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.


Surface acoustic waves (SAWs) propagating on piezoelectric substrates offer a convenient, contactless approach to probing the electronic properties of low-dimensional charge carrier systems such as graphene nanoribbons (GNRs). SAWs can also be used to transport and manipulate charge for applications such as metrology and quantum information. In this work, we investigate the acoustoelectric effect in GNRs, and show that an acoustoelectric current can be generated in GNRs with physical widths as small as 200 nm at room temperature. The positive current in the direction of the SAWs, which corresponds to the transportation of holes, exhibits a linear dependence on SAW intensity and frequency. This is consistent with the description of the interaction between the charge carriers in the GNRs and the piezoelectric fields associated with the SAWs being described by a relatively simple classical relaxation model. Somewhat counter-intuitively, as the GNR width is decreased, the measured acoustoelectric current increases. This is thought to be caused by an increase of the carrier mobility due to increased doping arising from damage to the GNR edges. Recent efforts, however, have concentrated on the interactions of SAWs with graphene27, a two-dimensional (2D) carbon allotrope with outstanding physical properties and numerous potential applications30.The transportation of charge in low-dimensional electron systems has attracted significant attention in recent years as a potential route towards nanoscale electronic devices. Surface acoustic waves (SAWs) propagating on piezoelectric materials provide a unique tool for studying and realising such systems. The strong interaction between the piezoelectric fields associated with a SAW, propagating on a piezoelectric substrate, and a charge carrier system is known as the acoustoelectric effect8, and we have previously studied the acoustoelectric response of large-area graphene sheets produced by chemical vapour deposition (CVD) transferred to LiNbO3 SAW devices at room temperature9, low temperature10, and under illumination11. Theoretical and experimental reports have examined DC-biased mono- and multi-layer graphene sheets for SAW amplification13, while others have studied the use of electrolytic solutions14, and gases15 to control the carrier concentration in graphene films, opening up possibilities for low-power chemical sensors. In contrast, by using an ion gel gate to modulate the conductivity of graphene, Bandhu and Nash16 demonstrated that graphene can be used to control the SAW amplitude and velocity shift. Optimization of this approach could lead to a practical, cost-effective voltage-controlled velocity shifter suitable for use in wireless sensor applications16.The ability of piezoelectric potentials to trap and transport charge at the speed of sound enables the generation of acoustoelectric currents. These have been measured in micron-scale graphene monolayers19, and several groups have demonstrated carbon monoxide, hydrogen20, and moisture24 detectors based on SAW-graphene systems.Theoretical studies predict a further variety of physical phenomena arising from SAW-graphene interactions31, there is an absence of research on SAW interactions with graphene nanoribbons (GNRs). It is a well-known disadvantage of graphene that its gapless band structure is not suited to integration with digital electronics, which typically require high on-off ratios32. Patterning graphene into GNRs can open a bandgap that varies as a function of ribbon width, with appropriate fabrication methods32. Additionally, the electric potential associated with SAWs propagating on piezoelectric materials enables the confinement of charge carriers in nearby electronic systems, that could have applications in metrology and quantum computing35.Despite several studies highlighting the potential advantages of incorporating graphene and other 2D materials with SAW devices3 delay lines. The acoustoelectric current was first studied in GNR arrays consisting of 500 nm-wide ribbons, and the measured linear dependence of acoustoelectric current on SAW intensity and frequency is consistent with the interaction between the SAWs and the graphene charge carriers being described by a relatively simple classical relaxation model. We have also investigated the width dependence of the acoustoelectric current in GNRs, with widths