Our result is two orders of a conventional surface plasmon resonance sensor. Furthermore, we find that when sensing medium is in the vicinity of water in THz, the sensitivity increases with increasing refractive index of the sensing medium.Graphene terahertz (THz) surface plasmons provide hope for developing functional devices in the THz frequency. By coupling graphene surface plasmon polaritons (SPPs) and a planar waveguide (PWG) mode, Fano resonances are demonstrated to realize an ultrasensitive terahertz biosensor. By analyzing the dispersion relation of graphene SPPs and PWG, the tunable Fano resonances in the terahertz frequency are discussed. It is found that the asymmetric lineshape of Fano resonances can be manipulated by changing the Fermi level of graphene, and the influence of the thickness of coupling layer and air layer in sandwich structure on the Fano resonances is also discussed in detail. We then apply the proposed Fano resonance to realize the ultrasensitive terahertz biosensors, it is shown that the highest sensitivities of 3260 RIU Optical sensors based on surface plasmon polaritons (SPPs) at noble metal (such as gold and silver) surfaces ,2 have bWhen the sensors are applied to biological detection, in contrast with traditional biosensors in the visible frequency, terahertz frequency biosensors are more suitable because their low-photon energy characteristics can excite the collective oscillatory mode of biomolecules and enhance the sensitivity of the biosensing molecules . In the Terahertz sensors based on graphene SPPs have been investigated by several authors ,16,17,18Here, we have proposed a THz biosensor based on the Fano resonance. The coupling between a broad resonance supported by FLG SPP and a narrow resonance supported by PWG mode leads to a sharp Fano resonance. We believe that the sharp line shapes can have a good potential as highly sensitive biosensors in the THz frequency range.pn = 3.41) at terahertz frequency. Graphene is marked by a thick red line.We plot A small air gap lays between the graphene and the coupling prism. Below the graphene, it’s a sensing structure formed by three layered PWG. The graphene is proposed to excite SPP mode, and the waveguide mode is supported by PWG. The structure we proposed makes it possible to couple SPP modes with PWG modes.3n is selected to be crystal quartz with 3n = 2.1. Cladding layer n2 is set to be polymethylpentene (TPX) with 2n = 1.46. We assumed the substrate to be water which is the sensing medium and follows a triple Debye function [1 = 10 μm, d2 = 50 μm and d3 = 100 μm, respectively.The PWG core function ,29:(1)εwGraphene is modeled as a surface conducting sheet and the surface conductivity of grapheme  formula ,31:(2)σiFE is determined by carrier density d and ɛ are the distance and electrostatic permittivity of the two electrodes and medium between the two electrodes respectively. Therefore Fermi energy can be dynamically tuned by gate voltage, where e is elementary, ħ is the reduced Planck’s constant, BK is the Boltzmann constant. τ is the phenomenological relaxation time which is assumed to be 1 ps, the temperature T = 300 K. And the effective thickness of monolayer graphene is usually taken to be dg = 0.34 nm.p-n1-FLG-n2 which consists of prism, air gap, few layer graphene and cladding layer of PWG as a system to match the boundary conditions. The SPP dispersion can be derived as:j = p, s, 1, 2, 3, 4, β is the x component wavenumber, In numerical calculations, we choose the nAs for the three layer waveguide structure, the dispersion of the PWG mode can be derived as: N = 3, the corresponding FE is small enough, so in the following analysis, the layer number of the graphene is considered to be N = 3.The black solid line is the effective refractive index of the PWG mode which is fixed. The red, blue, and green dashed line are the effective refractive indexes of monolayer graphene SPP, two layer graphene and three layer graphene, respectively. Obviously, they all can make the effective refractive index of graphene SPP and the PWG mode match and excite the Fano resonance. Monolayer graphene can only support SPP with very high Fermi energy. Although Fermi energy of 1 eV has been achieved experimentally ,33, loweN-layer model is used to solve the multilayer systems, shown in z-direction. The tangential fields at the first boundary Z = Z1 = 0 are related to those at the final boundary Z = NZ−1 as [U1 and V1 are the tangential components of electric and magnetic fields at the boundary of first layer respectively. NU−1 and NV−1 are corresponding fields at the boundary of Nth layer. M is known as the characteristic matrix of the combined structure.The transfer matrix method for the  ZN−1 as :(6)[U1V1FE = 0.37 eV of FLG is appropriate in It is known that z which is the distance from interface of Si prism and air. The related reflection spectrum is shown in In order to verify the two modes have been coupled together and further understand the origin of sharp Fano resonance, we plot sn) of the sensing medium would lead to a change in the angle-scan ATR resonance curve. Corresponding to the change in refractive index of sensing medium (sdn), the change of reflection is dR (sensing by intensity modulation). Then a sensitivity by intensity is given by:The characteristic line shape of Fano resonances make them have potential applications in highly sensitive sensors. The change in the refractive index (FE on sensitivity. FE from 0.33–0.43 eV. When FE = 0.37 eV, the lineshape of the resonance is nearly symmetrical, at this point, the sensitivity is the smallest one in FE is increased or decreased, the lineshape of the resonance becomes asymmetrical, and as FE continues to increase or decrease, the asymmetric lineshape of the reflection becomes sharper, and the sensitivity we calculate is larger. It’s easy to predict that a sharper asymmetric reflection lineshape leads to higher sensitivity.The Fermi energy is found to be an important factor affecting the sensitivity. We plot κ in the refractive index of the waveguide layer is introduced in our following calculation, which represents the loss of energy in the waveguide layers. We plot d2 on the sensitivity. In d2 increased, the slope of the reflectance curve become steeper. Corresponding to the change of the line shape, the value of the sensitivity becomes larger. We know that PWG mode excited by ATR method have radiation losses due to coupling between PWG and prism [Real waveguide layers produced in laboratories may have losses because of their different physical origins depending on the method and conditions of preparation. A nonzero imaginary part nd prism , and thed2, i.e., the distance between the PWG and the prism or graphene, the radiation loss and intrinsic loss of PWG mode decrease simultaneously, as a natural result the increase of the quality factor of PWG, and a narrower resonance is achieved, but with the resonance sharpened, we also see the degradation of resonance as d2 increases, i.e., as the coupling strength decreases at the same time. Therefore, we can see the sensitivity increases first and then decreases after getting peak values in When we increase d1 on the sensitivity. As d1 decreases, similarly radiation loss increases a little. The quality factor may become smaller, and the sensitivity is expected to decrease, but we note that when the air layer d1 is changed, the coupling state changes due to the varying dispersion of graphene SPPs. The lineshape becomes more asymmetric. Comparing the two effects, from We plot sn in We plot sn has a slight variation. This means that the proposed biosensor is very sensitive to the changes in the sensing medium. The electric field at the interface of crystal quartz/sensing medium will have an obvious change when sn. We can get that the sensitivity is improved as the refractive index of the sensing medium is increased. We compared our sensor with the SPP sensor excited by three layer graphene with FE = 1 eV and dair = 7 μm, which shows that the sensitivities of our proposed structure is two orders of magnitude larger than that with the conventional surface plasmon resonance sensor.We also study the sensitivity changes near the refractive index of water. −1. We believe that the biosensor we proposed will find good use in terahertz wavelength range detection.In this study, we have proposed a terahertz biosensor based on Fano resonance of a graphene/waveguide hybrid structure. The asymmetric lineshape of the Fano resonance is found to have a significant effect on the sensitivity. The sharper the asymmetric line shape of the Fano resonance is, the higher the achieved sensitivity of the biosensor is. The graphene intrinsic losses and radiation losses related to the distance between prism or FLG and the PWG influence the sensitivity a lot. Through the manipulation of structural parameters, we can get sensitivities as high as 3260 RIU


Examining interactions between nanomaterials and cell membranes can expose underlying mechanisms of nanomaterial cytotoxicity and guide the design of safer nanomedical technologies. Recently, graphene has been shown to exhibit potential toxicity to cells; however, the molecular processes driving its lethal properties have yet to be fully characterized. We here demonstrate that graphene nanosheets (both pristine and oxidized) can produce holes (pores) in the membranes of A549 and Raw264.7 cells, substantially reducing cell viability. Electron micrographs offer clear evidence of pores created on cell membranes. Our molecular dynamics simulations reveal that multiple graphene nanosheets can cooperate to extract large numbers of phospholipids from the membrane bilayer. Strong dispersion interactions between graphene and lipid-tail carbons result in greatly depleted lipid density within confined regions of the membrane, ultimately leading to the formation of water-permeable pores. This cooperative lipid extraction mechanism for membrane perforation represents another distinct process that contributes to the molecular basis of graphene cytotoxicity. Graphene’s remarkable physicochemical properties have long garnered favor among scientists seeking stable, electrically conductive, and optically active 2D nanomaterials. Numerous studies have demonstrated wide-reaching prospects for biomedical applications of graphene and graphene oxide (GO), particularly in biosensing134678101113in-vitro study of GO’s interaction with a lipid vesicle suggested potential damage of cell membrane18et al. found that GO could interact with Toll-like receptor 4 (TLR-4) and induce necrosis in macrophages by increasing the expression of TNF-αThe introduction of graphene-based nanomaterials into human-proximate systems has prompted efforts to understand graphene’s biocompatibility and cytotoxicity. Much of the existing literature attributes GO’s