Since its discovery, graphene has held great promise as a two-dimensional (2D) metal with massless carriers and, thus, extremely high-mobility that is due to the character of the band structure that results in the so-called Dirac cone for the ideal, perfectly ordered crystal structure. This promise has led to only limited electronic device applications due to the lack of an energy gap which prevents the formation of conventional device geometries. Thus, several schemes for inducing a semiconductor band gap in graphene have been explored. These methods do result in samples whose resistivity increases with decreasing temperature, similar to the temperature dependence of a semiconductor. However, this temperature dependence can also be caused by highly diffusive transport that, in highly disordered materials, is caused by Anderson-Mott localization and which is not desirable for conventional device applications. In this letter, we demonstrate that in the diffusive case, the conventional description of the insulating state is inadequate and demonstrate a method for determining whether such transport behavior is due to a conventional semiconductor band gap. The drive for the development of electronic devices using graphene has led to theoretical and experimental studies of methods for inducing a semiconductor gap in graphene. These schemes include exposure to reactive gas backgrounds and/or plasmas to alter surface chemistry12et al. have demonstrated that insulating samples result through exposure to ion damageet al. observed a large increase in room temperature resistance accompanied by a breakdown of the quasi-particle description as determined from photoemission in graphene exposed to atomic hydrogen6Previous work has shown that the transport properties of graphene are sensitive to disorder. It is known that weak-localization (WL) and enhanced electron-electron interactions (EEI) control the metallic transport properties in disordered conductors. For metallic graphene with moderately high mobility, there have been several studies reporting WL and/or EEI7891011121314In this report, we present transport data for graphene samples that were chemically modified via surface functionalization by exposure to low energy plasmas to induce a metal-insulator transition. These results show that increasing resistivity with decreasing temperature is not an adequate metric for determining whether a conventional band gap, and thus, band semiconductor behavior, is present in graphene. We also show that the temperature dependence of the mobility along with the presence of variable range hopping can be used to determine whether activated resistivity is caused by the presence of disorder rather than the existence of a band gap.The key to distinguishing between the two types of activated behavior is to contrast the scattering processes that influence transport in the two regimes. For high mobility conductors and semiconductors (which are narrow gap band insulators), the conductivity can be described by the familiar Drude expression:n is the carrier concentration, e is the electron charge, τ is the relaxation time for the dominant scattering mechanism, m is the effective electron mass, and μ is the mobility. Use of this expression is predicated on the assumption that the material is well ordered with a mean free path that is much longer than the inverse of the magnitude of the Fermi wave number (kFl ≫ 1). For applicable conductors, the high temperature conductivity is controlled by phonon scattering with the relaxation time, and hence the conductivity, increasing (i.e. decreasing resistivity) with decreasing temperature. At low temperature, temperature dependent scattering mechanisms become negligible  and static impurity scattering dominates, leaving the conductivity temperature independent with a μ that increases with decreasing temperature before saturating to a constant value. For a semiconductor, the Fermi energy falls within the band gap, resulting in an n and σ that exponentially decrease with decreasing temperature. Thus, for this situation, R ~ exp(1/T) and μ again increases with decreasing temperature before saturating at a constant valuewhere τel is the elastic scattering time, and with the density of states at the Fermi energy, N(EF). In this formulation the mobility is −1/4) in 3D and R ~ exp(T−1/3) in 2D. Therefore, it is necessary to examine the temperature dependences of the resistivity and mobility to determine whether a material is a band or disordered insulator.with the diffusivity, 2 or N2 to introduce oxygen- or nitrogen-functional groups24252 doses were administered to the nitrogen-functionalized samples while two O2 doses plus a final vacuum anneal were performed on the O2 functionalized sample.Epitaxial graphene samples for this study were grown via Si sublimation from nominally on-axis SiC (0001) substrates as described elsewhere2 nature of the graphene was recoverable after functionalization and vacuum annealing and that the integrity of its interfacial buffer layer remained intact. The Raman spectra confirmed that the characteristic 2D peak of graphene was present after each plasma dose and, as expected, a disorder-induced D peak emerged after the first dose that increased in intensity with increasing dosage. Furthermore, a peak in the XPS spectra associated with the interface layer was observed before functionalization and again after vacuum annealing.Raman spectroscopy and XPS were performed both before and after functionalization (see Supplementary information of ref. 23 for details) in order to ensure that the spHall, measurements in the conventional manner .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Our previous results show that M1, M2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M2 receptor is largely independent of both M1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination.The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M During the development of the nervous system, synapses are eliminated on a broad scale , adenosine receptors  and tropomyosin-related tyrosine kinase B (TrkB) receptors are involved in the control of synapse elimination in the mouse NMJ  can add their independent effect on axonal loss to the effect of the M1 muscarinic receptor, which leads to greater elimination because of the additive effect of the pathways  subtypes 16 Jrs/J mice were used . The mice express spectral variants of GFP (yellow-YFP) at high levels in motor and sensory neurons, and axons are brightly fluorescent all the way to the terminals.Levator auris longus (LAL) muscle. Neonatal pups of either sex (9 days) were obtained and the date of birth was designated postnatal day 0 (P0). We minimized the variability in our measurements by carefully monitoring the timing of conception. Also, the weights of the individuals were within 5% of the mean for a given day after conception. The mice were cared for in accordance with the guidelines of the European Community’s Council Directive of 24 November 1986 (86/609/EEC) for the humane treatment of laboratory animals. All experiments on animals have been reviewed and approved by the Animal Research Committee of the Universitat Rovira i Virgili (Reference number: 0233).Experiments were performed on the The newborn mice were anesthetized with 2% tribromoethanol . Mice pups received daily subcutaneous injections in the back of the neck beginning on postnatal day 5 of one or two substances . Under aseptic conditions, solutions were administered in 50 μl of sterile physiological saline by subcutaneous injection over the LAL external surface as described elsewhere  for 1 h at room temperature. Whole muscles were mounted in Mowiol with p-phenylenediamide (Sigma).NMJs were analyzed using an inverted Nikon TE-2000 fluorescent microscope  connected to a standard personal computer that was running image analysis software . The number of axons per endplate was counted. Because of the difficulty of determining the exact number of axonal inputs for each nAChR cluster, when more than two axons converged at the same synaptic site we classified the NMJs into three groups only: junctions that were monoinnervated, doubly innervated, or innervated by three or more terminal axons. These data enabled us to calculate the “average number of axonal inputs” and the “percentage of polyneuronal innervation” for all fibers receiving two or more axons.P < 0.05. The categories were scored and the counting was performed by a person with no knowledge of the age or treatment of the animals. The data are presented as mean ± SD.All NMJs visible in their entirety were scored, with a minimum of 100 per muscle. At least six muscles were studied for each age and condition examined. Fisher’s test was applied to compare percentages. The criterion for statistical significance was The stock solutions were pirenzepine (PIR) dihydrochloride  10 mM; methoctramine  1 mM; muscarinic toxin 3  50 μM. The working solutions used were PIR (10 μM), MET (1 μM) and MT3 (100 nM).The following stock solutions were used: recombinant human trkB/Fc Chimera , 100 μg/ml. Working solutions were trkB-Fc 5 μg/ml.1 blocker) and MET (M2 blocker), and the TrkB pathway blocker (a TrkB-Fc chimera) were applied once a day between P5-P8 on the LAL muscle surface, there was a notable delay in the transition to monoinnervation on the NMJ observed at P9 . The associations of the M4 blocker MT3  with the other substances has been represented separately in Figure 4 subtype is shown to have a complementary role.Our experiments were designed to investigate the effect on axonal loss of simultaneous incubation with two inhibitors (two antagonists of two different receptors) and reveal the possible occlusive or additive crosstalk effects between the corresponding pathways. Figures 1 and TrkB pathway inhibitors (PIR plus TrkB-Fc) results in a clear addition of their respective delaying effects on axonal loss. The percentage of the monoinnervated NMJ after simultaneous exposure to both inhibitors is significantly less (25% of single junctions) than after exposure to only PIR (39%) or only TrkB-Fc (43%). Interestingly, however, the individual effect of the TrkB-Fc does not add to the effect of MET and the result of this dual drug incubation is no different from the effect of MET by itself. The delaying effect of MET on axon loss is the most potent observed in the present experiments and is produced independently of the state of TrkB.The association of the mAChR blockers PIR and MET shows no additive effect or mutual occlusion in relation to axonal loss Figure . However4 blocker MT3 by itself has no effect on axonal loss at P9. Figure 4 on the other receptors. The presence of MT3 does not change the effect of PIR although some occlusion of the potent effect of MET is observed. MET still continues to significantly delay axon loss (the three-to-one