ranging from 200–600 nm, with GNRs of different widths all fabricated on the same SAW delay line. The measured increase in acoustoelectric current as the nanoribbon width decreases is attributed to additional p-doping of the GNR edges, raising the Fermi level in the samples and enabling conduction across grain boundaries and charge puddles arising from the polycrystalline nature of CVD graphene.In this manuscript, measurements of an acoustoelectric current generated in GNRs fabricated from monolayer CVD graphene transferred to LiNbO3-GNR SAW devices where the nanoribbons are all of 500 nm width. These are arranged in a 2 mm × 3 mm array, shown schematically in Fig. et al. in investigations of GNR arrays on SiO2/Si36. The use of double-digit interdigital transducers (IDTs) enables the efficient excitation of SAWs at a number of resonant frequencies. The IDTs were pre-defined on the surface by the manufacturer  of the LiNbO3 crystal with an acoustic aperture of 3.25 mm and separation distance 5.4 mm, allowing large-area CVD graphene monolayers to be easily positioned between the transducers without shorting them out. The fundamental frequency of 11 MHz (SAW wavelength of 361.7 μm) corresponds to a digit width of 45.2 μm.We first consider LiNbOae) in the GNRs between contacts A and B  as a function of SAW frequency and RF power, at room temperature under vacuum. Two devices of this design were studied (Devices 1 and 2), with these results obtained from Device 1. Continuous wave SAWs were excited in increments of 1 MHz in the range 1–500 MHz, and each data point represents the mean of five measurements of Iae. The measured current maxima in Fig. 9, confirming its acoustoelectric nature. Iae of up to ~5.5 μA was measured for a SAW frequency fSAW = 442 MHz and an applied RF power of +20.0 dBm. Acoustoelectric currents of the same order of magnitude were measured between contacts B and C, and C and D, similar to that reported by Okuda et al.14. This is significantly larger than values we have previously measured on unpatterned graphene samples, but this is due to improvements in the transfer method we use, leading to improved quality graphene film (which we believe results in a higher carrier mobility). This is reflected in room temperature current-voltage measurements of the GNR arrays on Device 1, under vacuum, which yielded values of sheet resistance of: 10.5 kΩ/□ (A to B); 23.7 kΩ/□; (B to C); and 56.8 kΩ/□ (C to D). These values compare to typical values of approximately 100 kΩ/□ we have measured previously11, including in GNR samples fabricated using our old transfer method The positive sign of Iae in the direction of SAW propagation is indicative of hole transportation, and is consistent with the CVD graphene being p-doped by PMMA residues left during the transfer process37. Raman analysis of Device 1 (Supplementary Information) showed the position of G peak at 1588 cm−1, a blue-shift also consistent with p-doped graphene38. Upon reversal of the SAW direction, the sign of the current also reverses, confirming that the current is induced by the SAWs. Measurements of Iae were found to be stable and reproducible over the course of several weeks. Qualitatively similar results were seen in Device 2, although, due to the significantly higher resistance, 323.6 kΩ/□ between contacts B and C , the acoustoelectric current was approximately 100 nA for the same SAW frequency and intensity. To rule out possible interference effects, measurements of the acoustoelectric current in Device 2 were repeated with RF frequency increments of 0.5 MHz, but the change in the measured current was negligible.Figure 11. A linear dependence of Iae on SAW intensity is confirmed via fitting (solid lines). The inset to Fig. ae on fSAW for a fixed intensity of 3 mW/m (dashed line), where the acoustoelectric current at each frequency has been extracted from the linear fits to the measured Iae in Fig. j is given by:μ is the charge carrier mobility, Q is the phonon pressure given by Q = IΓ/v, I is the SAW intensity, and v is the velocity of the SAW (3979 m/s in 128° YX LiNbO339). The attenuation per unit length Γ is given by a non-monotonic function of the diagonal component of the conductivity tensor σ2D 2:K2 is the piezoelectric coupling coefficient (0.056 in 128° YX LiNbO3), λ is the SAW wavelength, and the attenuation is maximised at a characteristic conductivity σM. Bandhu and Nash have reported σM = 