cytotoxicity to a secondary generation of reactive oxygen species (ROS)1516181920212223Accumulating experimental and computational evidence thus suggests that GO nanotoxicity is driven by multiple molecular processes. In that light, coarse-grained, mean-field simulations have also suggested the possibility of graphene-mediated perforation of cell membranes, a phenomenon likely to be cytotoxic−1, respectively31The morphologies of the GO nanosheets used in this study were first examined by atomic force microscopy (AFM). AFM images revealed a characteristic GO thickness of around 1 nm , implyin2931In previous work, we demonstrated that complete culture medium containing serum proteins can mitigate the cytotoxicity of GO261534353637In order to explore mechanisms of GO-related cytotoxicity, cellular morphology was first measured after GO nanosheet exposure. Interestingly, light spots were detected in the membranes of both A549  and Raw2Further analysis showed that GO-induced light spots appear even in the presence of serum protein medium (10% FBS), albeit after longer treatment times and at higher GO concentrations . HoweverTo better understand the origin of the light spots seen with inverted light microscopy, A549 cells transfected with green fluorescent protein (GFP) were probed with fluorescence microscopy. The light spots clearly correspond to dark regions under fluorescence-based imaging , suggestAfter excluding a relationship to autophagy, we hypothesized that the formation of light spots is related to cell membrane stress induced by GO. In order to validate this premise, GO-treated cell membranes were imaged by scanning electron microscopy (SEM). In a previous study by Zhang and coworkersAs mentioned above, our previous work revealed that strong dispersion interactions between lipids and graphene can result in an extraordinary extraction of phospholipids from cell membranes, providing a dominant mechanism for the death of cells bygraphene nanosheets18In vitro, and particularly at high graphene concentrations, multiple graphene nanosheets could certainly interact with a local membrane segment simultaneously. In principle, these cooperative interactions could result in a critical loss of membrane density, leading to perforations at lipid-depleted sites. Here, we indeed introduce a simple  system wherein multiple graphene molecules cooperate to weaken a membrane and induce pore formation at a nanosheet-interstitial site.This apparent discrepancy between past simulation results and our present observations of membrane perforation can perhaps be understood in terms of graphene nanosheet density. In our previous simulations, the effective graphene density was very low (projected across periodic boundary conditions), meaning extracted lipids could easily be replenished by neighboring molecules. 10O1(OH)1(COOH)0.5; i.e., 2 epoxy and 2 hydroxyl groups on both sides of the graphene basal plane and 1 carboxyl group on the edge of the graphene sheet per 20 carbon atoms, yielding a C:O ratio about 3:1), which represents a typical outcome of graphene oxidation reactions5152sp2-domains”)5154sp2-domains of GOs.Atomistic MD simulations, widely used to characterize interactions between and among both biomolecules4142434446474849Due to the significant computational resources required for atomistic simulations, we featured a much smaller graphene sheet (~10 nm) in our simulations than we used in our experiments (200–700 nm). As demonstrated by the data shown in We computed the vdW interaction energies between the graphene sheets and phospholipids , a quantTo further quantify this perforation mechanism, we calculated interstitial lipid densities during the three dynamical stages and projected these densities onto a designated y-axis see  is thinnIt is interesting to note that the pores observed here appear close to the edges of the graphenes in the quartet, suggesting this interstitial region is reliably weakened by lipid extraction. This edge-centric perforation mechanism is consistent with the previous experimental finding that nanosheet edges are important mediators of the toxical effects of graphenesThe observation of cooperative membrane perforation in our simulation system, of course, does not preclude the emergence of alternative perforation mechanisms driven by larger GO sheets. For example, if a single GO nanosheet were sized on the order of the linear dimension of a cell, that nanosheet might be capable of extracting a significant percentage of the phospholipids that make up an entire cell membrane. Establishing whether such isolated interactions could indeed cause membrane perforation, or whether some fundamental physical limitation prohibits this type of pore formation, represents and interesting subject of future study.In this work, we used both experiments and simulations to demonstrate that pristine graphene and graphene oxide nanosheets can induce pore formation on cell membranes. Electron micrographs show clear images of these pores, and molecular dynamics simulations reveal a molecular mechanism for perforation dependent on cooperative lipid extraction driven by several graphene nanosheets. The observed pore formation mechanism can roughly be divided into three stages: fast lipid extraction onto multiple graphene surfaces (Stage I); a metastable balance between dispersion forces and membrane tension in a region confined between these nanosheets (Stage II); and ultimately, perforation (Stage III). Lipids in the proto-pore region are transported onto graphene surfaces and into the inter-graphene area, critically depleting the membrane’s density at characteristic interstitial sites. A pore develops when the confined lipid supply is finally exhausted.To our knowledge, this cooperative, lipid-extraction-based pore formation process offers a unique perspective on the molecular mechanisms of graphene cytotoxicity. At low graphene concentrations, individual nanosheets should still be able to penetrate cell membranes and enter cell interiors. The damage caused by this graphene insertion  and Life Technologies Corporation , respectively.2.A549 and Raw 264.7 cells were maintained in RPMI 1640 and DMEM media, respectively , which were supplemented with 10% fetal bovine serum , 1% penicillin-streptomycin solution, 1% L-glutamine and 1% nonessential amino acids and cultured at 37 °C and 5% COCytotoxicity induced by GO was assessed by CCK-8 and live/dead assays. Briefly, A549 and Raw 264.7 cells were seeded on 96-well plates (Corning) at a density of 5,000 and 10000 cells per well, respectively, and cultured in complete culture medium containing 10% FBS. After 24 hours of incubation, A549 and Raw 264.7 cells reached ~80% confluence. Subsequently, for our CCK-8 experiments, A549 and Raw 264.7 cells were treated with GO in FBS-free medium at concentrations of 0, 1, 10, 50 and 200 μg/ml for 24 hours. Cells were then washed with PBS three times. CCK-8 was added to each well at a rate of 1:10 of the medium, and cells were subsequently incubated at 37 °C for another hour. Absorbance of the CCK-8 solutions was measured using a microplate reader at a 450 nm wavelength. A live/dead assay was also used to characterize the cytotoxicity of GO. Staining was performed following the protocol contained within the kit. Live (green) and dead (red) cells were observed via fluorescence microscopy.5/well). GO was dispersed in FBS-free medium at concentrations of 0, 10, 50 and 200 μg/ml and added to the 6-well plates. Cells were cultured at 37 °C for 2 hours and 6 hours, and cellular morphologies were imaged by microscopy. For live cell imaging, after 1 × 105 cells were seeded in confocal dish for 24 h, 50 μg/ml GO nanosheets were added. After GO treatment for 2 h, cells were imaged every 10 min for 990 min by a live cell imaging system (Olympus cell^R system).To perform cellular morphology observations after GO treatment, cells were seeded into 6-well plates . Then, 24 hours later, after achieving ~80% confluence, cells were treated in serum free medium (0% FBS) with or without GO (200 μg/ml) for 6 hours. Cells were then prefixed in 2.5% glutaraldehyde overnight at 4 °C followed by a postfix in 2% (vol/vol) aqueous osmium tetroxide for 1 hour. Cells were rinsed with PBS 3 times and then dehydrated in 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol. After dryness reached a critical point, the coverslips were coated using gold sputter deposition and imaged via field-emission SEM.2 of surface area is exposed to water. The force field used for graphene was adapted from our previous study+, 20 Cl−, and 10840 water molecules. The TIP3P water5760616263We carried out MD simulations for graphene nanosheets interacting with POPC  lipids that are present in eukaryotic cell membranes. The CHARMM force fieldThe NAMD2.9 How to cite this article: Duan, G. et al. Graphene-Induced Pore Formation on Cell Membranes. Sci. Rep.7, 42767; doi: 10.1038/srep42767 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Recent experimental evidence for and the theoretical confirmation of tunable edge plasmons and surface plasmons in graphene nanoribbons have opened up new opportunities to scrutinize the main geometric and conformation factors, which can be used to modulate these collective modes in the infrared-to-terahertz frequency band. Here, we show how the extrinsic plasmon structure of regular planar arrays of graphene nanoribbons, with perfectly symmetric edges, is influenced by the width, chirality and unit-cell length of each ribbon, as well as the in-plane vacuum distance between two contiguous ribbons. Our predictions, based on time-dependent density functional theory, in the random phase approximation, are expected to be of immediate help for measurements of plasmonic features in nanoscale architectures of nanoribbon devices. Quantized, coherent and collective density fluctuations of the valence electrons in low-dimensional nanostructures, better known as plasmons, have been attracting significant interest, due their capability to couple with light and other charged particles, thus paving the way to novel applications in a wide range of technologies, as diverse as biosensing, light harvesting or quantum information –5. On moA clear picture of confined edge (interband) and surface (intraband) plasmons in GNRs, as wide as 100–500 nm, has been achieved by infrared imaging measurements on the nanoscale . On the T = 300 K), including both intrinsic and extrinsic conditions. Different zigzag (Z) and armchair (A) configurations are examined, with the GNR-ends being passivated by hydrogen atoms, which mimics an ideal setup of long ribbons, with perfectly symmetric edges, suspended or grown on inert substrates. A specific focus is made on the 5AGNR, 11AGNR and 4ZGNR, 10ZGNR geometries that are, respectively, characterized by 4,10 zigzag-chains and 5,11 dimer-lines across the