transition), however. Interestingly, the presence of MT3 potentiates the delaying effect of TrkB-Fc on axonal loss, which indicates that the respective receptor pathways (M4 and TrkB) are cooperating.Figure 4 receptor by itself does not affect axonal elimination at P9 (black arrow). Interestingly, the association of the M1 and TrkB pathway blockers results in the addition of their respective delaying effects on axonal loss, which indicates that the corresponding receptors are cooperating (blue bond between these receptor pathways). However, the potent effect of the M2 cannot be modified with the simultaneous presence of the M1 or TrkB blockers. It seems that the M4 receptor, which does not by itself affect axonal elimination, cooperates positively with M2 (dotted green arrow). Also, M4 produces some occlusion of the TrkB pathway (red arrow) but does not cooperate with M1 (dotted black arrow).These data are represented in Figure 1, M2 and M4 mAChR subtypes and the TrkB signaling to modulate the conditions of the developmental axonal competition and loss. In a previous study we found that these receptors  separately contribute to accelerate synapse elimination around P9 in the mouse NMJ  and the TrkB-mediated signal (which may be originated by a postsynaptic-derived NT) have the same effect: namely, the acceleration of supernumerary nerve ending elimination. It seems that the outstanding regulatory resources in the NMJ synaptogenesis are committed to achieving monoinnervation. These presynaptic receptors converge in a common intracellular mechanism and a limited repertoire of effector kinases to phosphorylate protein targets and bring about structural and functional changes leading to axon loss. It is well known that in most cells M1 and TrkB operate by stimulating the phospholipase C gamma (PLC gamma) and therefore the protein kinase C (PKC) pathway along with the inositol triphosphate (IP3) pathway, whereas M2–M4 inhibit the adenyl cyclase (AC) and protein kinase A (PKA) pathway  in some nerve endings may have a leading role in synapse elimination. In this context, blockade of PKC in the newborn LAL muscle produces an initial blockade of synapse elimination and a subsequent delay  in the weakest axons during the competitive interactions. The neurotransmitter release capacity is an important factor in the competing capacity of the various nerve terminals in a NMJ. During development, in the polyinnervated NMJ several nerve endings with different levels of maturation and ACh release capacity get together and compete. The coupling to neurotransmitter release of the considered receptors and kinases is not the same in each of these various endings themselves and in the mature synapses in the adult  and PKC activity reduces ACh release in the weakest endings and promotes axonal loss. In fact, blocking mAChRs (M1- and/or M4-subtypes) or PKC or voltage-dependent calcium channels  can lead to similar percentage increases in the size of the synaptic potentials evoked by weak axons  on developing synapses  and a grant from Ministerio de Economía y Competitividad .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


By using CuO/graphene-oxide/CuO sandwich-like nanosheets as the building blocks, bulk nacre-inspired copper matrix nano-laminated composite reinforced by molecular-level dispersed and ordered reduced graphene oxide (rGO) with content as high as ∼45 vol% was fabricated via a combined process of assembly, reduction and consolidation. Thanks to nanoconfinement effect, reinforcing effect, as well as architecture effect, the nanocomposite shows increased specific strength and at least one order of magnitude greater recoverable deformation ability as compared with monolithic Cu matrix. This process technique realized simultaneous uniform dispersion and ordering of high content graphene in metal matrix. Its microstructure and mechanical properties were characterized and discussed, and the as-obtained bulk nano-laminated composite exhibited recoverable deformation strain at least one order of magnitude greater than that of pure copper as well as increased specific strength because of high content graphene.The challenges in fabricating graphene-metal bulk nano-laminated composite mainly lie in the following factors: homogenous dispersion and efficient ordering of graphene in metal matrix; consolidation of graphene-metal composite. These challenges will be much tougher when the graphene content is high. First, due to its large surface area and high surface energy that easily lead to agglomerations and restacking, as well as notable density difference between graphene and metals, it is extremely difficult to disperse graphene into metals homogeneously. Second, agglomeration and restacking of graphene also prevents consolidation of composite and results in a porous, fragile hybrid. Thirdly, no efficient method exists for odering graphene in metal matrix. Graphene could be incorporated into metal matrix in an ordered form by a modified powder metallurgy techonologyIn this work, we present a strategy to fabricate high content graphene-copper bulk nano-laminated composite by bottom-up assembly and reduction of CuO/GO/CuO sandwich-like nanosheets, by which the main obstacles mentioned above could be overcome to some extent. The whole fabrication process in this work was schematically shown in via sintering surface Cu layer of the Cu/rGO/Cu sandwich-like nanosheets, by which composites with a high relative density could be achieved. In contrast, in traditional powder-metallurgy processes, strong interfacial bonding between graphene and metal matrix is by no means easy because they are merely blended. Moreover, sintering could not proceed easily because the metal powders are separated from each other by graphene with large surface area.Pristine graphene is lack of enough active sites for anchoring and bonding metal ions and oxides, but GO is heavily oxygenated and is readily dispersed in water. GO can be synthesized from natural graphite flakes by a modified Hummers method. The GO played a role of template for fabricating CuO/GO/CuO composite powders. In order to assist the dispersion of GO in aqueous media and direct the deposition of CuO on the surface of GO, surfactant sodium dodecyl sulfate (SDS) was chosen to electrostatically adsorb and self-assemble onto the surface of GO. Cu cations were bound to the surfactant assembled onto the GO, forming CuO/GO/CuO sandwich-like nanosheets in alkaline solution with the decomposition of the added urea at elevated temperature. CuO was deposited on the both sides of the GO  and prevID/IG band ratio of 0.98 (The morphology of dried CuO/GO/CuO sandwich-like nanosheets was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) . As show of 0.98 . Wide-anvia vacuum filtration was confirmed by small-angle X-ray diffraction (SA-XRD)  , which ianalysis . The thianalysis , which canalysis  demonstranalysis , in whicanalysis .d-spacing of the nano-laminated structure was decreased to 1.8 nm because of reduction, and the fraction of rGO in Cu increased up to ∼13.5 wt. % correspondingly. Raman spectrum detected both D and G bands from rGO structure in the reduced film , but the bulk composite is near fully densified in view of high content rGO in the composite. The high density meets our anticipation as the consolidation that realized through the Cu surface layer of Cu/rGO/Cu sandwich-like nanosheets was similar with that of pure metal.Because separated by CuO and Cu on surface, GO in the assembled CuO/GO nano-laminated film could be reduced without restacking, and rGO-Cu nano-laminated film was obtained finally. The WA-XRD  and X-raced film . The ID/Nano-laminated structure was finally obtained in the bulk Cu/rGO composites. SEM image  demonstrThe mechanical properties along the directions both parallel (V-) and perpendicular (H-) to lamellae were investigated using compressive testing. Typical stress–strain curves of the composites are shown in ca 6%)The large recoverable deformation strain of the bulk Cu/rGO composite investigated in this work could be attributed to a combined effect: nanoconfinement effect induced by high content rGO and thus high interface density, reinforcing effect of rGO, as well as nano-laminated architecture. While bulk metals have high strength but limited ability to deform elastically, nanoscale metals could exhibit high elastic strain and even rubber-like behavior4547In summary, inspired by nature, we successfully fabricated a high content rGO (∼45 vol%) reinforced Cu bulk nano-laminated composite. The molecular level dispersing and ordering high content rGO were simultaneously realized by assembling and reducing CuO/GO/CuO sandwich-like nanosheets. This process is also expected to be applied to GO-ceramic nanocomposites by omitting the reduction step. Therefore, the unique strategy will greatly expand the application range of graphene or its derivates as a promising reinforcement in both metal and ceramic matrixes with a wide range of content. Thanks to the high content rGO, high interfacial density, as well as nano-laminated structure, the as-obtained nano-laminated bulk composite shows increased specific strength and at least one order of magnitude greater recoverable deformation ability as compared with the pure Cu. Combined with its high mechainical strength, such composites would be attractive as shock absorbers and energy storage devices.3)2•3H2O solution was added dropwise under vigorous stirring followed by the addition of 400 mL of 0.25 M urea solution and 300 mL of deionized water. The resulting mixture was further stirred in a sealed glass flask at 90 °C for 16 h, and CuO/GO/CuO nanosheet solution was obtained after the product was cooled to room temperature.In a typical procedure, 200 mg graphene oxide  was dispersed into 200 mL deionized water via ultrasonic under 500 W for 2 h, forming a GO aqueous dispersion with a concentration of 1 mg/mL, and then mixed with 75 mL of sodium dodecyl sulfate solution (0.5 M). The mixture was sonicated for 15 min. Then 100 mL of 0.2 M Cu and Raman spectroscopy . Phase and elemental analyses were performed by XRD  and XPS . The microstructures of the film and composite were observed by SEM (FEI SIRION 200) and TEM (JEOL JEM-2100F). For compressive testing, the obtained samples were cut using a wire electrical discharging machine along the direction both parallel and perpendicular to the layered direction and were polished to cylinder shape with a height of 3.5 mm and a diameter of 2 mm. The compressive testing was conducted using a servo-hydraulic materials testing system (Zwick/Roell Z020) at room temperature at a strain rate of 1 × 10How to cite this article: Xiong, D.-B. et al. High content reduced graphene oxide reinforced copper with a bioinspired nano-laminated structure and large recoverable deformation ability. Sci. Rep.6, 33801; doi: 10.1038/srep33801 (2016).


