadipocytes by increasing the expression of IGF-1. Additionally, ghrelin inhibited the differentiation of mouse and human preadipocytes by downregulating PPARγ2 and C/EBPα levels. As a result, lipid accumulation and lipogenic enzymes consistently decreased.All datasets generated for this study are included in the manuscript/HM did the cell experiments, analyzed data, and wrote the primary manuscript. HP supervised the experiments. LW and HY helped to analyze data. HZ designed the experiments and did the cell experiments. FG supervised the whole experiments and revised the primary manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dysregulation of TAR DNA-binding protein 43 (TDP-43) is a hallmark feature of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two fatal neurodegenerative diseases. TDP-43 is a ubiquitously expressed RNA-binding protein with many physiological functions, playing a role in multiple aspects of RNA metabolism. We developed transgenic mice conditionally overexpressing human wild-type TDP-43 protein (hTDP-43-WT) in forebrain neurons, a model that recapitulates several key features of FTD. After post-weaning transgene (TG) induction during 1 month, these mice display an early behavioral phenotype, including impaired cognitive and social function with no substantial motor abnormalities. In order to expand the analysis of this model, we took advantage of the temporal and regional control of TG expression possible in these mice. We behaviorally evaluated mice at two different times: after 2 weeks of post-weaning TG induction (0.5 month group) and after subsequent TG suppression for 2 weeks following that time point [1 month (sup) group]. We found no cognitive abnormalities after 0.5 month of hTDP-43 expression, evaluated with a spatial working memory task (Y-maze test). Suppression of TG expression with doxycycline (Dox) at this time point prevented the development of cognitive deficits previously observed at 1 month post-induction, as revealed by the performance of the 1 month (sup) group. On the other hand, sociability deficits  appeared very rapidly after Dox removal (0.5 month) and TG suppression was not sufficient to reverse this phenotype, indicating differential vulnerability to hTDP-43 expression and suppression. Animals evaluated at the early time point (0.5 month) post-induction do not display a motor phenotype, in agreement with the results obtained after 1 month of TG expression. Moreover, all motor tests  showed identical responses in both control and bigenic animals in the suppressed group, demonstrating that this protocol and treatment do not cause non-specific effects in motor behavior, which could potentially mask the phenotypes in other domains. Our results show that TDP-43-WT mice have a phenotype that qualifies them as a useful model of FTD and provide valuable information for susceptibility windows in therapeutic strategies for TDP-43 proteinopathies. Neurodegenerative diseases are incurable and debilitating conditions that arise as a consequence of the progressive degeneration and/or death of nerve cells. This heterogeneous group of disorders is characterized by behavioral changes that differ according to the disease entity. Among these, most forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) show clinic-pathological overlap. These diseases may represent a single pathological entity with diverse clinical manifestations , includeTDP-43 is a highly conserved and widely expressed RNA-binding protein (RBP) that normally resides predominantly in the nucleus of all cells. It has been described to be involved in different cellular processes, most conspicuously RNA metabolism, including RNA translation, splicing, and transport . DysreguGiven that multiple  forms of both sporadic and familial FTD/ALS cases of different genetic origin  converge in a common pathological presentation that involves TDP-43 dysregulation , there iUsing a mouse model of TDP-43 proteinopathies that conditionally overexpresses the wild-type human TDP-43 protein (hTDP-43-WT) in forebrain neurons and reproduces neuropathological changes of the FTD/ALS spectrum , we haveIn this work, we aimed to investigate how short-term transgene suppression affects the early behavioral phenotypes displayed by conditional TDP-43-WT mice . In partThese results emphasize the need to comprehensively evaluate the behavioral phenotypes in multiple disease models and suggest that the timing of treatment could widely influence the outcome of the different clinical manifestations of TDP-43 proteinopathies.ad libitum access to food and water. To produce hTDP-43 transgenic lines, as described previously . The protocol was approved by the CICUAL. Mice were kept under a 12-h light/dark cycle, with controlled temperature (23 ± 2°C) and humidity (40–60%) and had eviously , pronuclMice were treated with 0.2 mg/ml Dox  in drinking water to avoid prenatal and postnatal developmental effects of transgene expression. hTDP-43 expression was induced by switching mice to regular drinking water (without Dox) at weaning . Mice were analyzed at different time points .Mice were screened for the presence of the transgene using genomic DNA isolated from ear biopsies. PCR amplification was done with the following primers: TDP-forward (TTGGTAATAGCAGAGGGGGTGGAG), MoPrP-reverse (TCCCCCAGCCTAGACCACGAGAAT), Camk2a-tTA-forward (CGCTGTGGGGCATTTTACTTTAG), and Camk2a-tTA-reverse (CATGTCCAGATCGAAATCGTC) as previously described . To homoFor suppression experiments, mice were treated again with 0.2 mg/ml Dox in drinking water at 0.5 month after weaning to suppress transgene expression for 2 weeks, as indicated in All behavioral tasks were performed during the light phase  with the exception of the Y-maze spontaneous alternation, which was conducted during the initial dark phase (7:00 p.m to 9:00 p.m.) to maximize exploratory behavior and consistently obtain a high number of arm visits. Animals were allowed to habituate in the experimental room (with attenuated light and sound) for at least 1 h before the tests. All tests were recorded through a video camera mounted above the experimental room (unless noted) and mouse position was analyzed by automatic video tracking software . All mazes and objects used in behavioral analysis were cleaned with 10% ethanol between test sessions and sanitized with 70% ethanol at the end of the day.In agreement with what we previously demonstrated in experiments with TDP-43-∆NLS and TDP-43-WT transgenic mice , 2016, aThe horizontal Y-shaped maze consisted of three identical arms of transparent Plexiglas (43 cm × 4 cm × 12.5 cm) placed at 120° angles to each other . The tesThe test apparatus comprised a black Plexiglas rectangular box (40.6 cm × 15 cm × 23 cm) divided into three interconnected chambers. The floor of the apparatus was covered with clean bedding. The task was performed as previously described , 2016. FTo analyze general locomotion and exploratory behavior in a novel environment, we performed the open field test as previously described , 2016. TFor the assessment of motor coordination and balance, we used a rotarod apparatus . The test was performed as previously described . BrieflyThe presence of clasping was evaluated as previously described . hTDP-43Grip strength was studied using a standard wire cage and performed as previously described , 2016. BAll animals were deeply anesthetized with intraperitoneal administration of 5% chloral hydrate (1 ml/30 gr). Next, mice were perfused transcardially with ice-cold PBS  supplemented with 10 U/ml heparin. The brains were immediately extracted and fixed overnight by immersion in 4% paraformaldehyde (PFA), and then cryoprotected in 10 and 30% sucrose in PBS for immunofluorescence analyses.Fixed frozen hemispheres were cryosectioned (50 μm) on a sliding freezing microtome . The brain slices were stored at −20°C in cryoprotecting solution . Double immunofluorescence was performed as follows: the coronal free-floating sections were washed 2 × 5 min with PBS, permeabilized with 1% Triton X-100 in PBS for 1 h, and blocked for 1 h with 0.3% Triton X-100 and 5% goat serum in PBS. The primary antibodies (diluted in 0.3% Triton X-100 and 3% goat serum in PBS) were incubated overnight at 4°C with the indicated dilutions: polyclonal rabbit anti-TDP-43  two-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison post hoc test, when comparing three or more groups in the social interaction experiments. RM-ANOVA followed by Bonferroni’s multiple comparisons post hoc test was used for accelerated rotarod. Fisher exact test was performed for clasping analysis. When non-parametric tests were required (hanging wire grip test), Mann-Whitney U test was used. Statistical analysis of behavioral tests was performed using PRISM 6 (Graph Pad software) or Statistica 7 (Stat Soft). Data are presented as mean values ± SEM. A p < 0.05 was considered statistically significant.Statistical tests were performed as follows, as described in the text and figure legends for each dataset. Student’s In a recent work, we performed a detailed characterization of the behavioral changes occurring in hTDP-43-WT mice . Using atTA/WT12 bigenic mice or control littermates were induced at weaning  by removing Dox from their water supply . ParalleIn order to assess proper regulation of transgene expression within our experimental timeline, we performed double immunofluorescence studies –E. We ust(21) = 0.8096, p = 0.4273; t(21) = 0.2947, p = 0.7711; We have recently demonstrated that post-weaning overexpression of hTDP-43-WT during 1 month leads to cognitive deficits in our inducible tTA/WT12 mouse model . These pt test analysis established that bigenic animals from the 1 month (sup) group did not show significant differences compared with control mice (t(20) = 1.658, p = 0.1128; t(20) = 0.3583, p = 0.7238; To evaluate if short-term (2 weeks) transgene suppression could prevent the installment of this deficit, we reintroduced Dox in the drinking water of these animals after the 0.5 month time point and assessed Y-maze performance 1 month post-weaning, in a group termed 1 month (sup)  = 6.32, p = 0.019; F = 4.60, p = 0.042; F = 46.98, p < 0.0001; F = 0.57, p = 0.46, genotype F = 0.54, p = 0.47, interaction F = 0.51, p = 0.48, t(24) = 0.7137, p = 0.4823, Student’s t test) or to general deficits in novelty exploration  than with the inanimate object . Bigenic mice showed a deficit in social interaction evidenced by a significant decrease in time spent in direct social exploration of a conspecific demonstrator  = 6.41, p = 0.019; F = 27.76, p < 0.0001; genotype F = 5.60, p = 0.027; F = 4.75, p = 0.041; F = 2.76, p = 0.11; genotype F = 0.21, p = 0.64; interaction F = 0.02, p = 0.88; t(21) = 1.967, p = 0.063, Student’s t test). These results show that TDP-43-WT mice very rapidly develop alterations in social behavior, and these cannot be ameliorated by short-term suppression of transgene expression. Moreover, they support the idea that diverse behavioral domains present differential susceptibility to hTDP-43 overexpression and subsequent renormalization of TDP-43 levels.Next, we assessed the effect of turning off hTDP-43 expression on social behavior. Notably, transgene suppression in the 1 month (sup) group did not cause any improvement in social interaction in bigenic mice respective to controls  = 0.6150, p = 0.5444; t(24) = 0.7907, p = 0.4369; t test) between bigenic and control groups, including average speed  = 0.6294, p = 0.5351), maximum speed  = 0.5888, p = 0.5615), and percentage time in center  = 0.8465, p = 0.4057).We assessed general motor function and exploratory activity in the 1 month (sup) group using the open field test –D. In agF = 0.9200, p = 0.3470 for group; F = 44.09, p < 0.0001 for trial; F = 2.342, p = 0.0804 for interaction; Next, we evaluated motor coordination and balance in TDP-43-WT animals using the accelerated rotarod test. Both bigenic and control mice behaved similarly in the 1 month (sup) paradigm  motor abnormalities are not present after 0.5 month of transgene expression, consistent with our results after 1 month of hTDP-43 overexpression  and (2) In summary, we present evidence that defines the early time course of behavioral impairments and establish the susceptibility of different behavioral domains to transgene suppression in tTA/WT12 mice .Animal models are invaluable tools to understand the pathological basis of neurodegenerative diseases such as FTD and ALS. In this study, we explored the behavioral consequences of suppressing hTDP-43 expression in our inducible tTA/WT12 mice.The identification of disease-related molecules, the discovery of pathogenic pathways, and the targeting of pathogenic proteins are all critical steps to establish therapeutic strategies for incurable diseases, including neurodegenerative conditions. A major breakthrough was the discovery of TDP-43 as a pathological hallmark of ALS and FTD . MutatioC. elegans, Drosophila) and vertebrate  organisms (reviewed in TDP-43 belongs to a large family of RNA-binding proteins (RBPs) that have been shown to have multiple links with disease pathogenesis. Of specific relevance for neurodegenerative diseases, a prominent theory in the field states that neurons are particularly vulnerable to disruption of RBP dosage and dynamics . In partInducible animal models based on the tet-tTA system have been used to assess the neuropathological and behavioral impact of expression of different neurodegeneration-associated proteins, including TDP-43, tau, APP, α-synuclein, SCA1, SCA3, and huntingtin . HoweverTransgenic mice expressing TDP-43 carrying a pathogenic A315T mutation in CNS neurons display early motor and anxiety-like phenotypes that are reversible on Dox treatment, while memory impairments persist after transgene suppression . Our preOur results show that cognitive performance, as evaluated in the Y-maze test, remains unaltered after 2 weeks of wild-type hTDP-43 expression (0.5 month group). By contrast, we previously reported that expression of TDP-43-∆NLS using the same promoter system and induction protocol leads to a clearcut decrease in Y-maze alternation, reaching levels indistinguishable from chance and thus indicating severe deficits in spatial working memory . While tIn terms of social phenotype, our conditional hTDP-43-WT animal model recapitulates deficits that constitute a core feature of the clinical FTD/ALS spectrum, particularly in several subtypes of FTD . DecreasAlthough motor abnormalities develop after at least 6 months of transgene expression in TDP-43-WT mice , we sougA limitation  of this model is that, due to the pattern of expression provided by the driver transgenic line (CamKII promoter), which results in forebrain-enriched neuronal hTDP-43-WT expression, part of the phenotype can be restricted due to sparing of other regions . However, there are other rodent models available with pan-neuronal TDP-43 expression  as contributors to this phenotype.In summary, we show here that not only TDP-43-WT mice recapitulate several core behavioral features of FTD/ALS spectrum of human pathology, but also these behavioral domains display a different time course of onset and sensitivity to transgene suppression. This information is particularly relevant to understand both the time windows of efficacy for potential treatments and the selectivity/sensitivity to TDP-43 dysregulation of the neural circuits underlying the clinical phenotypes displayed by FTD/ALS patients. We also consider that our inducible model is especially relevant to explore the etiology of TDP-43-related FTD, due to the predominant social/cognitive phenotype with early sparing of motor symptoms  as required.PS and LI planned the design of the experiments and wrote the article. PS and GN carried out the experiments. PS and LI analyzed the data. All authors edited the manuscript. LI conceived and supervised all aspects of the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.