width of the GNRs. The dielectric properties of these systems are calculated in response to probe electrons or photons with incident energies, ω, smaller than 20 eV, and in-plane incident momentum q below 0.8 Å−1. For comparison purposes, the well-known intrinsic plasmonics of graphene are also reported.In this work, we extend the results of , to inclIn the following, we briefly account for the theoretical tools that we have used to explore the electronic structure and dielectric properties of 5AGNR, 11AGNR and 4ZGNR, 10ZGNR and graphene . Next, we will present a detailed analysis of the changes induced by the above mentioned parameters to the peak position, intensity, and dispersion of the GNR-plasmons .Our TDDFT approach is divided into two steps. First, the (ground-state) electronic properties of the different GNRs (and graphene) are obtained by DFT. Second, the basic equation of linear-response theory in the RPA is employed, with a corrected electron–electron interaction, to calculate the dielectric properties, and hence the plasmon structure, of the systems., which depend on the wave-vectors k of the first Brillouin zone (1st BZ), and the reciprocal lattice vectors G associated to the three-dimensional (3D) crystal of unit-cell volume Ω0. The ground-state electronic properties of the different GNRs (as well as graphene) are computed within the local density approximation (LDA), being defined by the Perdew–Zunger parameterization of the uniform-gas correlation energy [5 PWs.Density-functional calculations are performed using the plane-wave (PW) basis-set , i.e., tn energy . Norm-con energy . A cut-ok-points [k-points, including up to 120 bands, which is enough to explore the dielectric properties of the GNRs at probing energies below ≈20 eV. The IR to THz region is further scrutinized with a finer MP mesh of 2000 × 1 × 1 k-points, including up to 30 bands.In the GNR arrays, the C–C length is allowed to range from 1.414 to 1.426 Å, while the C–H bond length is fixed to 1.09 Å, with a bond-angle of 120 ° of around 0.7–2.2 nm are sorted out; (ii) zigzag and armchair edges are considered, to elucidate the role played by chirality; (iii) in-plane vacuum distances from 5 to 20 Å are tested; and (iv) different unit-cell extensions are simulated by changing the C–C bond length by about 0.5%, to account for stretching effects. As for intrinsic graphene, the C–C bond length and out-of-plane lattice constant are fixed to 1.42 Å and 15 Å, respectively. The self-consistent run is performed on a 60 × 60 × 1 MP-grid, and the KS electronic properties are then computed on a 180 × 180 × 1 MP-grid, including up to 80 bands.The simulations at hand explore ranges of different geometrical and conformation parameters, which will be used to characterize the tunability of the GNR plasmons. In particular, (i) GNR widths  cut-off, say, a truncated Coulomb potential is used [GG′ terms by the truncated Fourier integral:However, the long-range character of the Coulomb potential yields non-negligible interactions between replicas along out-of-plane direction  is used –49 to reg and Gz denote the in-plane and out-of-plane components of G, respectively.Here, With this 2D correction in mind, we can introduce the inverse dielectric matrix:The zeros in the real part of the macroscopic dielectric function (permittivity) provide the condition for a plasmon resonance to occur, stated as:The imaginary part of the inverse permittivity is proportional to so-called energy loss (EL) function, which provides the plasmon structure:ELOSS through G vectors for all GNRs (and ca. 51 G vectors for graphene), sorted in length and being of the form , give well-resolved and converged results in the sampled energy–momentum region, delimited by ω < 20 eV and q < 0.8 Å−1.Non-local field effects  are inclEF in the range of −0.2 to 0.2 eV.We proceed by clarifying the role played by the geometric and conformation parameters introduced above in the different GNRs, whose intrinsic response is shown in Independently on the width and chirality of the ribbons, all GNRs are characterized by two interband plasmons at excitation energies above about 2 eV. These excitations, shown in q→0 region, the π plasmon of all systems has a -like dispersion, while at q > 0.2 Å−1 it exhibits linear behavior. The intrinsic plasmons of 10ZGNR and 11AGNR appear in the same energy region as those of graphene, i.e., at ω ≈ 4–5 eV and ω ≈ 14–15 eV. On the other hand, they are red-shifted in 4ZGNR and 5AGNR, with the π plasmon having a peak at ω ≈ 2–3 eV and the π–σ plasmon lying at ω ≈ 13–14 eV. Furthermore, the π and π–σ plasmons detected in 4ZGNRs (w ≈ 0.9 nm) and 5AGNR (w ≈ 0.7 nm) exhibit markedly discontinuous dispersions, being split into more branches [The energy window displayed in branches . This isw→∞). Thus, semiconducting and semimetallic GNRs have plasmon resonances in the visible (VIS) to ultraviolet (UV) regime that may be controlled by the GNR width. This tunability feature is evidently absent in graphene.By increasing the GNR width (w > 1 nm), the number of bands increases, and less disjoint plasmon dispersions appear, which clearly tend to the continuous patterns of graphene  and a negative doping level (ΔEF = −0.1 eV). We see that 10ZGNR and 4ZGNR present similar plasmonic features, with the intraband plasmon resonance being blue-shifted by increasing the GNR width  semiconducting (armchair) and semimetallic (zigzag) arrays of GNRs, with ideal symmetric edges, passivated by hydrogen atoms.All the tested structures are characterized by two interband plasmons at energies larger than 2 eV, which are analogous to the π and π–σ plasmons of graphene. Their peak positions and dispersions are mostly influenced by the GNR width.At energies smaller than 2 eV, two more intriguing collective excitations appear, which correspond to recently reported edge and surface plasmons . These mIt is worth mentioning that some recent tight-binding models have investigated the role of edge roughness due to asymmetric defects –62, whicWe expect that our findings, combined with non-ab initio approaches suitable for the device scale, may open new strategies to construct materials with plasmonic resonances that will be tunable to a specific demand in both the UV–vis and THz regimes, by altering the chemical doping, electronic gating, and also by means of a careful choice of the geometry.


The observed layer-dependent etching rates reveal the relative strength of the graphene–graphene and graphene–substrate interaction and the resulting mode of adlayer growth. Scanning tunnelling microscopy and density functional theory calculations confirm a strong coupling between graphene edge atoms and platinum. Simulated etching confirms that etching can be viewed as reversed growth. This work demonstrates that real-time imaging under controlled atmosphere is a powerful method for designing synthesis protocols for sp2 carbon nanostructures in between graphene and graphite.In the transition from graphene to graphite, the addition of each individual graphene layer modifies the electronic structure and produces a different material with unique properties. Controlled growth of few-layer graphene is therefore of fundamental interest and will provide access to materials with engineered electronic structure. Here we combine isothermal growth and etching experiments with  Controlled preparation of few-layer graphene is a promising, yet challenging technological protocol. Here, the authors perform real-time imaging during chemical vapour deposition growth and hydrogen etching, to gain insight into layer-dependent growth mechanisms and graphene-substrate interaction. In the case of hydrogen plasma etching, incomplete understanding of the underlying reaction mechanisms limits its application to a trial-and-error approach in which the production of well-defined graphene edge structures without disturbance of the basal plane remains an unsolved problem. A control of the electronic states in graphene is also possible by taking advantage of interlayer interactions. Indeed, ordered structures consisting of two or more layers of graphene represent a broad class of materials, where the electronic structure and properties uniquely change with each additional layer789111213π electron dispersion and without the formation of new boundaries789151617181921et al.in situ low-energy electron microscopy and charge transport measurements. In contrast, Sun et al. using post-growth scanning electron microscopy (SEM) imaging found that multilayer growth on Pt is dominated by carbon precipitation below already formed layersin situ environmental SEM (ESEM) for studying the mechanistic details of graphene chemical vapour deposition (CVD) on Cu  and scanning tunnelling microscopy (STM)), high-resolution transmission electron microscopy and theoretical calculations. The ability to probe the interlayer interactions in graphene is important for developing key processing steps such as selecting the ideal substrate for facilitating SLG transfer and the tuning of the properties of FLG by controlling the sequencing of ALG stacking and the number of layers. The broader significance of this work is in demonstrating that etching in combination with direct imaging of in-plane dynamics in response to well-controlled experimental environments is a facile approach for deriving information about interlayer coupling that governs the vertical stacking behaviour of two-dimensional materials.2H4). After initial growth of FLG islands in a C2H4/H2 atmosphere, the C2H4 flow was turned off to perform isothermal etching in pure H2 at a total pressure of 25 Pa . We attribute the etching to carbon bond breaking by atomic hydrogen that is produced by dissociation of H2 on a Pt surface that is known to be a highly efficient catalyst for promoting H2 dissociationThe growth and etching of graphene were both performed in the chamber of an ESEM (see Methods section). The ESEM enables real-time imaging of the shape and size evolution of graphene islands in relation to the Pt grain structure and surface features. Observations can be performed as a function of the background atmosphere and temperature during both, growth and etching of graphenein situ SEM image in in situ SEM images of edge misalignment between mutual layers and individual sheets provide real-time information on the evolution of the rotation angle between growing layers, and formation of the stacking orderThe key for probing the dynamics of ALG during both growth and etching is the ability to differentiate individual layers stacked on top of each other . A wide in situ at 900 °C in an atmosphere of 25 Pa hydrogen. Designated areas in in situ SEM movie provided as The shape evolution of single-layer graphene, BLG and TLG during hydrogen etching is illustrated in 2 etching at 900 °C is plotted in −1 for the first, −16.01 nm