Various morphologies of the vertically-aligned graphene flakes were fabricated on the nanoporous templates treated with metal ions in solutions, as well as coated with a thin gold layer and activated in the low-temperature Ar plasma. The thickness and level of structural defects in the graphene flakes could be effectively controlled by a proper selection of the pre-treatment method. We have also demonstrated that various combinations of the flake thickness and defect levels can be obtained, and the morphology and density of the graphene pattern can be effectively controlled. The result obtained could be of interest for various applications requiring fabrication of large graphene networks with controllable properties. CarbOne of the techniques commonly used for the production of vertically-aligned carbon nanowalls is a simple and cheap chemical vapour deposition (CVD) method , which s+, Pd+, Au+). Also, a continuous gold film was applied onto one of the substrates to control the network morphology. The results of our experiments demonstrate a strong dependence of the carbon nanowalls network morphology on the pre-treatment technique.Here we report on a novel inductively-coupled plasma (ICP) based approach to grow the networks of vertically-aligned few-walled graphene flakes  on nanoporous alumina templates. To effectively control the morphology, thickness and level of the structural defects of graphene flake networks, various methods of pre-treatment were used including noble-gas plasma and solution-based treatments with various ions (Co2.2O3) templates using a simple and convenient anodization technique [μm). The anodization of the Al foil (thickness of about 300 μm) was conducted under direct current conditions, with the dc voltages of 40–60 V and electrolyte temperature of about 0 °C to achieve highly-ordered arrays [2, 0.1 M CoSO4, and 0.1 M PdCl2 solutions, respectively. Scanning electron microscopy (SEM) images of the samples pre-treated using Pd ions and Ar plasma are shown in figures −1. The gold-coated sample exhibited a continuous gold layer before the final plasma-based growth, and this layer fragmented into islands of 10–15 nm in size during the graphene growth in the CH4/Ar/H2 plasma described below. The samples treated in KAu(CN)2, CoSO4, and PdCl2 solutions did not show any observable changes.The process starts from fabrication of alumina , RF frequency 13.56 MHz, treatment time—6 min. Probe measurements show the following plasma parameters in the discharge: plasma density of about 1012 cm−3, electron energy of 2–3 eV [After that, all six samples were installed in the ICP chamber, and the network of vertically-aligned graphene flakes was grown under the following conditions : CHf 2–3 eV . The plaAfter deposition, samples were characterized using SEM, transmission electron microscopy (TEM) and Raman technique. To better characterize the morphology and structure of the graphene flake arrays, we have reconstructed the three-dimensional models using the half-tone SEM images  operated at electron beam energy of 1–5 keV with an InLens secondary electron detector. Figure μm2. To enhance the accuracy of measurements, Raman spectra from multiple spots of the samples were collected and averaged. Raman spectra for all the samples are also shown on figure −1 in the Raman spectrum of graphene is the G band, i.e., the in-plane vibrational mode that involves sp2 hybridized carbon atoms which comprise the graphene sheet. Other major bands (D at approximately 1328 cm−1 and 2D at approximately 2687 cm−1) were also found in the Raman spectra. Importantly, ratio of 2D and D bands indicates the number of layers of graphene. The ratio is larger for thinner flakes containing fewer graphene layers and smaller for thick flakes [4 (Co+ ions) for 24 h at a room temperature, the next are the plasma-treated sample and the sample treatedby the 0.1 M PdCl2 for 24 h at a room temperature. However, the thicker sample was grown on the substrate treated by 0.01 M KAu(CN)2, i.e., Au+ ion treatmentpromotes formation of many graphene layers. The reference sample, as well as samples coated with a 30 nm Au film, has demonstrated a largegraphene layer number.Micro-Raman analysis was performed using a Renishaw inVia spectrometer with laser excitations of 514 and 633 nm at a spot size of ∼1 k flakes . In our The summary of the Raman bands for all the samples studied is provided in table 4 solution has demonstrated the much lower levels of defects than other samples. Use of 0.01 M KAu(CN)2 solution to treat the template results in more defect. Coating the sample with Au film resulted in more defects in the graphene structure.Another very important factor to quantify the quality of graphene is the peak intensity ratio of D and G bands. In this case, the larger ratio indicates the presence of defects in the fabricated graphene. In our experiments the sample treated by CoSOThe summary of the sample treatments and characteristics is provided in table To additionally characterize the graphene structure, the grown flakes were observed with a JEOL 2100 TEM. An electron beam energy of 200 keV was used for the analysis. TEM analysis has shown that the flakes consist of several (up to 10–20) single graphene layers, see figure thickest sample (Au+ treated) demonstrates the low defect level, whereas the thinnest sample (Co+ treated) demonstrates the lowest level of the defects. On the other hand, Pd+ treated sample is thin and simultaneously, it has high level of the crystalline structure defects; and vice verse, the sample coated with 30 nm gold layer is thick and demonstrates the highest level of the structural defects  and edge states . Apparen013 cm−2 , 26. Thi+, Au+, Pd+) during the pre-treatment. We have conducted several experiments attempting to grow vertically-aligned graphene flakes  on flat alumina substrates . We have found that the treatment methods described in this work (Ar plasma pre-treatment and solution treatment) were not effective. Specifically, nucleation and growth of the vertical nanowalls on flat alumina, silica and silicon surfaces were observed only on (1) significantly structured (texturized) surfaces like nanograss array [The edges of the nanoporous template are also an important growth factor which is responsible for the graphene nucleation and, on the other hand, determine the vertical graphene flake pattern morphology via attachment of various ions (Coss array  or (2) oOne of the major findings described in our work is the possibility to control thickness and defect level of the carbon nanowalls by pre-treatment using different metals and methods. Let us consider some important features. Firstly, thick nanowalls with the 2D/G ratio of 0.56 were formed on the reference sample. Ar Plasma treatment resulted in significantly thinner nanowalls (2D/G = 0.75), and this could be attributed to sharpening the hole edges (see figure +) and thick (Au coating) nanowalls, which apparently could be attributed to very low carbon solubility in gold. On the other hand, the use of Co for pre-treatment resulted in the thinnest nanowalls. This is a quite expected result, since Co is one of the most popular catalysts used in the growth of carbon-based nanomaterials [Pre-treatment using Au ions and a 30 nm thick Au coating resulted in the thickest  ensuresThe defect level of the carbon nanowalls is a function of (i) nucleation conditions, and (ii) growth conditions. Considering the details we could point out that the lowest defect layer is a feature of the Co-treated sample. As we have mentioned and referenced above, Co is an efficient catalyst for carbon-based nanostructures , so the low defect level of the nanowalls grown with Co should be considered as a quite expected result. On the other hand, the highest defect level was found in the nanowalls grown on the Au-coated 30 nm) membrane. In this case the nanowalls were nucleated on relatively thick gold nanoislands which feature low carbon solubility. Besides, the lateral field caused by the non-uniform charging of the catalyst islands could play a significant role in the nucleation of nanowalls  nm membr, 32.The substrates during the plasma-enabled growth were insulated from the chamber, and thus they were under the floating potential . Under these conditions, the ion energy is usually too low to cause any significant damage to the graphene structure.4.In summary, we have reported on the fabrication of vertically-aligned graphene flakes of various morphologies on the nanoporous templates treated with various ions in solutions, as well as coated with a thin gold layer and activated in low-temperature Ar plasmas. We have demonstrated that various combinations of the flake thickness and defect levels can be obtained, and the morphology of the graphene pattern can be effectively controlled. The work could be of interest for various applications requiring fabrication of large graphene networks with controllable properties.


Antibody-based PD-1/PD-L1 blockade therapies have taken center stage in immunotherapies for cancer, with multiple clinical successes. PD-1 signaling plays pivotal roles in tumor-driven T-cell dysfunction. In contrast to prior approaches to generate or boost tumor-specific T-cell responses, antibody-based PD-1/PD-L1 blockade targets tumor-induced T-cell defects and restores pre-existing T-cell function to modulate antitumor immunity. In this review, the fundamental knowledge on the expression regulations and inhibitory functions of PD-1 and the present understanding of antibody-based PD-1/PD-L1 blockade therapies are briefly summarized. We then focus on the recent breakthrough work concerning the structural basis of the PD-1/PD-Ls interaction and how therapeutic antibodies, pembrolizumab targeting PD-1 and avelumab targeting PD-L1, compete with the binding of PD-1/PD-L1 to interrupt the PD-1/PD-L1 interaction. We believe that this structural information will benefit the design and improvement of therapeutic antibodies targeting PD-1 signaling. Pdcd1−/− mice that are genetically predisposed to systematic autoimmunity  families, etc.  to mediate respiratory tolerance  has approved two PD-1-targeted MAbs, nivolumab from Bristol-Myers Squibb  and pembrolizumab from Merck , for advanced melanoma, non-small cell lung cancer (NSCLC), and kidney cancer. In 2016, the US FDA gave accelerated approval to atezolizumab from Genentech  for the treatment of patients with locally advanced or metastatic urothelial carcinoma. Further, various MAbs targeting the PD-1 pathway are being developed and evaluated in numorous clinical trials involving thousands of patients  displays promising effects, with an ORR of 43% in PD-L1However, cases of ineffective PD-1 treatment have also emerged in the observation of clinical trials , PD-1 lacks a Cys in the stalk region, which prevents PD-1 homodimerization  domain and is structurally and functionally a monomer , and the cross-species binding affinities show no significant differences compared to the intra-species interactions  is 64%, while the identity between murine and human PD-L1 (mPD-L1 and hPD-L1) is 77% Fig. . Cross-sThree PD-1/PD-L1/L2 complex structures have so far been determined: mPD-1/hPD-L1, mPD-1/mPD-L2, and hPD-1/hPD-L1  competitive antagonist of PD-L1  and light chain (VL) of pembrolizumab are involved in contacting the C’D loop of hPD-1  complexed with hPD-L1 have been determined by Na et al.  and our et al. . The intet al. A left  loops, while VL contributes partial contacts by the CDR1 and CDR3 loops, leaving VL CDR2 without any binding to hPD-L1  of pembrolizumab to hPD-1 and avelumab to hPD-L1 are 27.0 pmol/L and 42.1 pmol/L, respectively . Whether these antibodies utilize the same blockade mode as pembrolizumab or avelumab remains undetermined. Moreover, whether there are hot-spots on PD-1 or PD-L1 to be targeted by different therapeutic antibodies requires further investigation. All of these findings would benefit the development of therapeutic agents targeting the PD-1 pathway to disrupt the PD-1/PD-L1 interaction.There are yet more therapeutic antibodies targeting PD-1/PD-L1/L2 in clinical use or clinical trials  interaction details are still needed to define the antibody targeting hot-spots and to better design PD-1/PD-L1/L2 antagonists for tumor treatment. Such efforts will pave a way to improve the efficacy of antibody targeting the PD-1 pathway and prolong survival in advanced cancer patients.


