The interconnected CFs are used as both current collector and electrode materials for flexible supercapacitors, in which the in-situ exfoliated graphene act as active materials and conductive “binders”. The in-situ electrochemical intercalation technique ensures the low contact resistance between electrode (graphene) and current collector (carbon cloth) with enhanced conductivity. The as-prepared electrode materials show significantly improved performance for flexible supercapacitors.Flexible energy storage devices are highly demanded for various applications. Carbon cloth (CC) woven by carbon fibers (CFs) is typically used as electrode or current collector for flexible devices. The low surface area of CC and the presence of big gaps (ca. micro-size) between individual CFs lead to poor performance. Herein, we interconnect individual CFs through the  A fast-growing market for electronic devices and the development of hybrid electric vehicles result in an ever increasing demand for environmentally friendly and low-cost energy devices1234578et al.et al.11With rapidly growing demand for personal electronics with small, thin, light-weight, flexible, and even roll-up characteristics, more and more attentions have been devoted to the flexible energy storage systems including flexible supercapacitors for these electronic devices111213ca. micro-size) between individual carbon fibers are generated, as observed by the scanning electron microscopy (SEM) images . During the production of carbon cloth, inevitably, big gaps  images  which wo4 as reference electrode, and tetramethylammonium perchlorate (TMAClO4) as electrolyte. We performed the cyclic voltammetry (CV) scanning to define a proper potential for the chronoamperometric (CA) mode to start the electrochemical cation intercalation process. As shown by the CV curves in 17in-situ exfoliated graphene, as shown in The electrochemical cation intercalation of carbon cloth was conducted by using a three-electrode system with carbon cloth as working electrode, Pt mesh as counter electrode, Ag/AgClO0 axis, indicating the microporous nature of of carbon fibers in CC3/g, much higher than that of CC (0.011 cm3/g). After the electrochemical cation intercalation, Ex-CC exhibited much higher surface area (68.5 m2/g) than CC without intercalation (11.5 m2/g). The enhancement of the surface area by the in-situ interconnected graphene could be clearly found.Based on the SEM observation, graphene was successfully exfoliated and acted as interlinkers to interconnect individual carbon fibers. It is well-known that graphene always has high surface area, therefore, it is expected that the interconnected carbon fibers by graphene, that is, Ex-CC would show much higher surface area than pristine CC. We performed the Brunauer Emmett Teller (BET) testing for Ex-CC as well as CC for comparison. D band and G band were located around 1330 and 1580 cm−1, respectively. It has been found that G band arises from the bonding stretching of sp2-bonded C-C pairs, while the D band is associated with the sp3 defect sites. In the Raman spectra of carbon-based materials, the ratio of ID/IG was usually used as an indicator of the defects level. It can be seen from D/IG ratio than pristine CC. For Ex-CC, graphite in carbon fibers was exfoliated to graphene, resulting in more exposure of edge defects and thus higher D band intensity in the Raman spectrum.The electronic properties of Ex-CC and CC were investigated by Raman spectra. Raman spectroscopy is an excellent tool for investigating the electronic structure and defect concentration in graphene2021222 and sp3 C1s, respectively3 C of Ex-CC increased relative to the pristine CC, indicating more edge defects exposed after exfoliation, in consistent with the observation of the Raman spectra as discussed above. It should be pointed out that no obvious oxygen-containing species were observed for Ex-CC, indicating the electrochemical cation intercalation is a non-oxidative route, which preserved the highly conductive properties of graphene.In order to monitor the change of the C bond configuration, fine-scan C1s spectra were collected from both CC and Ex-CC, as shown in 2SO4 solution as the electrolyte. For comparison, pristine CCs were also assembled to a symmetric flexible supercapacitor. The area of the devices is 2 × 2 cm2. CV measurements were first carried out to observe the electrochemical behaviour of Ex-CC and CC-based flexible supercapacitors. in-situ exfoliated graphene would enhance the conductivity of the composites for use as electrode materials. From the CV curves, it could be observed that Ex-CC showed much higher current than CC, which could be attributed to the unique structure of the interconnected carbon fibers through the in-situ exfoliated graphene.Based on the above physical and chemical characterizations, it