s−1 for the second and −11.76 nm s−1 for the third layer, and are thus different for different layers. With respect to the first layer, the etching speed of the second layer is higher by a factor of 2.74 and the one of the third layers by a factor of about 2. This implies different graphene-edge configurations and indicates a WC-like stacking. Indeed, in a WC configuration, graphene edge atoms are in direct contact with the Pt substrate only in the case of the first layer, while edge atoms of adlayers are located on top of a graphene sheet and are most likely hydrogen-terminated.The evolution of the perimeter and area of the graphene islands during Hr∼exp(−Ea/kBT), it should be possible to estimate the relative strength of the graphene interlayer coupling.Under the assumption that the removal of carbon atoms from the perimeter of a graphene sheet can be described by an Arrhenius-type rate Ea that is required for removing an edge atom by hydrogen etching contains several contributions. Amongst them, in-plane carbon–carbon bond breaking (EC–C) contributes the largest portion. However, there are also small contributions due to interlayer interactions between edge atoms and the graphene sheet underneath (ECoupling). Since edge atoms in the second and third layers are located on top of a graphene sheet in the WC configuration, they face a similar local environment and are exposed to the same hydrogen-rich atmosphere during etching. Thus, the product of the etching process should be the same for both layers. Differences in the activation energy might therefore give a hint on differences in the interlayer coupling strength. By forming the ratio between the experimental etching rates of the second and third layers using EC–C+unspecified contributions) should cancel out, leaving only terms due to different interlayer coupling:The apparent activation energy Using the experimentally determined etching rates for the second and third layers that are provided in the diagram of n-type doping of the graphene sheet, which can be detected by a corresponding shift in the position of the C1s peak in X-ray photoelectron spectran-type doping was also observed in the case of ruthenium substratesThe faster etching speed of the second layer compared with the third layer indicates that the van der Waals interaction between the second and first layer is weakened due to the interaction between the first layer and the Pt substrate. In the case of copper substrates, it has previously been shown that coupling between SLG and the substrate induces A closer scrutiny of in situ grown SLG  surface. From the DFT calculation it follows that down-step etching is slower because it requires breaking a 3.2 eV C–Pt bond compared with the up-step etching that needs to break only a 1.4 eV C–C bond . The large difference in step-up and step-down etching speed is thus a consequence of the different bonding types at the step edges.where in situ SEM images shows that the size of the lighter grey outer layer decreases with time by hydrogen etching, while the size of the smaller darker patch exhibits no detectable change. This behaviour indicates that the large outer layer is being etched because it is a topmost layer that is directly exposed to hydrogen. In contrast, the small darker patch appears to be effectively sealed off from the hydrogen atmosphere. Indeed, closer inspection reveals that it actually grows due to segregation of C from the Pt as shown in Up to now we have discussed the stacking sequence in FLG grown by isothermal CVD. However, in the case of Pt, the formation of ALG can also occur by segregation of dissolved from the polycrystalline Pt foils. We obtained precipitation growth after the termination of an isothermal CVD growth process, during a subsequent cooling step. A typical example for FLG structures that form by segregation during cooling in pure hydrogen atmosphere is shown in The subtle features in In this work we demonstrate that real-time imaging by ESEM is a versatile and powerful method for the generation of mechanistic insight that is required for the controlled production of FLG with defined number of layers. Direct observation of the shrinking behaviour of individual layers during isothermal etching in pure hydrogen atmosphere provides the missing clue for unravelling the order of layer stacking in FLG growth. In isothermal CVD growth, new layers grow on the topmost layer, while they are inserted between the substrate and already grown layers by C segregation during cooling. The etching rates reveal that the first layer is strongly coupled to the Pt substrate and provides an estimate for the interlayer coupling strength between the second and third layers. By combining theoretical simulations and STM imaging data we show that the coupling of the first layer involves C bonding at the graphene edges, where terminal carbon atoms bend down towards a flat Pt surface or attach laterally to Pt step edges.in situ SEM imaging for developing a real-time feedback loop for controlling coverage uniformity in FLG growth by isothermal CVD. It is possible to grow multilayers, etch some of them and then continue growth of the remaining layers under conditions where no new nucleation events occur and thus, to obtain FLG with a desired number of layers. Finally, this work demonstrates that observation of in-plane dynamics in response to well-controlled experimental environments can provide information about the vertical stacking behaviour of two-dimensional materials and more generally, the capabilities of in situ SEM for the study of surface dynamics under controlled environments.The etching of vacancy islands exhibits anisotropy that depends on the density and orientation of Pt step edges. Anisotropic etching serves as further evidence for strong interactions with the Pt surface. We conclude that C bonding at the edges is the decisive factor determining the observed layer stacking. It impedes diffusion, intercalation and transport of reactants and products during graphene growth and etching. It hinders both, underlayer growth and etching, but promotes growth and etching of the topmost layer. Moreover, considering the graphene–metal interaction, we propose that the stacking sequence of FLG during isothermal CVD growth is the same on substrates that are characterized by a similar or stronger graphene–metal interaction and comparable carbon solubility such as Pd, Ru, Ir and Rh. In the case of CVD growth on copper in hydrogen-rich atmospheres, the reversed stacking sequence might be a consequence of hydrogen-terminated edge atoms, as recent DFT calculations have predictedIn situ CVD growth experiments were performed inside the chamber of a commercial ESEM (FEI Quantum 200). The vacuum system of the ESEM was modified and upgraded with oil-free pre-vacuum pumps. The instrument is equipped with a home-made heating stage, a gas supply unit (mass flow controllers from Bronkhorst) and a mass spectrometer (Pfeiffer OmniStar) for the analysis of the chamber atmosphere. The ESEM is not ultra-high-vacuum capable. Owing to the use of rubber O-rings for sealing and the fact that the chamber cannot be baked out, the base pressure of the instrument is around 2 × 10−5 Pa, with a residual gas composition mostly comprising water, N2 and O2  and C2H4 (99.95% purity). Samples of sizes ranging from 3 × 3 to 5 × 5 mm were cut from a 0.25 mm-thick polycrystalline Pt foil (99.99% purity) purchased from Alpha Aesar. Before all CVD growth experiments, the chamber of the ESEM was plasma cleaned. The foils were annealed at 1,000 °C under a hydrogen flow of 10 s.c.c.m. at 25 Pa for 1 h inside the chamber. The temperature was measured via a B-type thermocouple that was spot-welded onto the substrate and simultaneously served to ground the sample. CVD growth was performed at 900 °C using a flow of 10 s.c.c.m. H2 and 0.1 s.c.c.m. of C2H4 at a total chamber pressure of 25 Pa. Hydrogen etching was performed under 10 s.c.c.m. H2 at 900 °C at 25 Pa. During the experiments, the microscope was operated at an acceleration voltage of 5.0–7.5 kV. Images were recorded by a large field detector during CVD growth and etching. No influence of the electron beam on the growth and etching process could be observed. The imaged regions and their respective surroundings showed similar behaviour, as evidenced by changing the magnification or by moving the sample under the beam. Furthermore, no electron beam induced contamination was observed at elevated temperatures.2 and O2 . In the Raman spectroscopy was performed using a Horiba/Jobin-Yvon T64000 spectrometer  with a Coherent Innova 400  argon-ion laser operating at 514.5 nm for the excitation. The Raman signal was collected with a multi-channel charge-coupled device detector. A laser power of 20 mW at the sample and an objective with a × 100 magnification were used. Measurements were performed in confocal mode to reduce the background scattering with respect to the graphene signal. To obtain a satisfactory signal-to-noise ratio, the spectra were recorded with integration times of 60 s, and a total of 10 accumulations.f0=40–75 kHz and spring constants k=0.32 Nm).AFM images were recorded on a Bruker Sharp Nitride Lever probe (SNL-10). Imaging was done in tapping mode using a V-shaped cantilever probe B .Supplementary Figures 1-5, Supplementary Notes 1-4 and Supplementary ReferencesIn situ SEM movie showing the growth of multi-layer graphene on Pt in a flow of 10 sccm H2 and 0.1 sccm of C2H4 at 25 Pa and a substrate temperature of 900 °C. Images for this movie were recorded at a scan rate of one frame per 10 s.In situ SEM movies showing the shrinking behavior of a SLG and of a WC stacked FLG during etching in 25 Pa H2 at 900 °C. Etching rates abstracted from this video are discussed in detail in the main manuscript. Images were recorded at a scan rate of one frame per 90 s.In situ SEM movies showing the shrinking behavior of a larger number of WC stacked FLG domains during etching in 25 Pa H2 at 900 °C. Etching rates abstracted from this video demonstrate that second layers consistently etch faster than third layers. Images were recorded at a scan rate of one frame per 90 s.In situ SEM movie showing the evolution of a vacancy island on a faceted substrate grain during graphene etching at 25 Pa H2 900 °C. Due to the large number of steps, the etching shows a pronounced anisotropy. Images were recorded at a scan rate of one frame per 90 s.In situ SEM movie showing the evolution of a vacancy island during graphene etching on a smooth substrate grain at 25 Pa H2 900 °C. In this case, etching gives rise to more regularly shaped holes. Images were recorded at a scan rate of one frame per 90 s.In situ SEM movie showing the shrinking behavior of IWC stacking FLG during etching in 25 Pa H2 at 800-900 °C. Images were recorded at a scan rate of one frame per 90 s.