Tabby is a pattern of short irregular stripes, usually related to domestic cats. We have produced Tabby patterns on graphene by attaching fluorine atoms running as monoatomic chains in crystallographic directions. Separated by non-fluorinated sp2 carbon ribbons, sp3-hybridized carbon atoms bonded to zigzag fluorine chains produce sp2-sp3 interfaces and spin-polarized edge states localized on both sides of the chains. We have compared two kinds of fluorinated graphite samples C2Fx, with x near to 1 and x substantially below 1. The magnetic susceptibility of C2Fx (x < 1) shows a broad maximum and a thermally activated spin gap behaviour that can be understood in a two-leg spin ladder model with ferromagnetic legs and antiferromagnetic rungs; the spin gap constitutes about 450 K. Besides, stable room-temperature ferromagnetism is observed in C2Fx (x < 1) samples: the crossover to a three-dimensional magnetic behaviour is due to the onset of interlayer interactions. Similarly prepared C2Fx (x ≈ 1) samples demonstrate features of two-dimensional magnetism without signs of high-temperature magnetic ordering, but with transition to a superparamagnetic state below 40 K instead. The magnetism of the Tabby graphene is stable until 520 K, which is the temperature of the structural reconstruction of fluorinated graphite. Thus, low dimension magnets have the advantage of bulk materials in providing sufficient intensity for experiments investigating the thermodynamic and spectroscopic characterization of magnetism. Most studies of low dimensional magnetism concentrate on molecular magnets based on organic radicals (see e.g.4) or Cu and Ni compounds which have spins ½ or 1, correspondingly5. We have recently synthesized a novel graphene derivative decorated by monoatomic fluorine chains running in crystallographic directions, and have observed clear signs of one-dimensional-like magnetism in this two-dimensional material6. Nanoscale magnetic activity of pure graphene is controlled by the edge geometry. In the present study, graphene fluorination, instead of breaking of the carbon-carbon bonds, is used as an efficient approach to generate edges and, therefore, correlated magnetic states in this material. The fluorine chain running in zigzag direction induces strong spin polarization with a mixed ferro-antiferro-magnetic coupling between locally emerged magnetic moments.Magnetism in restricted dimensions can be studied in real bulk crystals if the exchange interactions are much stronger in one or two spatial directions than in the remaining onesTabby stripes. Tabby is a pattern of a cat’s coat with tiger stripes and leopard spots. Ideally, if the sp2-sp3 interfaces were parallel and evenly spaced lines, they would form embedded graphene nanoribbons. The stoichiometry C4F yields a 3-carbon atom wide nanoribbon, C3F is a 2-atom wide one, and C2F produces monoatomic chains  in Fig. 0 + χCurie + χspin into a temperature-independent term χ0, a small Curie-like part χCurie = C/T identified by the low-T upturn, and a spin contribution χspin. The term χspin(T), that is the nontrivial part of the magnetic response, shows a non-monotonous temperature dependence with a broad maximum. This feature cannot be understood in the frames of independent magnetic moments. As shown in Fig. spin is the hallmark of low dimensional magnetism and is typical for both chain- and ladder-structured materials14. The position of the maximum in the plot of χspin (T) establishes an energy scale related to the strength of antiferromagnetic (AF) spin-spin interaction which favours spin pairing, and therefore, opposes the spin alignment parallel to an external magnetic field15.The linear magnetic susceptibility χ, the Hatfield model17 of a 1D spin chain with modulated coupling, a dimerised chain (Bleaney-Bowers18), and a spin ladder with antiferromagnetic interactions (Troyer-Tsunetsugu-Würtz19). However, none of these models which assume various modifications of only antiferromagnetic couplings, could give satisfactory agreement with our experiment. Indeed, the picture of the spin interactions that was predicted by the discoverers of the peculiar edge states of zigzag graphene nanoribbons in the seminal work by Wakabayashi et al.20, is characterized by ferromagnetic exchange coupling JFM ~ 103 K within a zigzag edge and an antiferromagnetic edge-edge coupling of the order JAFM ~ 10–100 K, depending on the nanoribbon width21. It is therefore reasonable to interpret the experimental results in the frames of the spin ladder model with ferromagnetic legs (JFM) and antiferromagnetic rungs (JAFM). We calculated the magnetic susceptibility of this model for the number of spins in a leg, Ns, changing from 1 to 126, and found that this model could be used successfully to fit a large batch of samples. As an example, the solid line in Fig. s = 3 and Ns = 8.To extract the strength of the interaction from the data and to estimate the amount of interacting spins, one may think in terms of localized spins with the Heisenberg Hamiltonian, H = ΣT = 200–280 K, the exchange interaction strength (JAFM) value is in the range JAFM = 300–450 K22; (ii) the concentration of the exchange-coupled spins is rather large, being in the range 1–20% per CnF structural unit; (iii) the residual concentration of isolated spins ½ does not exceed 0.1%. The JAFM value confirms quantitatively that the AFM interactions within the graphene planes are much stronger than those in the organic radical crystals , making the Tabby graphene an ideal candidate for 2D behaviour studies of pure organic materials.On the basis of the analysis of the susceptibility curves obtained on the different samples, we have arrived at the following quantitative conclusion: (i) with the maximum at Tabby graphene is the development of macroscopic magnetic order during ageing. Figure J = S = ½ (free isolated electron spin). The saturation value in the magnetization curve shown in Fig. A remarkable property of the Tabby graphene has pure organic origin and is determined by the fluorine arrangement on the basal plane.As the samples were purely organic, their magnetism was prone to thermal effects. We observed a sharp drop of paramagnetic response Fig.  and satuH) loops and plotting the saturated magnetization at each temperature point  were prepared by using similar technological procedures as in the case of C2Fx, x < 1 (see the supplementary information for more details). All samples were Curie-like paramagnetic at room temperatures without any sign of ferromagnetism. However, on cooling, a strong increase in the magnetic moment was observed, pointing out at a ferromagnetic-like transition near 40 K , and at H = 1000 Oe the difference between the ZFC and FC magnetizations was undetectable  isothermal dependencies  asymmetric broad lines with g ~ 2. The total spin densities were found to be 1018–1020 spin/g with only 1–2% contribution coming from the narrow signals. We focused the attention on the broad lines. For all samples, doubly integrated intensities for the broad lines which corresponded to EPR susceptibility followed the trends for SQUID susceptibility  for this paramagnetic entity was determined by the origin of the entities (g-factor) and the internal magnetic fields (like hyperfine one etc.). Thus, the developing temperature shift of Hrbroad manifested progressive strengthening of the internal magnetic field. It is worth mentioning here that the resonant field of the narrow EPR line, attributed to fluorinated carbon, remained the same within the entire temperature region of 4–300 K. This may have reflected both the inhomogeneity of the samples and the local nature of magnetic ordering in the corresponding entities.The EPR technique, which allows selective tracking of different magnetic entities, diagnoses the magnetic units responsible for the described phenomena. For all the studied samples, the room temperature EPR spectra revealed two main constituents: (a) narrow lines within the region of see Fig. , while t2Fx(x ≈ 1). There we observed clear transformation of the same broad EPR signal that was observed at room temperatures, into a multicomponent ferromagnetic resonance (FMR) signal , the sp2-sp3 interfaces created by the fluorine chain play the same role as zigzag edges. The graphene bipartite lattice consists of inequivalent A and B sublattices. In the case of a zigzag chain, one expects a set of localized spin states in sublattice A on one side of the nanochain and sublattice B on the other side. Density functional theory (DFT) calculations for well-separated fluorine chains clearly reveal the emergence of magnetism in fluorinated graphene6. The calculated complex magnetic configuration combines the strong ferromagnetic interaction between the local magnetic moments of C atoms within each side of the CF chain with the antiferromagnetic coupling between the magnetic moments of C atoms located on the opposite sides of the CF chain.Fluorination is well known to be an effective method for introducing localized spin centres into graphene. When the attached fluorine atoms are arranged as monoatomic chains running in crystallographic directions , or “digging” the wide-gap derivatives, removing extra atoms and forming one-dimensional graphene regions: point defects, quantum dots, nanoribbons, superlattices.Several graphene derivatives are known, e.g. graphene oxide, hydrogenated graphene, fluorographene, and chlorographene. Functionalization opens the band gap of graphene, which is desirable for on/off electronics. However, at full graphene coverage, the possibilities for manipulation of electronic properties are limited because the π electrons are already spent to the attached atoms. Extensive studies are done on releasing the π electrons by producing single-side CTabby graphene, the method of slow fluorination provides conditions where adsorbates tend to align in a chain sequence in which F adatoms are located on alternating sides of the graphene plane. Fluorine has the highest electronegativity of all elements. If the synthesis is made slowly at low temperature, quasi-equilibrium conditions allow fluorine to attach to the thermodynamically preferable places. Fluorinated carbon tends to separate spatially from non-fluorinated carbon. If one F is attached to the graphene, the next F will most preferably attach close to it, to another side in the ortho-position. The adsorption of an odd number of F atoms disturbs the π-electron system, so the adjacent atom attaches as close as possible, but to another side due to strong repulsion. The meta-position is unfavourable because it creates unpaired radicals.In the synthesis of the 2Fx (x < 1) samples after prolonged ageing . During the gas-phase synthesis, simultaneous F adsorption at multiple sites resulted in seeding of the chains in a random distribution across the graphene planes. Apparently, the chains were not long enough to produce the zigzag-inherited edge states. The honeycomb lattice of graphene is a bipartite