10−7 Ω−1 in graphene-lithium niobate hybrid systems16.In Fig. 40, and it is likely that the lithium niobate also impacts the charge carrier mobility. In previous work in similar samples the mobility μ was estimated as ~5–10 cm2/V.s11. In Device 1 the relatively high conductivity compared to σM (σ2D/σM~1000) is expected to yield relatively low acoustoelectric attenuation of SAWs, via Equation  with the periodicity (10 μm) of the perpendicular graphene bridges in the array. This is similar to the recent study by Mayorov et al.25, in which IDTs were formed from graphene on LiNbO3 for the purpose of SAW generation and detection. This coupling could explain the extremely low SAW intensity and relatively large acoustoelectric currents measured for fSAW = 356 MHz in Devices 1 and 2. Iae measured between contacts B and C of a third device fabricated without the perpendicular bridge structures did not exhibit such behaviour  in the device without bridge structures , as shown schematically in Fig. 28. Subtle differences in fabrication processes can lead to different concentrations of etchant salts and PMMA residues between devices37, which can further affect performance. The fabrication of multiple nanoribbon arrays on the same LiNbO3 SAW delay line both reduces the variability in quality between different width ribbons, but also ensures that the different GNR arrays experience the same SAW intensity at any frequency. Arrays were defined in the acoustic beam path, with a width of 400 μm and a lateral spacing of 20 μm from any neighbouring arrays. All arrays shared a common electrical contact, A, in Fig. To investigate the effect of the GNR width on the acoustoelectric interaction, multiple nanoribbon arrays were fabricated on the same LiNbOae from these arrays are therefore not presented. Arrays of 200, 350, 400, 500, and 600 nm-wide GNRs had sheet resistance of 15.1, 3.7, 3.8, 5.4, and 7.0 kΩ/□ respectively.Current-voltage measurements at room temperature, under vacuum, revealed that contacts A and H were open circuit, and that the array between contacts A and G had a relatively high sheet resistance of 38.8 kΩ/□. Measurements of IfSAW = 356 MHz, consistent with the measurements in Figs λSAW coincides with the periodicity of the perpendicular bridge structures in the arrays. This is the case even without accounting for the frequency response of the IDTs. For SAW frequencies of 11, 33, 97, and 119 MHz the acoustoelectric current generally decreases as the ribbon width increases, so that the largest acoustoelectric current is measured in the smallest ribbons. At SAW frequencies above 205 MHz Iae, decreases significantly when the ribbon width is decreased from 350 nm to 200 nm. A Raman map of the graphene 2D and G peaks taken at a random location in the array of 200 nm-wide GNRs revealed spatial discontinuities of ~15 μm across (see Supplementary Information). As the attenuation of the SAWs by the charge carriers is determined by the conductivity of the graphene on the scale of half the SAW wavelength40, and for fSAW = 205 MHz the SAW wavelength is ~20 μm, the inhomogeneous conduction path in the 200 nm array leads to lower conductivity, lower SAW attenuation, and correspondingly lower acoustoelectric currents at high SAW frequencies, where the dimensions of the inhomogeneities are similar to the SAW wavelength. At frequencies below 205 MHz, where the SAW wavelength is much larger than the scale of the discontinuities, the acoustoelectric current increases when the ribbon width decreases from 350 nm to 200 nm, consistent with the overall trend of increasing current with decreasing ribbon width .In Fig. fSAW = 33 MHz, is plotted as a function of σ2D/σM (solid orange line), where we have taken a value of σM = 10−7 Ω−1, determined for graphene-LiNbO3 hybrids16. The symbols in this plot correspond to the attenuation calculated for each GNR, again using Equation In Fig. 43. In this study, significant p-type doping was observed as the width of the nanoconstriction was decreased, which was attributed to the oxidation of the edges of the constrictions caused by the fabrication process. The increase in the p-type doping was reflected in a shift in the Dirac voltage, with a shift of approximately 3 volts seen between 600 nm and 300 nm wide constrictions. Using the standard capacitor model for graphene on Si/SiO2, this shift in the Dirac point corresponds to a significant increase in the Fermi