Alzheimer's disease (AD) and other forms of dementia create a noncurable disease population in world's societies. To develop a blood-based biomarker is important so that the remedial or disease-altering therapeutic intervention for AD patients would be available at the early stage.TDP-43 levels were analyzed in postmortem brain tissue and platelets of AD and control subjects.We observed an increased TDP-43 (<60%) in postmortem AD brain regions and similar trends were also observed in patient's platelets.Platelet TDP-43 could be used as a surrogate biomarker that is measurable, reproducible and sensitive for screening the patients with some early clinical signs of AD and can be used to monitor disease prognosis. In this study, we explore to identify an Alzheimer's disease (AD)-selective phospho-specific antibody that recognizes the diseased form of TDP-43 protein in patient's blood-derived platelets. Our results suggest that selective antiphosphorylated TDP-43 antibody discriminates AD from non-demented controls and patients with amyotrophic lateral sclerosis. Therefore, platelet screening with a selective antibody could potentially be a useful tool for diagnostic purposes for AD. More than 5 million Americans are living with Alzheimer's disease (AD) and this number is projected to rise to 13.8 million by 2050. As the world population gets older, the incidence of AD is rising. Unfortunately, only one in four people with AD have been diagnosed ; however® analysis . Due to its 43 kDa size, the acronym TDP-43 will be used throughout this paper. Substantial research has been conducted to decipher the role of TDP-43 in different cellular events as well as its role(s) in neurodegenerative disease states . TDP-43 analysis A. In neuMonitoring disease-relevant biomolecules in the CNS is a challenge due to unfeasibility and invasiveness of taking repeated samples from brain and spinal cord tissues. Hence, sampling blood platelets may serve as a feasible media where the aberrant TDP-43 is detectable. Platelets are anuclear blood cell fragments that are derived from megakaryocytes  and sharWe chose platelets for identifying and measuring both total and phosphorylated TDP-43 protein derivatives in AD for the following reasons: platelets are easy to repeatedly obtain acessible human tissue  from the patients with minimal distress; their life span is short (7–10 days)  which wi2PO4, 7.5 mM Na2HPO4, 4.5 mM sodium citrate and 2.4 mM citric acid, pH 6.5) [Anti-hTARDBP polyclonal antibody  and phosphorylated derivatives of the pTDP-43 antibodies  ; Sigma-Aldrich,  MO, USA),  Cat# T1705, SAB4200225; Biolegend Inc, CA USA, Cat# 829901) were commercially purchased. Citrate wash buffer   and platHuman blood-platelet samples were obtained from the following sources; the Bio-specimen Bank of University of Kansas Medical Center (KUMC); platelets were previously collected from AD patients and age-matched with otherwise healthy subjects (non-demented) and stored at -80°C and ALS clinic at the University of Kansas Medical Center, Kansas City. ALS patient platelets were utilized as a negative disease control for identifying a selective antibody for AD patient platelets. AD patients in this study were not classified early onset (EOAD) versus late onset (LOAD). The genotyping for APP and Presenilin (PSEN) associated EOAD was not performed. All AD patients (n = 10) were categorized based on age-relevant cognitive symptom onset. The baseline evaluation included a thorough clinical examination by a trained clinician was conducted. This evaluation included a clinical dementia rating to exclude the presence of dementia was performed according to clinical and cognitive variables and descriptive data collected from AD Centers . A trainPlatelet donors comprised five female AD patients with average age of 75.2 ± 10.5 and five male AD patients with average age of 75.8 ± 8.6. Non-demented control subjects comprised two males with average age of 77.5 ± 2.13 and eight non-demented females with average age of 73.6 ± 3.5.g for 20 min. The washed platelet pellet was ruptured, sonicated in 0.6 ml of rupturing buffer with protease and phosphatase inhibitors, and subjected to high speed centrifugation  to obtain platelet cytosol. Protein concentrations were determined by the bicinchoninic acid spectrophotometric method [Platelets were isolated from freshly drawn blood into ACD containing vacutainer tube from clinically diagnosed patients and otherwise healthy subjects according to a standard two-step low speed centrifugation method adopted by Bio-Specimen bank facilities of the University of Kansas Medical Center. The platelet pellets were gently washed with 1 ml of citrate was buffer  and re-cc method . The sam4, 10 mM epsilon-caproic acid, 0.1 mM EGTA, protease inhibitor cocktail 0.1% v/v, 10 mM HEPES, pH 7.4). Tissue: homogenate buffer ratio was kept at 1:20 for achieving efficient solubilization The homogenization was carried out in an ice bucket with 8–10 strokes. The homogenate was aliquoted and stored in -80°C until use. Protein concentrations were analyzed by the bicinchoninic acid method [The human brain tissue samples from postmortem AD patients and age-matched control subjects were obtained from the Bio-Specimen bank of the University of Kansas Medical Center. The 100–200 mg samples of excised tissue from three brain regions  were received and homogenized in a Teflon-pestle glass homogenizer containing. Ice-cold buffer  were resolved in 12% SDS-PAGE and 4–20% SDS-PAGE, respectively under the reducing conditions. The proteins were transferred onto a polyvinylidene floride (PVDF) membrane and subsequently the membrane was probed with both pan anti-TDP-43 and several antiphosphorylated TDP-43 antibodies listed in reagents section. The protein bands were visualized by enhanced chemiluminescence and infrared dye-based fluorescence methods, and they were analyzed by NIH's ImageJ (V.1.46r) and Image Studio™ Lite software (V. 4.0).The platelet lysates from AD, ALS patients and otherwise healthy subject cohort were analyzed by a simple western system, a new technology developed by ProteinSimple, Inc., USA. This technology does not require classical SDS/PAGE and western blotting components. It uses very little sample-mix volume (∼3–5 μl) and provides high-throughput style sample run. The platelet lysate proteins (0.2 mg/ml) were analyzed in duplicate and both capillary electropherogram and pseudo protein bands were generated and analyzed by the system software .Mann–Whitney U rank sum test was employed for statistical analysis.In early stages of this work, we have shown that total TDP-43 protein levels were increased in the brain regions of postmortem AD patients (n = 3). The most noticeable TDP-43 increase was observed in the hippocampus while the frontal cortex and cerebellum reflected a slight TDP-43 increase as compared with nonsymptomatic control subjects A. Total ® VL3-BA) was employed to predict 152 aa long regions of disorder that were characterized by other methods . First, we have employed a computer-based Predictor of Natural Disordered Region (PONDR) algorithm using TDP-43 sequence (NCBI accession code: Q5R5W2.1). Disordered Enhanced Phosphorylation Predictor analysis predicted 28 potential phosphorylation sites and a majority of them were Ser amino acid enriched on the C-terminus aa 369–410) A. Anothe methods B. NMR st0 A. Anot methods A and fro methods B. A prom methods A.Misfolded aberrant protein aggregation is frequently observed in neurodegenerative diseases . PatholoIn this preliminary study, we have tested our hypothesis and provided some new findings that human blood-derived platelet TDP-43 and its derivatives may reflect the changes in the TDP-43 profile in human AD brain. There are several reports in the literature supporting the link between TDP-43 pathology and diagnosed AD cases ,49–51. IIn the next stage of this study, we tested whether AD patient brain TDP-43 protein profile could be observed in peripheral tissues such as blood-derived platelets. If we could demonstrate the same or similar profile in platelets, then we will have an assay platform by which TDP-43 and its derivatives can be easily measured and analyzed in platelet-lysate during the disease prognoses. Intracellular TDP-43 species such as aggregates, truncated TDP-43 fragments, and post-translationally modified TDP-43 have been found in neurodegenerative diseases . The chaTo identify an AD-selective antibody was the major undertaking in this study. Eight antibodies raised against different regions of TDP-43 as well as phosphorylated derivatives of TDP-43 were tested. They were purchased from different vendors . We have identified an antiphospho (S409/410–2) TDP-43 antibody purchased from ProteinTech (Cat#22309–1AP) as an AD-discriminating antibody for AD-platelet lysate phosphorylated TDP-43 content. This antibody did not well-recognize platelet lysate phosphorylated TDP-43 in another neurodegenerative disease, ALS. We have used ALS patient platelet lysate as a negative disease control for testing the selectivity of the antibody that recognized the high levels of pTDP-43 in AD platelet lysate. This observation was unexpected and requires more in depth analysis in larger patient cohort. The alternative explanation would be that AD-selective antibody-producing clones would differ in recognizing the specific modification in TDP-43 in AD platelet lysate.TDP-43 proteinopathy is characterized by decreased solubility, hyper-phosphorylation and the generation of a 25 kDa C-terminal fragment ,56–58. Tet al., have reviewed the recent findings about the prion-like characteristics of TDP-43 propagation and offered the concepts of contiguous and noncontiguous propagation of misfolded proteins including TDP-43 [How does aberrant brain TDP-43 appear in peripheral blood cells? One explanation might be that the TDP-43 protein has a C-terminus Q/N rich region ; therefog TDP-43 . Consideg TDP-43 , it is cg TDP-43 . We are AD-selective antiphospho (S409/410-2) TDP43 antibody screening could be utilized as part of a diagnostic panel rather than standalone signature marker at this stage. We do not clearly know the pathobiology of TDP-43 in early stages of AD and other relevant diseases that eventually lead to AD such as mild cognitive impairment, mild dementia and frontal lobe dementia. It would be desirable to obtain platelet samples from patients with AD-relevant diseases and to analyze the appearance of phosphorylated TDP-43 in platelets, which not only help to monitor the disease prognosis but also contribute to the early diagnosis.In this study, we have provided a trend of TDP-43 profiles in AD and age-matched healthy subjects A & C. ToIn vitro biophysical studies in cell culture and mouse brain have suggested that TDP-43 naturally tends to form a dimeric protein as cited in a recent review [et al., cell-based TDP-43 aggregation and modifications model is a powerful tool [It is a well-known fact that protein aggregations occurs long before the clinical manifestations are revealed . In vitrt review . Could wful tool  to test In the near future, research teams may consider some therapeutic approaches by which cell permeable chemical chaperons that bind to misfolded protein and stabilize the folded state reduce protein misfolding . In normFinally, our results suggest that peripheral blood-derived platelets could be used a venue to identify AD-relevant biomarker candidate proteins. We have identified an AD-selective antiphospho (S409/410–2) TDP-43 Ab in platelet lysates of AD patients. In future studies, we would like to utilize this antibody as a screening tool in large AD patient population and in addition to other AD-relevant diseases such as mild cognitive impairment, mild dementia and frontal lobe dementia to validate that phosphorylated TDP-43 could be a reliable biomarker candidate. The challenge is to identify a reliable and validated blood-based biomarker(s) during next 4–6 years.TDP-43 and its phosphorylated derivative can be measured in platelet lysate.A-Phospho (S409/410-2) TDP-43 is identified as a selective antibody for Alzheimer's disease (AD) patients that discriminates AD from non-demented control and amyotrophic lateral sclerosis (based on platelet analysis of phospho-TDP43.This AD-selective antibody may be utilized as a screening tool to strengthen AD diagnosis along with cognitive tests.Patient populations with mild cognitive impairment, mild dementia and frontal lobe dementia need to be assessed for the phosphorylated TDP-43 profile.