lattice. Sublattices A and B are identical in a single layer. In a bilayer, atom attachment on B sites is energetically more preferable than on A sites. The chain grown in a certain direction of a graphite layer may dictate the rule for the pattern formation in the adjacent layer. The C−F bonds in graphene are dynamic, with a low energy barrier for the migration of F atom to the nearest C atom. The fluorine chains repeal and arrange in two neighbouring layers like the teeth of a comb. This creates an additional discriminating mechanism leading to the formation of regular Tabby patterns where crossing and branching are suppressed, which is presumably favourable for magnetic coupling.We observed 2D spin gap -activated magnetism of CTabby graphene are substantially spaced, as in C2Fx (x ≈ 1), the compound can be considered as a stack of monolayers. In this case the discriminating mechanism for chain formation is absent. Although fluorination does produce localized spins, the chain length is not enough for the development of zigzag edge states with measurable magnetic interactions on the 2D plane, or the development of high-temperature long-range magnetic ordering. High-temperature long-range magnetic ordering was absent in the samples of our study. Instead, we observed transition to 3D superparamagnetic behaviour at low temperatures. A low temperature ferromagnetic transition was seen in the multicomponent FMR signal, with the features typical for partially oriented ferromagnetic subsystems having different magnetic anisotropies. Ageing of the samples led to 50-fold reduction in the 3D magnetism, whereas transition to 2D behaviour was detected from the broad maximum and low-temperature activated behaviour of magnetic susceptibility.When the layers in the Tabby graphene. The sp2–sp3 interfaces produced by the fluorine atoms attached in the zigzag directions gave rise to numerous magnetic phenomena. Graphene is a two-dimensional material, whereas the fluorine chain patterns reduce the effective dimensionality to 1D. Magnetic susceptibility shows a behaviour typical for low-dimensional quantum spin-ladder systems, which is characterized by spin ordering along the zigzag edges and their antiparallel alignment between opposite zigzag edges. The existence of a gap in the spin excitation spectra of graphene ribbons with zigzag edges has been theorized since their discovery in 1998. We produced magnetically active fluorinated graphite samples and showed that this type of low-dimensional magnetism can be realized experimentally in a graphene-based system. We observed magnetic dimensional crossover with changes in the fluorine loading and interlayer distance, the parameters that can be controlled by synthesis conditions, sample ageing, and annealing. The Tabby graphene is a promising material with tuneable electronic and magnetic properties, and it provides a playground for the exploration of new quantum many-body states as well.To sum up, fluorination of graphene in such a way that the attached fluorine atoms formed monoatomic stripes running in crystallographic directions produced a special type of graphene derivative, which we call the 2Fx with the fluorine content x, 0.5 ≤ x ≤ 1, were produced by room temperature synthesis27. The starting material for the synthesis of the fluorinated graphite samples was natural graphite from the Zaval’evo deposit (Ukraine). To remove metal and silicate impurities, the material was purified by double acid treatment (HNO3:HCl 1:3 and concentrated HF). The grain size of the samples was about 100 × 100 × 20 μm, and the content of 3d metal impurities was below 1 ppm. Detailed description of sample preparation can be found in Supplementary Information.Samples of fluorinated graphite CT < 300 K were carried out by using a Bruker EMX-200 X-band (ν~9 GHz) EPR spectrometer equipped with Oxford Instrument ESR900 cryostat and Agilent 53150 A frequency counter at microwave power (PMW) levels ranging from 50 µW to 200 mW. The structure and composition of the fluorinated graphite samples were studied by means of X-ray diffraction (XRD) on a DRON-SEIFERT-RM4 diffractometer by using CuKα radiation and X-ray photoelectron spectroscopy (XPS) on a Phoibos 150 SPECS spectrometer using monochromatized AlKα radiation with the energy of 1486.7 eV. The results of structure and composition analysis are given in Supplementary Information.DC magnetic measurements were performed at a Quantum Design SQUID magnetometer (MPMS-XL-1) in -1 T–+1 T magnetic fields. The DC magnetic susceptibility data were collected in the 1.76–400 K range in a 10 mT magnetic field. Electron Paramagnetic Resonance (EPR) measurements within the temperature range 4 K < All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


Triple negative breast cancer patients have a poor course of disease not least because of limited treatment options however immunotherapy by targeting the PD-1/PD-L1 checkpoint system is a promising strategy to improve the outcome. Here we systematically investigated the expression of PD-1 on tumor infiltrating lymphocytes and PD-L1 on both tumor and infiltrated immune cells. Moreover, the PD-L1 gene status in tumor cells was assessed.in-situ hybridization was applied to the PD-L1 gene and centromere region of chromosome 9. The disease free and overall survival rates were determined as a function of the PD-1/PD-L1 status.103 tissue microarray samples derived from triple negative breast cancer specimens were immunohistochemically stained against PD-1 and PD-L1. Dual marker fluorescence A slight gain of the PD-L1 gene region was found in 55% of all samples but an elevated PD-L1/cen9 ratio was rather rare (7%). An increased gene dose is not associated with an enhanced protein expression and the PD-L1 expression only weakly correlates with the amount of immune cell infiltration. Instead, we found an association of PD-L1 expression on tumor and immune cells, respectively. Notably, the PD-1 expression on immune cells is associated with a favorable disease free and overall survival. PD-1 expression indicates an enhanced immunological anti-tumor activity and represents a favorable prognostic impact. A deeper understanding of factors that affect the regulation and function of the PD-1/PD-L1 system is required to establish predictive variables and to utilize the system for therapeutic intervention of triple negative breast cancer patients. Triple negative breast cancers (TNBC) account for 10–17% of all breast cancers (BC), tend to grow more aggressively than other subtypes, show relatively early recurrence and intrinsically have poor prognosis [Immunotherapy research is trying to overcome the cancer’s ability to resist the immunological tumor defense and to stimulate or to reactivate mechanisms that result in regaining immunological effectiveness against cancer. To this end different strategies are being developed, amongst them a specific targeting of molecules that are involved in curbing immune cells. Programmed cell death protein 1 (PD-1) expressed on (activated) T-cells and the corresponding programmed death ligand 1 (PD-L1)expressed on immune and tumor cells represent a prominent inhibitory immune checkpoint system that has been demonstrated to play a major role for example in malignant melanoma , 3 and shttps://clinicaltrials.gov/ct2/show/NCT02685059). Interestingly, even patients with PD-L1-negative scored tumors seem to benefit from an anti-PD-L1 treatment. Even though it is known that PD-L1 expression can be triggered as response to a T-cell attack, the underlying molecular / cellular mechanisms contributing to the treatment response require elucidation. It appears plausible, however, that an efficient inhibition of an immunological tumor defense by PD-L1-positive tumor cells requires interaction with PD-1-positive lymphocytes. Thus, a systematic assessment of both parts of the PD-1/PD-L1 system on tumor cells and TILs will shed light on the tumor tissue related immune status and might reveal a valuable prognostic or predictive impact.Due to its higher genetic instability, an enhanced mutational load, and the appearance of neoantigens PD-L1 expression is more frequently found in HER2-positive and triple negative BCs than in other BC sub-entities  . Moreovein-situ-hybridization (FISH). We associated and correlated these parameters to each other and retrospectively analyzed the overall and progression free survival  of TNBC patients as a function of PD-1 and PD-L1. Overall, we found a favorable outcome of TNBC patients with PD-1 positive TILs compared to those patients who had tumors with lymphocytes expressing low levels or no PD-1.Here we scored the amount of TILs in 103 TNBC samples and immunohistochemically evaluated the expression of PD-L1 on tumor cells and lymphocytes. In addition we analyzed the PD-1 expression on TILs and quantified the PD-L1 gene copy number in tumor cell nuclei by fluorescence-r = 0.652; p < 0.001; Figure p = 0.87) or PFS (p = 0.62) could be revealed  of all specimens. However, in 37/55 of all positive samples the frequency of positive cells was below 10%. Notably, no correlation was found between the PD-L1 gene copy number and PD-L1 expression  or the PFS  of the patients. Complementary, the PD-L1 expression on TILs does also not correlate with OS (p = 0.31) or PFS (p = 0.14) as shown in Figure p = 0.17) or PFS (p = 0.13). Only nine samples with low infiltration (Score 1 = 1–9 TIL/HPF) but 43 tissues scored 2 (10–49 TIL/HPF) and 50 scored 3 (>50 TIL/HPF) were identified. Overall, 93/102 (91%) of all samples showed high (Score 2) or a very high (Score 3) immune cell infiltration. Therefore, only a trend towards a better outcome of disease for Score 3 cases (compared to Score 1/2 cases) could be revealed  correlation between PD-L1 expression on tumor cells and on TILs , n = 99) and between PD-1 and PD-L1 expression on TILs ; n = 101). On the other hand, and even more importantly, the PD-1 expression on TILs has a favorable impact on the OS  and especially on the PFS . The PD-1 expression turned out as the strongest prognostic marker that determines the outcome of disease.Despite the fact that the TIL score could not be correlated to PD-L1 expression nor to the patient’s OS and PFS the presence of PD-1 positive TILs favorably affects the outcome of disease: On the on hand we found a direct correlation of PD-1 expression on TILs and PD-L1 expression on tumor cells , which does not result in an increased PD-L1/cen9 ratio, indicates the presence of (low grade) polysomy 9. Moreover, the missing correlation between PD-L1 gene copy numbers and PD-L1 expression on tumor cells suggests that the PD-L1 gene dose does not determine the degree of protein expression. In other words an enhanced PD-L1 protein expression was found independently from the PD-L1 gene copy number and the PD-L1/cen9 ratio. Unlike as, for example, the expression of the HER2 receptor protein, which (in BC) is strongly determined by the her2 gene copy number/ gene amplification, the PD-L1 