is expected that Ex-CC would have attractive electrochemical performance in supercapacitors. Since the Ex-CC with high surface area originates from CC and preserves the well-defined cloth structure with good mechanical strength, Ex-CC could be an excellent candidate as an advanced binder-free electrode for flexible supercapacitors. The symmetric flexible supercapacitors were constructed using two Ex-CC samples as both positive and negative electrodes, Whatman filter membrane as the separator, and 1.0 M HIR drop of the Ex-CC is much smaller than CC. The IR drop is caused by the equivalent series resistance (ESR), which includes electrode resistance and electrolyte resistance. The smaller IR drop on Ex-CC is attributed to the existence of various pore structure and high conductivity of the in-situ exfoliated graphene. The specific capacitance in this work was calculated from the CD curves. In addition, the in-situ exfoliated graphene filled the gaps between individual carbon fibers in Ex-CC samples; therefore, it is interesting to investigate the contribution of the in-situ exfoliated graphene to the area-normalized capacitance. So, in this work, the specific capacitances were described with “mF cm−2”. According to the calculation, the area-normalized capacitance of Ex-CC at the discharge current of 3 mA is 64.5 mF cm−2, much high than that of CC (17.1 mF cm−2). The higher area-normalized capacitance of Ex-CC may be attributed to the presence of the in-situ exfoliated graphene with high surface area and high conductivity. Furthermore, we investigated the durability of the Ex-CC electrode using continuous charge/discharge cycles at a constant current load of 5 mA. In order to further investigate the performance of the two electrodes, galvanostatic charge-discharge (CD) experiments were carried out with the voltage windows the same as for the above CV analysis. As shown in the CD curves , the disFinally, we examined the electrochemical performance of the as-fabricated flexible supercapacitor based on Ex-CC as the electrode materials under bending conditions. Based on the above analysis, it could be found the individual carbon fiber in the carbon cloth could be efficiently interconnected by the electrochemically exfoliated graphene. The morphology characterizations demonstrate the ideal structure for the potential applications in supercapacitor due to more exposed active sites for charge storage. On the other hand, the enhanced capacitance as demonstrated by the electrochemical testing may also be attributed to the increased conductivity of the composite. The exfoliated graphene could significantly decrease the contact resistance between individual carbon fibers by linking each together.in-situ electrochemical exfoliation method. The electrochemical exfoliation leads to generation of graphene without de-attachment from carbon fiber under mild exfoliation conditions and preserves the high conductivity of graphene without the formation of oxygen-containing species. The exfoliated graphene, which remains in the matrix of carobn fibers, acted as conductive “binders” to enhance the conductivity and to increase the surface area of the composites2627In summary, we successfully interconnected carbon fibers of CC with graphene through the 4 as reference electrode in 0.1 M TMAClO4 in NMP as electrolyte. First of all, Voltammetry was performed as a scan rate of 20 mV/s. The potentials observed in the cyclic voltammetry (CV) were used to define the potential set in the chronoamperometric mode to control the mild intercalation process. Subsequently, −2.5 V was chosen for the chronoamperometric mode to realize the mild electrochemical exfoliation for 10000 s. Following the electrochemical exfoliation, the as-obtained Ex-CC was washed with acetone thoroughly.The electrochemical exfoliation of carbon cloth was conducted in a three-electrode system with carbon cloth as working electrode, Pt mesh as counter electrode and Ag/AgClO2 were used as the positive electrode and negative electrode, respectively. Whatman membrane was used as the separator and 1 M H2SO4 in aqueous was used as electrolyte. For comparison, CC based supercapacitors were also assembled in the similar way.To construct Ex-CC based flexible supercapacitor device, two pieces of Ex-CC electrodes with the size of 2 × 2 cm2).Cyclic voltammetry and galvanostatic charge/discharge tests of assembled two-electrode supercapacitors were carried out using an Autolab potentiostat/galvanostat. CV measurements were conducted in the applied voltage window of 0–1 V. Galvanostatic charge/discharge tests were operated under a constant charge/discharge current of 3 mA within an applied voltage window range from 0 to 1 V. The specific capacitance was calculated from the galvanostatic charge/discharge curves normalized by the surface area of the devices .