The role of a series of functionalized graphene additives in power cable insulation in suppressing the growth of electrical treeing and preventing the degradation of the polymer matrix has been investigated by density functional theory calculations. Bader charge analysis indicates that pristine, doped or defect graphene could effectively capture hot electrons to block their attack on cross-linked polyethylene (XLPE) because of the π–π conjugated unsaturated structures. Further exploration of the electronic properties in the interfacial region between the additives and XLPE shows that N-doped single-vacancy graphene, graphene oxide and B-, N-, Si- or P-doped graphene oxide have relatively strong physical interaction with XLPE to restrict its mobility and rather weak chemical activity to prevent the cleavage of the C–H or C–C bond, suggesting that they are all potential candidates as effective additives. The understanding of the features of functionalized graphene additives in trapping electrons and interfacial interaction will assist in the screening of promising additives as voltage stabilizers in power cables. Howevcarriers . Besidescarriers –12,17–222, MgO and ZnO [2 nanocluster as a stabilizer in trapping electrons, preventing the cleavage of C–C bond and restraining the mobility of polyethylene chains by hydrogen bonds [Recently, progress has been made towards the wide experimental observation of nanosize particle fillers in suppressing the growth of electrical treeing, such as SiO and ZnO –31, toge and ZnO –34. As iet al. prepared graphene-enhanced low-density polyethylene by pretreating and the melt-compounding method, and revealed its thermal stability and improved mechanical properties by scanning electron microscopy, Raman spectra etc. [et al. explored the advancement of antioxidant functionalized graphene oxide filler in promoting the thermal stability and retaining the electrical insulating properties of low-density polyethylene [Graphene, as one of the most popular two-dimensional materials, has been widely applied in the fields of photocatalysis, pharmacology, solar cells etc. –41. The tra etc. . Bu et aethylene . However2.2.1.−5 eV and 0.05 eV Å−1, respectively. The wave function at each k point was expanded in plane wave basis sets with a kinetic cut-off energy of 450 eV. The integration of the Brillouin zone was conducted with a 2 × 2 × 1 Monkhorst–Pack grid centred at Γ-point. For minimizing periodic interactions, a vacuum layer as large as 15 Å was set in the perpendicular direction. The DFT-D2 approach [Spin-polarized density functional theory (DFT) calculations with the Perdew–Burke–Ernzerhof (PBE) functional  were perapproach  was perfapproach  was emplapproach  method w2.2.8H18 (i.e. CH3CH2CH2CH(CH3)CH2CH2CH3) organic molecule was used to simulate the chemical activity of the polyethylene chain on the surface of the graphene series sheet. The nearest distance of two C8H18 molecules in the neighbouring lattice is more than 5.6 Å so that the weak van der Waals interaction between them can be reasonably ignored. The present theoretical model is mainly concerned with the simulation of C–H bond-breaking behaviour of C8H18 by the catalytic action of graphene series, and the local interaction between them, which could effectively reflect the main interaction between the XLPE and graphene fillers in the real physical problem.A (6 × 6) supercell was constructed to model the pristine graphene sheet (G). Based on it, models of graphene oxide (GO), GO doped with B, N, Si or P atoms , as well as single-vacancy graphene (SVG) and SVG doped with B or N atoms  were constructed and optimized, as shown in 3.As predicted, the addition of nanosize particle fillers in power cable insulation was able to change the charge distribution and produce the interfacial interaction with XLPE. Therefore, the role of graphene-based series additives will be discussed below with respect to their abilities of trapping hot electrons, restricting the mobility of the polyethylene chains and preventing the C–H or C–C bond cleavage of XLPE.3.1.In general, the charge carriers are produced from the system or injected from the electrode. If the nanosized particles existing in the system could constrain the flow of charge carriers by accumulating electrons on their surfaces, the resulting negative charge will make the electric field of the cathode weaker and then suppress the continued injection of electrons from the cathode, so as to reduce the amount of space charge in the system.8H18 molecule adsorbed upon a series of functionalized graphene surfaces. When 1 or 2 extra electrons are injected into the systems, denoted as the Ion1− and Ion2− case, respectively, the charge is redistributed among the atoms. By summarizing the charge of atoms belonging to the C8H18 molecule and graphene series, respectively, we could determine the location of extra electrons in the systems. One can see that the charge values at the graphene series are up to −0.89 and −1.86 for the Ion1− and Ion2− case, respectively, whereas those at the C8H18 molecule are close to zero. Thus the electron density is mainly concentrated on the graphene series, not on the C8H18 molecule. The strong ability of capturing electrons is mainly generated by the π–π conjugated unsaturated structures of the graphene series, which is consistent with a previous report for polycyclic additive [1− and Ion2− case, which indicates that they have almost equal ability to trap electrons. So we could infer that the graphene-based particles, which include not only the nine patterns in this study but also other defect or doped graphene, are all very good candidates as additives for power cable insulation in the general sense of effectively trapping hot electrons to suppress the growth of electrical treeing. The mechanism of using unsaturated electronic structures of additives to trap extra electrons could extend to more broad cases. It could help us to understand why most of the nanosize particles could act as good stabilizers, because they usually have unsaturated surface structures with similar properties to the graphene series we studied.additive . It sugg3.2.3.2.1.8H18 molecule. As typical two-dimensional materials, the pristine and functionalized graphene sheets could produce an adsorption effect for the C8H18 molecule, which could be seen from the calculated adsorption energies defined as follows:E(C8H18/graphene) is the total energy of the pristine or functionalized graphene sheet with the adsorbed C8H18, and E(graphene) and E(C8H18) are the energies of pristine or functionalized graphene sheet and free C8H18 molecule, respectively.The interaction between the additive and XLPE in the interfacial region is very important for evaluating the mobility and stability of the polyethylene chains. In the present theoretical models, the interaction of the additive with the XLPE could be evaluated by the adsorption ability of the graphene series to the C8H18 among the graphene-based models studied in the present work. As we know, heteroatom doping or defect in graphene will induce charge redistribution and increase dipole moment. The Bader charge distributions at each atom for all the nine patterns are depicted in 30, C43) possess highest positive charges of 0.374 and 0.302. The enhanced dipole–dipole interaction with C8H18 strengthens the adsorption ability. Stronger physical adsorption activity usually means a stronger ability to restrict the mobility of polyethylene chains. So the heteroatom doping and defect are effective functionalization ways to enhance the physical interaction of graphene-based additives and XLPE, and thus improve the stability of the XLPE.As listed in 3.2.2.8H18 to the graphene-based sheets is studied with the conditions of neutral and anion (Ion1− and Ion2−) states by injecting 0, 1 or 2 electrons to the systems, respectively. The transition states involved in the reaction pathways of H migration are located. Based on the energy barrier (ΔE = ETS − ER) and the reaction energy (ΔH = EP − ER), listed in E and ΔH are low, it means the H atom of XLPE could move easily to the graphene-based surface to build a new chemical bond with the atom in it, simultaneously resulting in the formation of a C8H17 radical. The radical is so active that it could cross-link with other carbon chain radicals, not beneficial for the stability of XLPE. On the other hand, the radical could react with other molecules in its environment, which will probably lead to the breaking of the C–C bond and the growth of electrical treeing. On the contrary, the graphene-based fillers with relatively high ΔE and ΔH for H migration reaction will be suitable additives to block H migration and further protect the XLPE.Besides the van der Waals interaction in the interfacial region discussed above, there may be stronger chemical interaction between the additive and XLPE. In the latter case, some chemical reactions can be involved, such as the H migration process. Owing to the doping or defect, there are some active sites in the functionalized graphene sheet as mentioned above, and these active sites probably possess the ability to attract the H atom of XLPE. As a following step, the H migration will probably induce the C–H or C–C bond cleavage of XLPE, and finally result in electrical treeing. Therefore, the reaction mechanism for H migration from Ca, the H atom in C8H18 transfers to the pristine graphene, forming a new C–H bond. In Figure 4b, the O–H bond is produced in the graphene oxide series . In figure 4c, the H atom prefers to bond with the C atom at the edge of the single-vacancy area of SVG, B-SVG and N-SVG. The key values of bond distances and angles for reactants, transition states and products on the H migration reaction pathways for neutral and ionic states can be seen in electronic supplementary material, table S1.The geometrical structures of reactants, transition states and products on the H migration reaction pathways are shown in figure 41− and Ion2− state, respectively. In particular, the H migration products are more stable than the reactant as seen by the negative ΔH values for SVG in E and negative ΔH indicate that SVG can promote H migration from the XLPE to the filler, and make the XLPE unstable. However, the circumstances are improved by B or N doping to SVG. Especially for N-SVG, the ΔE and ΔH increase to be more than 1.63 and 1.15 eV, respectively, which could effectively prevent the H migration process and protect the XLPE. Furthermore, it is worth noting that the ΔE and ΔH of ionic states are usually higher than those of the neutral state for most of the cases we have studied above. It tells us that the H migration reaction from XLPE to graphene-based fillers is even more difficult with the involvement of hot electrons, which is beneficial for protecting the XLPE. In summary, because of the relatively strong ability to hinder the H migration reaction and keep the stability of XLPE, the graphene-based particles studied in this work can be promising candidates as additives in power cable insulation, except SVG. Among all the considered potential candidates in this study, N-SVG could be the best one because of its strongest adsorption ability to XLPE (−0.49 eV) and relatively high barriers (more than 1.63 eV) and reaction energies (more than 1.15 eV) in the H migration reaction. In contrast with that, SVG without doped atoms is the weakest candidate as an additive in power cable insulation.Owing to the highest barriers and reaction energies for both neutral and ionic states, as presented in 3.3.2 with an exposed O atom, they will easily induce the H migration reaction and destroy the stability of XLPE. It is expected that the information above will be useful for the screening and development of promising additives in power cable insulation.With the discussion and information above, we could have a better understanding of the role of graphene-based fillers in power cables. More importantly, it could guide the rational design of and screening for potential additives in power cable insulation. The strong ability to capture electrons is firstly essential as a property for a promising additive in power cables. By eliminating the attack of hot electrons, the growth of electrical treeing could be suppressed significantly. So the nanosized particles, graphene or nanotubes-based carbon materials, polycyclic organic molecules, as well as others with π–π conjugated structures are all potential candidates. The second factor which should be examined is the strong physical interaction between the additive and polyethylene to constrain the mobility of polyethylene chains. As calculated in this paper, the proper adsorption energies between them should be in the range −0.16 to −0.49 eV. It is also related to the electronic properties of the additives. Those polar particles with relatively big dipole moments usually show many advantages through the dipole–dipole interaction with XLPE. That is the reason that doped or defect graphene has stronger van der Waals interaction with XLPE than pristine graphene. Thirdly, the inert chemical activity of the additive in the interfacial region should be guaranteed, i.e. a high reaction barrier of around or more than 1.0 eV in the H migration reaction. If the particles have obvious dangling bonds or electron-defect atoms, such as SVG with dangling C or SiO4.DFT calculations are performed to evaluate the role of pristine and functionalized graphene-based additives in power cable insulation. It is predicted that the graphene series or others with π–π conjugated unsaturated structures have a general ability of effectively trapping hot electrons to suppress the growth of electrical treeing. By calculating the adsorption energy and reaction pathway of H migration, it is found that N-doped single-vacancy graphene, graphene oxide, and B-, N-, Si- or P-doped graphene oxide possess relatively strong physical interaction with XLPE and quite weak chemical activity, which are promising additives to suppress electrical treeing and the degradation of the polymer matrix. The studies of the features of the trapping electrons, the physical interaction and chemical activity in the interfacial region provide further insights into the design of promising additives for power cables.