expression seems not to be chromosomally determined. This is consistent with other studies in which only a weak correlation of PD-L1 transcripts and no correlation of PD-L1 protein content with PD-L1 gene copy number aberrations were reported [We assessed the PD-L1 gene and cen9 copy numbers in tumor cells of 103 TNBC tissues by dual marker FISH and interrelated the cytogenetic data to the respective PD-L1 expression. We found 59/103 (57%) events with a rather moderate increase of PD-L1 gene copy numbers. Not more than 7/103 (7%) specimens showed an elevated PD-L1 gene copy number only  which results in a (slightly) enhanced PD-L1/cen9 ratio. In contrast, 96/103 samples (93%), including those with moderately enhanced PD-L1 gene copy numbers, come without an increase of the PD-L1/cen9 ratio which suggests the absence of PD-L1 gene amplification in these cases. Since the PD-L1 gene region is located very much distal on the short arm of chromosome 9  and far away from the cen9 region a common amplification of both regions can be excluded in samples that show both moderately enhanced PD-L1 and cen9 copy numbers. Instead, the correlation of simultaneously elevated PD-L1 gene and cen9 copy numbers  positive TILs in 15% of TNBCs and an association of PD-1 expression with the presence of CD8+ cytotoxic T-cells [The presence of TILs in particular in HER2-positive and TNBC samples has been repeatedly associated with a favorable prognosis , 33 and, T-cells , whereas T-cells –41. Consregs). It has been reported that PD-1-pos. Tregs represent impaired activity [regs can cause an increased activity of effector T-cells and consequently stimulate the systemic immune response. A tumor cell associated PD-L1 and Treg associated PD-1 interaction, which would impair a Treg mediated inhibition of e. g., cytotoxic T-cells, could explain the favorable impact of PD-1 which entails an improved outcome of disease [We could not reveal a correlation of an increasing amount of TILs (expressed by the TIL score ranging from 0 to 3) with the PFS or OS. This is probably due to the fact that most of the tumor samples came with a rather enhanced immune cell infiltration  samples had an infiltration score of 2 or 3). Tissue specimens without the presence of TILs (Score 0) were not observed at all. In this study we did not differentiate lymphocyte subsets. However, PD-1 expression can basically be found on T-, NK-, and B-cells, but also on monocytes and even regulatory T-cells  since immune cell infiltration can be heterogeneous and vary amongst different tissue areas. Consequently, the size of specimens that undergoes the investigation potentially plays a role for proper evaluation of immune cell infiltration and the estimation of PD-1 and PD-L1 expression . One migOverall, the prognostic value of both PD-L1 and PD-1 expression in TNBC (and probably other taxonomic BC entities) remains uncertain and requires further investigation. Notwithstanding, data supporting either a favorable or an adverse effect of the PD-1/PD-L1 system on the course of disease should not necessarily be contradictory since different effects of PD-1/PD-L1 might be elicited by the environment and the type of immune cell that express this receptor and its ligand. Hence, it is rather unlikely that the assessment of PD-L1 only (expressed on tumor cells) will decisively facilitate a patient stratification in respect of eligibility for a checkpoint treatment. Instead, the data heterogeneity amongst a great number of studies suggests that multifactorial analyses are required to understand the impact of PD-1/PD-L1 positive cells with tumor tissues on immunological defense, tumor growth and progression and finally the course and outcome of disease. Further studies that comprise not only the overall-evaluation of TILs and the degree of PD-L1 expression on tumor and immune cells but also include the differential analysis of PD-1 expression on immune cell subpopulations  will specify and thus considerably enhance the diagnostic and prognostic significance of immune cell analyses . Moreove103 TNBC tissue samples were derived upon surgery and were recruited between the years 2004 to 2015. 90.5% of those samples, which were derived from cytotoxically treated patients, were taken at the non-pretreated stage . The PDCD1LG2 specific probes were labeled with SpectrumGreen and the cen9 specific probe with SpectrumOrange. PDCD1LG2 hybridization spots reflect the PD-L1 gene copy number whereas the cen 9 spots are considered to reflect the number of chromosome 9 within a cell nucleus.Fluorescence Sealed slides were imaged with an AxioImager Z1 fluorescence microscope  equipped with specific filter sets for DAPI fluorescence , SpectrumGreen , SpectrumOrange . Hybridization signals in 25 non-overlapping cell nuclei per specimen were quantified by two independent observers, and count values were averaged. If necessary, brightfield microscopy was used to verify the presence of either malign or benign breast tissue in the visual field. Analyzes were performed using AxioImager-Z1 (Zeiss) and the hybridization signals of 50 non-overlapped nuclei were manually counted on single cell basis. Results are presented as PD-L1 gene signals per one cell and calculated as FISH ratio .All specimens were acquired from the tissue archive of the Institute of Pathology, University of Regensburg (Germany). The embedding procedure was performed as described elsewhere . ImmediaTissue microarrays (TMAs) were generated as described previously  and were1.5 µm paraffin sections were prepared from the embedded tissue blocks. Specimens were deparaffinized and pretreated by microwave heating for 30 min at 320 W in 0.1 M citrate buffer adjusted to pH 7.3. The immunostaining was automatically performed on a Ventana Nexes autostainer  by using the streptavidin– biotin peroxidase complex method and 3,3′-diaminobenzidine (DAB) as chromogen. The autostainer was programmed based on the instructions given by the iView DAB detection kit (Ventana). The mouse monoclonal anti-PD-1 antibody NAT105 (ab52578) and the rabbit monoclonal anti-PD-L1 28–8 (ab205921) were used . The specimens were microscopically analyzed using a Zeiss Axiovert 200 instrument (Zeiss). The degree of immmune cell infiltration and the frequency of positive immune/tumor cells were scored by percentage or by number of cells/high power fields (HPF) and translated into a score system ranging that covers the scores 0, 1, 2, and 3 Tables .P-values were two-sided. P values less than 0.05 were considered significant. All statistical analyses were performed using R version 3.3.3  or GraphPad Prism .Survival curves were estimated using the Kaplan–Meier method, and the log-rank test was used to test for differences between the groups. To calculate the association between parameters, Pearson or Spearman correlation coefficient analyses were applied. All reported


Alzheimer’s disease (AD) is characterized by progressive decline of memory and cognitive functions. Deep magnetic stimulation (DMS), a noninvasive and nonpharmacological brain stimulation, has been reported to alleviate stress-related cognitive impairment in neuropsychiatric disorders. Our previous study also discovered the preventive effect of DMS on cognitive decline in an AD mouse model. However, the underlying mechanism must be explored further. In this study, we investigated the effect of DMS on spatial learning and memory functions, neurogenesis in the dentate gyrus (DG), as well as expression and activity of the cholinergic system in a transgenic mouse model of AD (5XFAD). Administration of DMS effectively improved performance in spatial learning and memory of 5XFAD mice. Furthermore, neurogenesis in the hippocampal DG of DMS-treated 5XFAD mice was clearly enhanced. In addition, DMS significantly raised the level of acetylcholine and prevented the increase in acetylcholinesterase activity as well as the decrease in acetyltransferase activity in the hippocampus of 5XFAD mice. These findings indicate that DMS may be a promising noninvasive tool for treatment and prevention of AD cognitive impairment by promoting neurogenesis and enhancing cholinergic system function. Alzheimer’s disease is the most prevalent form of dementia among the elderly and is an emerging global epidemic event . AlthougNeurogenesis, an important mechanism for brain development, is closely associated with hippocampus-mediated learning and memory in the adult DG. These neurons also receive abundant cholinergic innervation, and cholinergic neurotransmitters released from cholinergic neurons are known to play a key role in memory-related circuits in the brain. One such neurotransmitter, Ach, is synthesized from choline and acetyl coenzyme A by ChAT and plays a critical role in learning and memory . The durSince reduced Ach levels were found in the basal forebrain and hippocampus of AD patients and thought to be associated with dementia , inhibitDeep brain magnetic stimulation, a novel noninvasive approach of brain stimulation, was developed to generate pulses of low-intensity magnetic field (only 1/500 TESLA) by passing a local variable electric current through an inductive coil, which thus could reach the hippocampus directly theoretically due to whole-head penetration  under transcriptional control of the neuron-specific mouse Thy1 promoter. The 5XFAD mice were originally obtained from Jackson Laboratory (number 006554) and maintained by crossing heterozygous transgenic mice with C57BL/6J wild-type breeders. Genotyping was performed using PCR analysis of tail DNA. Female transgenic mice at 4 months of age were utilized, and wild-type littermates served as controls. Mice were housed in cages (4 per cage) in a standard environment  with free access to food and distilled water. All animal care and use procedures were in strict accordance with the national regulations concerning animal protection and were approved by the animal ethics committee of China Capital Medical University. In sum, all efforts were made to minimize animal suffering and use.APP/PS1 (5XFAD) double transgenic mice coexpress the human APP and PS1 transgenes, which contain 5 familial AD mutations , WT with DMS , 5XFAD , and 5XFAD with DMS . The specific parameter settings were based on a previous study  dissolved in physiological saline to make a 10 mg/mL solution was intraperitoneally injected daily for 5 day to label newborn cells in the DG. Behavioral assessments were carried out 24 h after the last DMS treatment. A timeline of experimental procedures is shown in Cognitive function was assessed using the MWM test as previously described . BrieflyAfter completion of the behavioral tests, mice were fully anesthetized with 6% chloral hydrate  and perfused transcardially with cold saline solution. Brains were rapidly removed and bisected into each hemisphere through the midsagittal plane. The right hemisphere was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 48 h and then transferred sequentially to 20% and 30% sucrose