energy of approximately 15 meV. The conductivity of transferred CVD graphene is known to be limited by grain boundaries, tears, wrinkles and non-homogenous impurities42, and in previous temperature dependent measurements of the acoustoelectric current10, we showed that the mobility was thermally activated, with an activation energy of approximately 60 meV. Although we are unable to directly measure the carrier concentration or Fermi energy in our measurements, an increase in the Fermi energy of 15 meV would cause the mobility to more than double (taking the activation energy to be 60 meV), when the ribbon width is reduced from 600 nm to 300 nm. This is broadly consistent with our measurements, where an increase in the mobility of this order is required to explain the observed increase in the acoustoelectric current. Lee et al.44 also showed how a non-uniform carrier concentration in the graphene can be used to explain the origin the dependence of the mobility on channel width.The increase in conductivity with decreasing GNR width Fig.  is consiμ ~ 5–10 cm2/V.s11. Finally, we believe that the relatively large acoustoelectric currents measured in the array of 200 nm-wide GNRs arise from the dominant effect of poor conductivity leading to increased momentum transfer from the SAW to the graphene charge carriers, rather than the increased mobility arising from increased p-doping seen in the other arrays.Although we cannot directly measure the mobility in these devices, the magnitude of the currents observed in Device 3 Fig.  are simi16, and better understand the role of GNR edges on the mobility and conductivity of these systems. The charge carrier conduction mechanism in graphene arises from several competing mechanisms, and the role of the LiNbO3 substrate in this process is not yet fully understood. We also intend to investigate the effects of changing the bridge periodicity on the acoustoelectric current, tailoring it to coincide with other SAW wavelengths, for example.Further work is now underway to examine the width dependence of the acoustoelectric effect in graphene nanoribbons that are electrostatically gated3 SAW delay lines, with widths up to 600 nm, is consistent with the description of this interaction by a relatively simple classical relaxation model. In 300 μm × 3 mm arrays composed of 500 nm-wide GNRs, an acoustoelectric current of up to ~5.5 μA was measured at a SAW frequency of 442 MHz. Enhanced acoustoelectric currents were observed when perpendicular bridge structures were inserted into the nanoribbon array with a period similar to the SAW wavelength. The increasing acoustoelectric current with decreasing nanoribbon width is thought to be caused by increased doping along the damaged, rough edges introduced by the fabrication process. We believe these measurements provide a stepping stone towards the eventual use of the acoustoelectric effect in graphene nanoribbons for such applications as sensing and metrology.In conclusion, we have demonstrated that the acoustoelectric effect in monolayer CVD graphene nanoribbons on LiNbO45, monolayer graphene produced by chemical vapour deposition on ~5 mm × ~5 mm copper foils (Graphene Supermarket) was transferred in-between the interdigital transducers (IDTs) of commercially available 128° YX LiNbO3 SAW delay lines. These had identical input and output aluminium IDTs defining an acoustic beam width of 3.25 mm and a path length of 5.4 mm. Patterning of the graphene was undertaken using electron beam lithography and an argon (10 W) reactive ion etch. GNRs were aligned such that their long axes lay parallel to the SAW wave vector. For all devices, 20 μm-wide Cr/Au (5 nm and 50 nm respectively) electrical contacts were deposited by thermal evaporation.The devices measured here were fabricated as follows: Via the poly(methyl methacrylate) (PMMA) transfer techniqueDevices supporting 500 nm-wide GNRs only  are shown in the Supplementary Information).The presence of monolayer graphene on the substrate was confirmed using a confocal Raman microscope (WITec Alpha300) equipped with a thermoelectrically cooled CCD detector. A 532 nm wavelength laser was used for the excitation, and a 50x objective lens was used for backscattered light collection with a lateral resolution of 388 nm. The laser power incident on the sample was approximately 2.3 mW, and the 2D and G peaks were located at 2696 cm−6 mbar) with continuous pumping to reduce the accumulation of dopants, at room temperature. Continuous wave SAWs were excited at the input transducer using a Hewlett-Packard 8648C RF signal generator, while Iae was measured using a Keithley K2400 source-measurement unit. Two-terminal current-voltage measurements were used to determine the GNR array conductivities. GNR arrays that were not being characterised were electrically grounded to reduce interference. The SAW amplitude at the output IDT was measured using a LeCroy WaveRunner 204Xi-A digital oscilloscope, and used to estimate SAW intensity.All measurements were made under vacuum (chamber pressure approximately 6 × 10Supplementary materials