The RNA-binding and -processing protein TAR DNA-binding protein 43 (TDP-43) is heavily linked to the underlying causes and pathology of neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In these diseases, TDP-43 is mislocalized, hyperphosphorylated, ubiquitinated, aggregated and cleaved. The importance of TDP-43 cleavage in the disease pathogenesis is still poorly understood. Here we detail the use of D-sorbitol as an exogenous stressor that causes TDP-43 cleavage in HeLa cells, resulting in a 35 kDa truncated product that accumulates in the cytoplasm within one hour of treatment. We confirm that the formation of this 35 kDa cleavage product is mediated by the activation of caspases. Inhibition of caspases blocks the cleavage of TDP-43, but does not prevent the accumulation of full-length protein in the cytoplasm. Using D-sorbitol as a stressor and caspase activator, we also demonstrate that the A90V variant of TDP-43, which lies adjacent to the caspase cleavage site within the nuclear localization sequence of TDP-43, confers partial resistance against caspase-mediated generation of the 35 kDa cleavage product. TAR DNA-binding protein 43 (TDP-43) is a ubiquitously expressed nuclear RNA- and DNA-binding protein , 2. It iIn brain tissues of FTLD-U patients, CTFs of 20–25 kDa are found in insoluble protein fractions , and 35 Here, we use the sugar alcohol D-sorbitol as a stressor to induce the formation and cytoplasmic accumulation of the 35 kDa fragment of TDP-43 in a cell culture system. We show that the formation of this cleavage product, but not the mislocalization of uncleaved TDP-43 to the cytoplasm, is abrogated by caspase inhibition. We also show that an alanine to valine amino acid substitution at position 90 (A90V) confers partial resistance to caspase cleavage at the Asp89 site.TARDBP, wild-type reference sequence NM_007375.3) were purchased from Origene. The presence of the A90V (c.269C>T) and M337V (c.1009A>G) mutations was confirmed by DNA sequencing performed by GENEWIZ . NLS-EGFP was a gift from Rob Parton (Addgene plasmid # 67652) [HeLa cells  were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum (FBS), 100 u/ml penicillin and 100 μg/ml streptomycin. For immunofluorescence analysis, cells were grown on coverslips in 24-well plates (Cellstar). For stress experiments, cells were treated with either 10 μM N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal  for 4 h, 1 μM thapsigargin (Sigma) for 60 min, 0.5 mM sodium arsenite (Sigma) for 30 min, 1 mM hydrogen peroxide for 60 min or 0.4 M D-sorbitol (Sigma) for 60 min. Cells were transiently transfected using Fugene HD (Promega), with a Fugene:DNA ratio of 3:1, for 24 hours. Plasmids harboring N-terminally myc-tagged wild-type or mutant human TDP-43 sequences (# 67652) .In a subset of D-sorbitol stress experiments, HeLa cells were pre-treated with 50 μM of the irreversible pan-caspase inhibitor Z-Val-Ala-Asp-(OMe)-Fluoromethyl Ketone (Z-VAD-(OMe)-FMK, Cayman Chemical) or DMSO solvent control. After 30 min of caspase inhibition, 0.4 M of D-sorbitol or an equivalent volume of fresh medium was added and cells were incubated for a further 60 min before processing for immunofluorescence staining or fractionation of cells into enriched nuclear and cytosolic fractions.To assess the rescue of TDP-43 and HuR cellular localization, cells were treated with 0.4 M D-sorbitol for 1 h and then allowed to recover in normal medium for 0 min, 15 min or 60 min.et al. [For analysis of total protein, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer  supplemented with protease inhibitors , phosphatase inhibitors  and 2 mM ethylenediaminetetraacetic acid (EDTA). Cellular debris was removed by centrifugation at 16’000 x g for 15 min. Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Cellular fractionation experiments to obtain enriched cytosolic and nuclear fractions from cells grown on 60 mm dishes (Costar) were performed using a cell fractionation kit  according to the manufacturer’s standard protocol. RIPA-soluble and–insoluble protein fractions were extracted according to a protocol adapted from Cohen Protein lysates and fractionated samples were separated on Tris-glycine SDS polyacrylamide gels (polyacrylamide concentrations ranging from 10–15%) and transferred onto polyvinylidene difluoride  membranes. Blocking solution and antibody dilutions were made up in 5% skimmed milk in Tris-buffered saline and 0.1% Tween-20 (TBS-T). Western blots were developed with enhanced chemiluminescent substrates . Digital images were acquired with a Chemidoc MP imaging system (BioRad). Where required, blots were stripped with stripping buffer for 15 min at room temperature (Thermo Fisher Scientific), blocked in 5% milk in TBS-T and reprobed with appropriate loading control antibodies.Following stress treatments, cells were washed with phosphate-buffered saline (PBS) and fixed for 15 min with 4% paraformaldehyde (in PBS) before solubilization with 0.25% Triton-X (in PBS) for 10 min. Cells were washed again and blocked for 1 hour in 10% normal goat serum in PBS (Abcam). The fixed cells were incubated in primary antibody overnight at 4°C in a humidified chamber, and in secondary antibody for 1 hour at room temperature. Coverslips were mounted on microscope slides with Prolong Gold antifade with DAPI (Thermo Fisher Scientific) or counterstained with Hoechst 33342  and mounted with Prolong Gold antifade (Thermo Fisher Scientific). Cells transfected with NLS-EGFP were counterstained with Hoechst and mounted with Prolong Gold antifade. Images were acquired using a Nikon A1 confocal/Eclipse Ti inverted microscope system and NIS Elements software (Nikon).We used the following antibodies for detection of proteins by Western blot: rabbit carboxy- and amino-terminal TDP-43 , mouse α-tubulin , mouse GAPDH , rabbit GAPDH , rabbit Lamin B1 , mouse histone H3 , rabbit caspase-3 , mouse caspase-9 , mouse Nup153 QE5 . Anti-mouse and anti-rabbit horseradish peroxidase-coupled secondary antibodies were obtained from Jackson Immunoresearch . For immunofluorescence staining, we used rabbit carboxy- and amino-terminal TDP-43 (1:150 and 1:500), mouse myc 9E10  and mouse HuR . Secondary anti-mouse and anti-rabbit Alexa-488-, Alexa-555- and Alexa-568-coupled antibodies were purchased from Thermo Fisher Scientific.Western blot band densities were quantified with Image Lab 5 (BioRad). For immunofluorescence signal quantification of the cytosolic and nuclear TDP-43 ratios we used the Fiji open source program . For sta2+-ATPase inhibitor thapsigargin  [2,6 = 13.16, P = 0.0064, followed by Tukey’s multiple comparisons test)  , the oxi 60 min) , 29 and  60 min) . However 60 min) , 29, 42.ns test) . These ff TDP-43 . In addi2,9 = 19.87, P = 0.0005, followed by Dunnett’s multiple comparisons test) and an increase in the 35 kDa cleavage product  .We quantified both full-length TDP-43 and CTF35 in untreated, sorbitol- and hydrogen peroxide-treated HeLa cells and found that sorbitol led to a significant reduction in full-length TDP-43  (one-way ANOVA: F 0.0044) . We obseBy fractionating untreated and stressed HeLa cells into cytoplasmic and nuclear components, we detected CTF35 by Western blot in the cytoplasmic fraction in sorbitol-treated samples, while full-length TDP-43 in the nuclear fraction was reduced , Fig 4C.et al. also observed poly ADP ribose polymerase (PARP) cleavage in HEK293T cells treated with sorbitol, suggesting that these cells are undergoing apoptosis and thus caspase-3 is activated, which might in turn be responsible for the formation of the 35 kDa fragment of TDP-43 [3,16 = 26.95, P < 0.0001, followed by Tukey’s multiple comparisons test), indicating that sorbitol induces the activation of caspases, which in turn cleave full-length TDP-43. Using a caspase-3-specific antibody, we confirmed that caspase-3 is activated by sorbitol, and that pre-treatment with ZVAD-FMK inhibits its activation   (We then performed immunofluorescence staining of HeLa cells treated with sorbitol in the presence or absence of ZVAD-(OMe)-FMK in order to determine whether caspase inhibition can also rescue the TDP-43 relocalization from the nucleus to the cytoplasm . Howeverns test) . In ordens test) . While wns test) .2,8 = 7.754, P = 0.0134, followed by Dunnett’s multiple comparisons test).The nuclear envelope is the cellular barrier that demarcates the nucleus. During apoptosis, nuclear permeability increases due to the breakdown of the nuclear pore complex (NPC), which regulates traffic of large macromolecules between the cytosol and nucleus –48. As a2,6 = 119.7, P < 0.0001, followed by Dunnett’s multiple comparisons test: 0 