solutions in PBS. The brains were sectioned along the coronal plane on a freezing microtome at a thickness of 30 μm in each region (bregma -1.8 mm to -3.24 mm) and stored at -20°C in cryoprotectant solution for staining, including BrdU, BrdU/NeuN, and doublecortin (DCX). The left hemisphere was isolated, and the hippocampus was dissected and stored at -80°C to quantify cholinergic levels, including Ach, ChAT, and AchE.BrdU, which is incorporated into DNA during the S-phase, is commonly used as a marker for mitotic cells. Injection of BrdU can be used to evaluate survival time and track the fate of dividing cells and their progeny . Immunod+ cells were analyzed as described previously (+ cells colabeled with NeuN (BrdU+/NeuN+ cells). The number of BrdU+ cells or BrdU+/NeuN+ cells in every sixth section (180 mm apart) was obtained from eight sections, multiplied by 6, and then multiplied by 2 to account for both hemispheres.We used the stereological techniques to count the same one side, the number and phenotype of BrdUeviously . Briefly2O2) was carried out for 30 min. Quantification of DCX+ cells in sections of each group was carried out using Image Pro Plus 6.0 software.DCX, a microtubule-stabilizing factor expressed early in neuronal differentiation, is a reliable marker of newly migrating neuroblasts in the adult DG . BrieflyCholinergic markers were measured as described previously study . Brieflypost hoc test to compare differences between groups, and p < 0.05 was considered statistically significant. Other data (when comparing three or more groups) were analyzed by one-way ANOVA followed by Bonferroni post hoc test using Prism 5.0 (GraphPad Software).All values are shown as mean ± standard error of mean (SEM). All data were performed by an investigator who was blinded to the experimental conditions. The following tests were used as required: Data recorded in the training trail (escape latency and swimming speed) were analyzed by two-way analysis of variance (ANOVA) with repeated measures using SPSS 13.0 software , followed by LSD p = 0.538, Figure 2A). Subsequently, in the acquisition phase, mice from all groups showed a progressive decline in escape latency during training , and this decline was more significant as the training days progressed in the DMS treatment group. Mice in the Tg group exhibited prolonged escape latencies during all sessions compared with the control mice, while mice in the Tg + DMS group significantly decreased in escape latency compared with the Tg group from the fourth day (p < 0.05). However, the average swimming speed did not differ significantly  among the groups in this trial, which indicated that DMS treatment substantially improved learning deficits. In addition, to evaluate spatial memory retention, a probe trial was performed Figure 2D. Similarly, mice in the Tg + DMS group crossed the former platform location more often and spent more time in the target quadrant than those in the Tg group , suggesting that DMS effectively reversed spatial memory impairment in AD mice.To determine whether DMS alleviates Aβ-induced cognitive impairment, we tested spatial learning and memory ability of 5XFAD mice in the MWM following DMS treatments. In the visible platform test, there was no group difference in swimming strategies . Compared with the WT group, mice in the Tg group showed not only a decreased number of surviving BrdU+ cells  but also a decreased number of new neurons  in the DG . However, marked increases in the number of BrdU+ cells  and BrdU+/NeuN+ cells  were observed in the Tg + DMS group compared with the Tg group, suggesting that DMS could promote differentiation of newborn cells into mature neurons.The thymidine analog BrdU was injected to label dividing neural progenitor cells. After 8 weeks of DMS treatment, BrdU+ cells in the DG was markedly reduced in the Tg group compared with the WT group , while DMS treatment dramatically increased the number of DCX+ cells compared with that of the Tg group , indicating that DMS treatment effectively promoted neurogenesis in the hippocampus of AD mice.In addition, the number of DCXFigures 4A,C, compared with the WT group, the content of Ach  and ChAT  significantly decreased to about 24.07 and 33.57%, respectively, and the activity of AchE markedly increased in the Tg group . However, compared with the Tg group, mice in the Tg + DMS group showed partial restoration of Ach levels  and ChAT activity , as well as inhibition of AchE activity , demonstrating that DMS treatment effectively regulated cholinergic system balance in AD mice.To determine whether DMS could regulate the activity of cholinergic system, we tested some cholinergic associated factors in the hippocampus of 5XFAD mice following DMS treatments. As shown in In the current study, we verified therapeutic effects of DMS, a novel and promising therapeutic approach, on cognitive impairment in an AD mouse model and investigated underlying mechanisms. Consistent with our previous observations, DMS effectively rescued learning and memory in 5XFAD mice. Furthermore, DMS clearly promoted neurogenesis and restored cholinergic system balance in the hippocampus of 5XFAD mice, both of which are critical to hippocampal learning and memory.Several researchers have reported that 5XFAD double transgenic mice showed significant cognitive decline at 4–5 months of age . Once in+/NeuN+ cells as well as the number of DCX+ cells in the DG of 5XFAD mice, suggesting that deficits in proliferation and differentiation of neural progenitor cells in AD transgenic mice could be rescued by DMS. Thus, DMS may improve cognitive function at least in part by promoting neurogenesis in the hippocampus.Overwhelming evidence shows that the adult brain of humans and other mammals continuously generates new neurons throughout life , which iHow does DMS treatment promote hippocampal neurogenesis in 5XFAD mice? According to previous studies, the cholinergic system plays a pivotal role in promoting survival of neuronal progenitors in adult neurogenesis. Moreover, Aβ oligomers are considered a primary cause of impairment of the cholinergic system in the brains of AD patients, which show multiple aspects of cholinergic system dysfunction such as decreases in Ach levels, low activity of choline synthetase, and a reduction in the number of cholinergic receptors . In addiFurthermore, several researches reported the prospects of modulating Wnt signaling as a strategy for neuroprotection . This wiThe limitations to our study are that we have not performed dynamic observations of DMS effects on hippocampal neurogenesis and cholinergic systems in AD mice, and we have not explored critical signals regulating neurogenesis .Our results suggest that DMS may effectively improve learning and memory performance in 5XFAD mice by restoring cholinergic activity and promoting hippocampal neurogenesis. Furthermore, our findings indicate that DMS could be a new noninvasive therapeutic strategy for AD and other aging-associated cognitive impairments. In the further study, we will expore the probable molecular mechanism of DMS in regulating the neurogenesis.This study was carried out in accordance with the recommendations of National Institutes of Health guidelines for care and use of laboratory animals and with the European Communities Council Directive of 24 November 1986 (86/609/EEC).JZ and XW contributed to the design, acquisition of the data, and the draft of the manuscript. YQ, JF, HA, and WW performed the whole experiments and helped acquire and interpret of the data. RS contributed to analyze the data and discuss the results. YZ and XW provided intellectual contribution during editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Atmospheric water vapour is an essential ingredient of weather and climate. The key features of its distribution can be represented by kinematic models which treat it as a passive scalar advected by a prescribed flow and reacting through condensation. Condensation acts as a sink that maintains specific humidity below a prescribed, space-dependent saturation value. To investigate how the interplay between large-scale advection, small-scale turbulence and condensation controls moisture distribution, we develop simple kinematic models which combine a single circulating flow with a Brownian-motion representation of turbulence. We first study the drying mechanism of a water-vapour anomaly released inside a vortex at an initial time. Next, we consider a cellular flow with a moisture source at a boundary. The statistically steady state attained shows features reminiscent of the Hadley cell such as boundary layers, a region of intense precipitation and a relative humidity minimum. Explicit results provide a detailed characterization of these features in the limit of strong flow. The interaction between the subsequent transport and condensation of this evaporated water gives rise to intriguing distributions of water vapour in the atmosphere: for example, persistent relative humidity minima are observed in the subtropics ,2, and bA framework to explain the key features of the atmospheric humidity distribution is the advection–condensation model ,6. In thet al. . At t=0+, the air parcels start to move in the counterclockwise sense along the circular streamlines of  with small random fluctuations induced by the Brownian motion. The parcels that move in the −y direction are entering regions where qs(Y)>Q, thus no condensation occurs and Q remains constant. On the other hand, for parcels moving in the +y direction along a streamline of radius r, condensation starts immediately. These parcels continue to lose water vapour as condensation goes on until they reach ≈ and Q≈qs(r)—the minimum of qs on the streamline. By the timeb). The rapid initial drying is best exhibited by the decay of the global specific humidity defined asN air parcels. Assuming all parcels have the same air mass, t=0 tot≈ta. Further drying from this time on relies on the Brownian motion of the parcels and corresponds to the slow stochastic drying phase. Figure 2a shows this transition for different Ω including the case without a vortex (Ω=0); other simulation parameters are the same as in Initially, at h parcel b. The ra(b)r1 that wanders onto another streamline of radius r2>r1 is being quickly advected into the region of y≈r2 with lower saturation specific humidity. Rapid condensation within this region reduces the specific humidity of the parcel from Q=qs(r1) to Q=qs(r2). Our primary goal in this section is to calculate the resulting PDF of Q. Following previous work . Since qs decreases monotonically with y, this implies that the random variables Q and Λ are related byC of Λ for an air parcel located at x at time t. Suppose there is an absorbing barrier at y=λ. We follow a parcel backward in time according to (X(t) hits the absorbing barrier at some t>0. It then follows thatE1 conditioned upon event E2, and χS is the indicator functionu,  satisfies the backward Kolmogorov equation × with reflective