We aimed to analyze the frequency and distribution of PD-L1 expression in specimens from prostate cancer (PC) patients using two different anti-PD-L1 antibodies .PD-L1 immunohistochemistry was performed in a tissue microarray consisting of 82 castration-resistant prostate cancer (CRPC) specimens, 70 benign prostate hyperplasia (BPH) specimens, 96 localized PC cases, and 3 PC cell lines, using two different antibodies . Staining images for CD4, CD8, PD-L1, and PanCK of a single PD-L1 positive case were compared, using a newly developed dot-wise correlation method for digital images to objectively test for co-expression.Depending on the antibody used, in tumor cells (TC) only five (E1L3N: 6%) and three (SP263: 3.7%) samples were positive. In infiltrating immune cells (IC) 12 (SP263: 14.6%) and 8 (E1L3N: 9.9%) specimens showed PD-L1 expression. Two PC cell lines  also displayed membranous immunoreactivity. All localized PCs or BPH samples tested were negative. Dot-wise digital correlation of expression patterns revealed a moderate positive correlation between PD-L1 and PanCK expression, whereas both PanCK and PD-L1 showed a weak negative Pearson correlation coefficient between CD4 and CD8.PD-L1 was not expressed in localized PC or BPH, and was only found in a minority of CRPC tumors and infiltrating immune cells. Protein expression maps and systematic dot-wise comparison could be a useful objective way to describe the relationship between immuno- and tumor-related proteins in the future, without the need to develop multiplex staining methods. The therapeutic options for castration-resistant prostate cancer (CRPC) have increased dramatically in the past years. Recent clinical trials have led to the FDA approval of cabazitaxel , abiratein vitro, and mediates clinical anti-tumor activity [Programmed Death Receptor 1 , a member of the extended family of T cell regulators, is expressed on the surface of activated T cells, B cells, and macrophages . Its ligactivity –20. A coactivity –21. PD-Lactivity . In the activity . Recentlactivity . Tumor tactivity .The first aim of this study was to systematically describe the expression of PD-L1 in benign prostatic hyperplasia (BPH), localized prostate cancer (PC), and CRPC using two anti-PD-L1 antibodies. The second aim was to observe if PD-L1 status was dependent on previous treatment modalities. The third aim was to develop a new method to describe the relationship between immune cells and tumor-related proteins using pseudo-colored protein expression maps, and to use dot-wise correlation coefficients of superimposed images as an objective co-location measurement.versus SP263. The comparison of PD-L1 expression (%) with clone SP263 versus E1L3N showed significant correlation coefficients  and 3 PC cell lines. There was no expression of PD-L1 observed in either BPH or localized PC samples (0%). Examples of PD-L1 tumor cell (TC) and immune cell (IC) staining in CRCP specimens and cell lines (clone SP263 and E1L3N) are given in Figure s Figure . No signs Figure , phosphos Figure , phosphos Figure , and Ki-s Figure .n = 10), brain (n = 1), lung (n = 1), or lymph node (n = 1) metastases  was present in 5 of 81 (6%) cases. All five positive specimens were retrieved by palliative transurethral resection of the prostate (TURP), and no PD-L1 expression was found in bone . All patients were treated in different hospitals with the available options: surgical ADT (orchiectomy), chemical ADT ), chemotherapy (docetaxel) or radiotherapy. Inclusion criteria for this retrospective analysis required tissue collection during transurethral resection of the prostate (TURP), or tumor metastasis resection, and rising PSA or clinical progress under androgen deprivation therapy. The first TMA included 78 