vs. 15 min p = 0.001, 0 vs. 60 min p = 0.001). This decrease in the cytoplasmic-to-nuclear TDP-43 ratio was paralleled by an increase in nuclear TDP-43 signal   , S5B Fig2,9 = 5.989, P = 0.022, followed by Tukey’s multiple comparisons test) and sorbitol-treated cells    (The caspase cleavage site resulting in the formation of the TDP-43 cleavage product CTF35 lies after aspartic acid 89 . Introduns test) . Furtherns test) . As we dns test) . We thus2,12 = 4.758, P = 0.03, followed by Dunnett’s multiple comparisons test). These results are in line with published literature showing cytoplasmic accumulation of TDP-43 in A90V- and M337V-expressing cells in vitro [2,12 = 0.664, P = 0.533) . Sorbitol treatment caused a decrease in RIPA-soluble TDP-43 and an increase in TDP-43 in the urea fraction, confirming that sorbitol promotes the aggregation of overexpressed TDP-43 regardless of mutation. Last, we investigated stress granule formation in HeLa cells overexpressing TDP-43. We detected TDP-43-positive stress granules in a very small number of untreated cells (<4% of transfected cells). Sorbitol treatment caused an increase in the number of cells with stress granules (>8% of transfected cells). However, we found no difference in the number of cells with TDP-43-positive stress granules between genotypes under either control or stress conditions  . However= 0.533) . To conf= 0.533) . We dete= 0.533) . There wnditions . HoweverWhile the occurrence of C-terminal fragments of TDP-43 is a common pathological hallmark in ALS and FTD pathology, the significance of TDP-43 truncation for disease pathogenesis remains unclear. It is unknown whether TDP-43 fragments are disease-relevant species or a by-product of the cellular protein degradation machineries’ attempt to get rid of toxic full-length TDP-43 species. For example, whether C-terminal fragments affect cell viability is uncertain, with various papers suggesting reduced , similaret al. that describes the use of high concentrations of D-sorbitol, a sugar alcohol, as an osmotic and oxidative stressor in HEK293T cells that drives TDP-43 cleavage, resulting in the generation of a 35 kDa fragment [et al. showed that cleavage of Poly(ADP-ribose) polymerase (PARP), a known substrate of caspase-3, preceded cleavage of TDP-43 in sorbitol-treated HEK293T cells [et al. This is in line with the literature showing that CTF35 is formed in response to caspase activation [In the current study, we have adapted a protocol by Dewey fragment . D-sorbi3T cells . However3T cells , 57. By tivation .By fractionating cells into nuclear and cytoplasmic fractions, we showed that CTF35 accumulates in the cytoplasm of sorbitol-treated HeLa cells, while full-length protein in the nucleus is depleted. However, while pre-treatment of cells with caspase inhibitor prevented the cleavage of TDP-43, we found that it did not rescue the relocalization of full-length TDP-43 from the nucleus to the cytoplasm. This result suggests that C-terminal protein truncation is not necessary to trap TDP-43 in the cytoplasm and that the cytoplasmic relocalization of TDP-43 likely precedes its caspase-dependent cleavage. We also found that HuR, another nuclear-cytoplasmic shuttling protein, relocalizes to the cytoplasm in response to sorbitol, as does NLS-EGFP, albeit with a smaller effect size than seen with TDP-43 and HuR. The accumulation of TDP-43 in the cytoplasm is thus likely the result of sorbitol-induced leakiness of the nuclear envelope. One of the factors known to regulate nuclear permeability is the nuclear pore complex component Nup153, which is degraded in apoptotic cells , 48, 49.in vitro study has shown that A90V-TDP-43 protein incubated with purified caspase-3 is less efficiently cleaved than WT-TDP-43 [We also used sorbitol to investigate the cleavage propensity and cytoplasmic accumulation of the A90V variant of TDP-43. This variant lies within the bipartite nuclear localization sequence of TDP-43 directly adjacent to the caspase-3 cleavage site after Asp89. Genetic studies have detected the A90V variant both in ALS cases as well as in healthy control individuals, leaving the impact of this single amino acid mutation on disease pathogenesis unclear , 62–64. T-TDP-43 . We addrT-TDP-43 , 65. ThiT-TDP-43 , 66. ThiIn summary, we have described here the use of D-sorbitol as an inducer of caspase-dependent cleavage of TDP-43 to investigate the relationship between TDP-43 cleavage and cytoplasmic relocalization. We also looked at the impact of these phenotypes in the context of the TDP-43 mutants A90V and M337V and observed cleavage resistance of the A90V variant in a cellular context.in vitro [At the time this study was conducted, the cleavage resistance of the A90V variant at the Asp89 cleavage site was not published. However, a recent paper published by Chiang et al. demonstrated this resistance against caspase-3-mediated cleavage in vitro .S1 Fig+/HuR+ stress granules (N = 4). Results represent mean number of cells with stress granules ± S.E.M.; * p < 0.05, ** p < 0.01; one-way ANOVA  followed by Tukey’s multiple comparisons test.HeLa cells were either pre-treated with pan-caspase inhibitor Z-VAD-FMK or DMSO control before 0.4 M D-sorbitol was added as a stressor for 1 h. Control cells were not treated with either sorbitol or Z-VAD-FMK. (A) Example of immunofluorescence staining of untreated and sorbitol-stressed HeLa cells. Stress granules (indicated by arrowhead) are defined as cytoplasmic puncta co-stained with TDP-43 (green) and the stress granule marker HuR (red). Scale bar = 30 μM. (B) Quantification of cells with a minimum of three TDP-43(TIF)Click here for additional data file.S2 Fig2O2, 1 μM thapsigarin (TH) or 0.4 M D-sorbitol. Full-length TDP-43 and its 35 kDa cleavage product (marked with asterisk) were detected in the RIPA-soluble protein fraction (R), but not in the RIPA-insoluble, urea-soluble fraction (U). Arrowhead denotes unspecific band.HeLa cells were treated with 1 mM H(TIF)Click here for additional data file.S3 Fig2O2), 1 μM thapsigargin (TH) or 0.4 M D-sorbitol. The generation of CTF35 as well as the activation of caspase-3 was observed in HeLa cells after 1 hour of D-sorbitol treatment, but not in SH-SY5Y cells. B) Prominent formation of HuR-positive stress granules in SH-SY5Y cells in response to sorbitol (indicated with arrowhead). Blue = DAPI, green = TDP-43, red = HuR. Scale bar = 30 μm.A) HeLa cells and undifferentiated SH-SY5Y cells were treated with 0.5 mM (SH-SY5Y) or 1 mM (HeLa) hydrogen peroxide (H(TIF)Click here for additional data file.S4 Fig2,8 = 7.754, P = 0.0134) followed by Dunnett’s multiple comparisons test.(A) Immunofluorescence images of HeLa cells transfected with EGFP fused to a nuclear localization sequence. After 24 h, cells were treated with DMSO or Z-VAD-FMK for 30 min, followed by regular medium or 0.4 M sorbitol for 60 min. Scale bar = 20 μm. (B) Quantification of nuclear NLS-EGFP signal as a proportion of total cellular EGFP signal (N = 3–4). Results represent mean percentage of nuclear GFP signal ± S.E.M.; * p < 0.05; one-way ANOVA (F(TIF)Click here for additional data file.S5 Fig2,6 = 10.34, P = 0.0114; HuR: F2,6 = 18.72, P = 0.0026) followed by Dunnett’s multiple comparisons test.HeLa cells were exposed to 0.4 M D-sorbitol for 1 h, then left to recover for 0 min, 15 min or 60 min. (A) Nuclear TDP-43 and (B) HuR signals were quantified. Results represent mean nuclear immunofluorescence per cell ± SEM; ** p < 0.01 (N = 3); one-way ANOVA (TDP-43: F(TIF)Click here for additional data file.S6 FigTDP-43 was expressed in HeLa cells for 24 h before sorbitol was added for 1 h. Overexpressed myc-TDP-43 forms a granular staining pattern in the nucleus indicative of protein aggregation both in cells with (blue rectangle) and without (orange rectangle) cytoplasmic TDP-43 relocalization (WT-transfected example).(TIF)Click here for additional data file.S7 Fig2,12 = 1.098, P = 0.365; +Sorbitol: F2,12 = 0.3245, P = 0.729).HeLa cells were transfected with WT, A90V or M337V TDP-43. After 24 h, cells were either left untreated or were treated with 0.4 M sorbitol for 1 h. (A) Immunofluorescence staining of transfected HeLa cells with or without additional sorbitol treatment. Green = myc, red = TDP-43. Arrowheads indicate stress granules. (B), (C) Quantification of proportion of transfected cells with TDP-43-positive stress granules in control conditions (B) or after sorbitol stress (C). Results represent mean percentage of transfected cells with TDP-43-positive cytoplasmic granules ± SEM. one-way ANOVA followed by Tukey’s multiple comparisons test (-Sorbitol: F(TIF)Click here for additional data file.