boundary condition; see x and y are re-scaled to have the same typical length L as discussed near the end of §y=0: the specific humidity Q of air parcels hitting (and reflecting on) the bottom boundary is reset to qmax, the saturation value there  contribute to precipitation at time t. The amount of condensation from one such parcel between time t and t+Δt isx,y) is thusRnet(x) by integration over y to findG given by qmin/qs(πL/2); the inset in figure 5c shows that it decreases towards this minimum as b the mean relative humidity as a function of y along a fixed x=πL/2. These profiles are obtained from Monte Carlo simulations by averaging Q/qs(Y) over parcels as well as over time. Figure 7b shows that the relative humidity decreases from y=0, reaches a minimum, then increases with y, approximately as qmin/qs(y). When the circulation strength U increases with κ fixed, the minimum relative humidity decreases and its location shifts towards y=0, or the direction of increasing qs.When evaporation balances condensation, the system reaches a statistically steady state and the mean moisture distribution has a steady profile. The snapshot in ϵ→0 since the area of the source and condensation boundary layers (where Q≠qmin) tends to 0 with ϵ. The leading-order correction is controlled by the solution near the corner of the domain at =, where the moisture content is at its largest. A computation detailed in appendix D givesκ, that is, with the intensity of small-scale turbulence, and decreases as the strength of the large-scale circulation U increases. The inset in figure 7b confirms this result.It is interesting to assess the dependence of the total water-vapour content, as estimated by the global specific humidity Q¯ in , on the 5.Motivated by the importance of synoptic-scale moisture transport in the atmosphere, we have studied two idealized problems based on the advection–condensation paradigm. The key element in both cases is that the advecting velocity has a large-scale coherent component in addition to small-scale white noise. The analytically tractable models introduced here capture some of the essential processes that control the large-scale dynamics of atmospheric water vapour, enabling us to examine the three-way interaction between large-scale advection, small-scale turbulence and moisture condensation.We first study in §x,y)-plane with a moisture source at the boundary y=0. This can be interpreted as a large-scale circulating flow on an isentropic surface if we take x and y as the zonal and meridional directions, respectively. Alternatively, this set-up is a crude representation of the Hadley cell if we interpret x as latitude and y as altitude. This simple model produces some interesting features reminiscent of the atmosphere. First, a boundary layer near the source is formed as a result of the balance between large-scale and small-scale motions. This layer roughly mimics the atmospheric boundary layer whose role in moisture transport has been investigated using idealized simulations with full physics [x=0—the tropics of the model—where intense precipitation occurs. Second, we find that the moisture distributions inside both boundary layers are bimodal. The dry peak is a consequence of the subsidence of parcels with low humidity originated from the top of the cell. Satellite measurements indeed show that the PDF of relative humidity over the whole tropics is bimodal although the PDF within a subregion could be unimodal [et al. [For the steady-state problem, we consider the single overturning cell  on the , which only affects the spatial distribution of rainfall. It is known that changes in global-mean evaporation and precipitation with surface temperature are strongly constrained energetically [There is a continuous interest in how climatological and seasonal variations in the strength and width of the Hadley cell ,38 affecre input , and as re input  depend oetically . How welPrevious work using simplified one-dimensional models has established the Lagrangian formulation of the advection–condensation paradigm as a promising strategy to investigate atmospheric water vapour. The present study provides a step forward in this direction through the analysis of a stochastic Lagrangian model that incorporates the dynamics of a two-dimensional large-scale circulation. An important extension in the future is to include the effects of latent heat by making temperature a dynamical variable and the saturation profile temperature dependent. As it is often difficult to untangle the many interacting processes in full general circulation model simulations, idealized models such as the one introduced here can help to reveal the role of specific processes in controlling the distribution of water vapour in the atmosphere.


Recently, rocuronium with subsequent use of sugammadex was proposed for electroconvulsive therapy (ECT) as an alternative to succinylcholine. Because sugammadex is cleared via the kidney with no metabolism, it is unknown that rocuronium-sugammadex use is safe in hemodialysis patients who received ECT.In this case report, we used rocuronium with subsequent administration of sugammadex in a 69-year-old female, hemodialysis patient, scheduled for ten ECT sessions for severe major depression. In the initial eight sessions, we tested the feasibility of rocuronium-sugammadex use for ECT. During the series of four ECT sessions, we measured plasma concentrations for the sum of sugammadex and sugammadex-rocuronium complex and observed whether possible residual sugammadex affected muscle relaxation during subsequent sessions of ECT. The results showed the feasibility of rocuronium-sugammadex use as muscle relaxants for ECT in patients undergoing hemodialysis. However, an accumulation of sugammadex did occur even after two sessions of hemodialysis, and residual sugammadex decreased the effect of the rocuronium given in the subsequent ECT sessions. Rocuronium-sugammadex was successfully utilized as muscle relaxants for ECT in this patient.Our experience in this case may indicate that if succinylcholine is contraindicated, rocuronium-sugammadex can be an alternative method for muscle relaxation during ECT in patients undergoing hemodialysis. When this rocuronium-sugammadex procedure is used, the effect of residual sugammadex after hemodialysis on the subsequently administered rocuronium should be considered. Depression is common in patients undergoing hemodialysis . SeveralA 69-year-old female patient, 133 cm in height and weighing 25 kg, was scheduled for ten ECT sessions for severe major depression. She had a history of chronic renal failure, and hemodialysis was induced 6 months before consultation to the psychiatric department of Kure Medical Center. She had a history of bronchial asthma. Brain MRI showed brain atrophy and old fine cerebral infarctions. She was diagnosed with depression with a mixed type of cognitive impairment. Psychiatric medical therapy was started, but her depressive state worsened, and she was admitted by legal control. After admission, she had hemodialysis three times a week, basically on Monday, Wednesday, and Friday. Every hemodialysis was performed for 3 h using a high-flux membrane . In spite of medical therapy, her symptoms deteriorated. Therefore, ten sessions of ECT were planned after obtaining informed consent from her family. Because of possible succinylcholine-induced hyperkalemia, we initially applied a rocuronium-sugammadex procedure for muscle relaxation of ECT. We obtained informed consent on the following anesthesia protocols from the patient’s family and approval of our IRB (IEC201504). All sessions of ECT were performed in the operating room. Blood pressure, heart rate, and oxygen saturation were monitored using anesthesia monitor . The adductor pollicis train of four (TOF) ratio using kinemyography  was also monitored.We determined the appropriate doses of rocuronium and sugammadex required for ECT during the initial four sessions. ECT was performed once a week on Wednesday morning, where the patient had hemodialysis in the afternoon, and on Friday and the Monday before the next ECT session. Before induction of anesthesia, surface electrodes were positioned over the ulnar nerve at the wrist. The MechanoSensor™ was attached between the thumb and index finger with a piece of tape. Anesthesia was inducted by propofol with a target propofol concentration in the blood of 2 μg/mL. After automatic calibration, supramaximal TOF stimulation was measured at 20-s intervals. Rocuronium was given with an incremental dose of 5 mg to obtain the TOF ratio of 0 %, and assisted mask ventilation was initiated with 100 % oxygen, followed by a bilateral electrical stimulation of a brief-pulse wave for approximately 5 s . Immediately after seizure stopped, sugammadex was given with an incremental dose of 25 mg for antagonizing rocuronium to achieve recovery of the TOF ratio of 100 %. Hypertension induced by ECT was treated with intravenous nicardipine. We titrated the doses of rocuronium and sugammadex during these sessions. Throughout these four sessions, 20 mg rocuronium and 75 mg sugammadex were found adequate to obtain 0 % and 100 % of the TOF ratios, respectively.g, and 2 mL of plasma was cryopreserved at −20 °C. Later, plasma concentrations for the sum of sugammadex and its rocuronium complex were determined using validated liquid chromatographic assay methods with mass spectrometric detection at the Institute of Applied Medicine Inc., Hokkaido, Japan. The lower limit of quantification for the assays was 0.1 μg/mL.During the fifth to eighth sessions, ECT was given twice a week on Tuesday and Thursday as ordinary procedures . ChangesChanges in plasma concentrations for the sum of sugammadex and its complex are shown in Fig. Ten sessions of ECT were performed without complications, and her depressive state dramatically improved. There was no recurarization, muscular weakness, or respiratory problems encountered after ECT during the sessions.Although not recommended for use in patients with severe renal impairment , 10, sugThe prevention of bone fractures during ECT is especially important in hemodialysis patients because of the prevalence of osteopenia in such patients . A twitcAlthough we confirmed that the combination of rocuronium and sugammadex is a feasible alternative muscle relaxation for ECT even in patients with hemodialysis, the high costs of sugammadex may be a challenge economically . The posWe successfully used the rocuronium-sugammadex procedure over eight sessions as muscle relaxation for ECT in a hemodialysis patient. If succinylcholine is contraindicated, the rocuronium-sugammadex combination may be an alternative method for muscle relaxation during ECT in patients undergoing hemodialysis. When this alternative procedure is used, neuromuscular monitoring such as quantitative measurement of the TOF ratio should be used to titrate the dose of rocuronium while considering the effect of residual sugammadex after hemodialysis.