formalin-fixed paraffin embedded tissue specimens of patients, which were sampled after the diagnosis of CPRC. Suitable areas for prostate tissue retrieval were marked on routine H&E-stained sections. A section of the paraffin block, 0.6 mm in diameter, was punched out of the suitable area and inserted into the recipient block using a tissue arrayer . The whole tissue microarray preparation was accomplished on the paraffin blocks using cores from two different areas of each tumor or control tissue to account for tissue heterogeneity.n = 70), localized PC (n = 96), the androgen-resistant human PC cell lines (PC3 and DU145) and the androgen-sensitive PC cell line (LnCaP). The detailed characteristics of this TMA have been reported previously [The second TMA consisted of BPH samples , deparaffinized, rehydrated and stained with hematoxylin–eosin using standard histological techniques. For immunohistochemical staining of both TMA and large sections, the Ventana Benchmark automated staining system and Ventana reagents  were used. After deparaffinization in xylene, slides were rehydrated in decreasing concentrations of ethanol. Endogenous peroxidase was blocked using the Ventana endogenous peroxidase blocking kit after a rinse with distilled water. For antigen retrieval, slides were heated with cell conditioning solution  according to the manufacturer's instructions.For the detection of the PD-L1 protein in BPH, PC, and cell line specimens, one anti-human PD-L1 rabbit monoclonal antibody was used: E1L3N . For the detection of the PD-L1 protein in CRPC, two anti-human PD-L1 rabbit monoclonal antibodies were used: E1L3N , SP263 . A multi-tumor TMA and a tonsillectomy specimen were used as positive and negative controls to establish the staining protocol for the PD-L1 antibodies. A dilution of 1:1000 resulted in a strong membranous signal without non-specific background staining in positive controls (squamous epithelium of tonsillectomy specimen). PD-L1 negative lung cancer cases were used as negative controls. For the expression maps, primary antibodies against CD4 , CD8 , and PanCK  were applied and adjusted to the Ventana Benchmark system after performing titrations. Immunohistochemistry with antibodies against Phospho-ERK1/2, Phospho-mTOR, Phospho-4E-BP1, and Ki-67 (MIB1) was performed, as previously described .An experienced pathologist (PJW) evaluated all scanned and digitized tissue microarray spots . A second independent observer (CDF) re-evaluated the TMAs. In discrepant cases, the two observers achieved consensus after detailed case discussion. Percentages of PD-L1 positive tumor cells and the staining pattern were evaluated for each punch. PD-L1 expression was recorded if a distinct membranous staining signal on the tumor cell surface was observed. In analogy to the scoring guidelines for non-small cell lung cancer , we usedThe staining images for CD4, CD8, PanCK, and PD-L1 were aligned, superimposed, and normalized for the generation of a pseudo-colored expression map. Whole slide images of one case, and of all performed stainings were digitized  at 40x magnification (approximately 2k-3k x 2k-3k pixels per image). The images of consecutive tissue slices were pre-processed and aligned for dot-wise correspondence in four steps. First, the original images were white-balanced to remove grayish background. Second, the tissue regions were cropped and rotated, translated and rescaled for rigid alignment of the consecutive slices to the middle slice. The background was then removed to exclude background artifacts that could disturb the following finer alignment process. Third, to refine the alignment and to address for morphological changes of the tissues in consecutive slices, we incorporated a non-linear SIFT flow algorithm . For SIFIn summary, the scarce PD-L1 expression in CRPC and absence in localized PC indicates that advanced PC patients may have to be selected carefully when offered checkpoint inhibition. However, prospective clinical trial data is needed. Our results do not confirm that PD-L1 expression can be induced by orchiectomy, LHRH-agonists, anti-androgens, radiation therapy or chemotherapy. Neither did we find an association with PD-L1 expression and ERK- or mTOR signaling activity. Protein expression maps and dot-wise comparison could be a useful objective method to describe the topographical relationship between immunological and tumor-related proteins, in general.