Proton transfer across single-layer graphene proceeds with large computed energy barriers and is therefore thought to be unfavourable at room temperature unless nanoscale holes or dopants are introduced, or a potential bias is applied. Here we subject single-layer graphene supported on fused silica to cycles of high and low pH, and show that protons transfer reversibly from the aqueous phase through the graphene to the other side where they undergo acid–base chemistry with the silica hydroxyl groups. After ruling out diffusion through macroscopic pinholes, the protons are found to transfer through rare, naturally occurring atomic defects. Computer simulations reveal low energy barriers of 0.61–0.75 eV for aqueous proton transfer across hydroxyl-terminated atomic defects that participate in a Grotthuss-type relay, while pyrylium-like ether terminations shut down proton exchange. Unfavourable energy barriers to helium and hydrogen transfer indicate the process is selective for aqueous protons. Proton transfer across graphene is associated with large computed energy barriers and is thought to be generally unfavourable. Here, the authors observe aqueous proton transfer through graphene subjected to pH cycling, suggesting that it is due to transfer through rare, naturally occurring atomic defects. As shown in 141519a,eff values of 3.5(1) and 8.3(2), which fall within the reported literature values  images of graphene single layers deposited on fused silica windows show a low density of macroscopic pinholes and that the graphene is free of cracks or folds . Two-dims−1 refs , 38, 39.− groups . This re−5 cm2 s−1, or just half of that of bulk water, once protons arriving through any opening within the graphene sheet interact with the hydroxylated portion of the surface that is located behind the reaction front. To conservatively assess an upper bound limit for our estimations, we calculated the proton mean-square displacement using a D-value of 1 × 10−6 cm2 s−1. The probability of placing our laser beam within the propagating reaction front emanating from a given macroscopic pinhole was then estimated to be 4% and 21% for 1 and 10 s SHG jump times, respectively  was then used to search for atomic defects using annular dark field (ADF) STEM imaging at 60 kV (see Methods). The majority of the images show perfect six-fold symmetry in the position of the carbon atoms and vast areas that lack grain boundaries and atomic, or vacancy, defects . Neverth44To elucidate the mechanisms for proton transfer, we discuss findings from density functional theory (DFT) calculations  and Reax50We find that the main restriction for aqueous proton transfer through pristine, defect-free graphene is the energy required to push the proton through the center of an aromatic ring in the hydrophobic graphene layer as shown in The basal planes of pristine graphene can, and do, contain rare atomic-scale defect sites comprised of carbon atom vacancies, as shown in et al.)All of the defect terminations considered are energetically favourable as compared with the bare quad-vacancy system . The rem2 transfer requires barriers exceeding 1.9 eV  to fill ~1 cm2 with a single layer. Following annealing in a flow of 4% H2 in Ar for 30 min at 300 °C, vibrational sum-frequency generation spectra showed no evidence for CH stretchesWe used graphene having a grain size of ~100 μm ref.  grown on54SEM images were collected from the center of the graphene film. Graphene on an optical window was imaged using a Hitachi S-4800 scanning electron microscope (SEM) operating at 2 kV with a probing current of 10 μA and an Everhart–Thornley detector. Copper tape was used to reduce charging effects. Individual images were taken at × 1,200 magnification with 1,280 × 960 resolution. An array of 5 × 5 images  was stitched together using Adobe Illustrator. Automatic brightness and contrast adjustment on each frame was carried out using the ‘auto adjust’ feature in Preview . No other postedit feature or change was applied.−5 torr) for 8 h to remove surface contamination. Following heating in vacuum, the specimen was immediately transferred to the UltraSTEM for ADF STEM imaging. The surface of the graphene still retains residual PMMA that was used in the transfer processes to the transmission electron microscopy grid as shown in To confirm the single-layer nature of graphene synthesized using the CVD method2 and NaCl . The concentration of NaCl was confirmed using a conductivity meter . Solution pH was adjusted with minimum amounts of dilute solutions of ~1 M NaOH  and HCl (EMD ACS grade). The pH-jump experiments were carried out using a fused silica hemisphere  pressed against either a fused silica window  or a CVD-prepared graphene film transferred onto a silica window in an experimental setup previously reported13The aqueous solutions were prepared with Millipore water, prepared the day before an experiment and left open to air overnight to equilibrate with atmospheric CO−1 flow using variable flow peristaltic pumps as previously reported13As shown in 134 μJ cm−2 per pulse with a 30 μm focal spot, which is well below the damage threshold of graphene as previously reported1313A detailed description of our SHG setup has been described previously606162Ab Initio Simulation Package64x and y dimensions. A 15 Å gap was inserted between the graphene layer perpendicular to the surface. The gap was subsequently filled with enough water molecules to match the overall density of water at 1.0 × 103 kg m−3. The initial simulations were carried out with water on both sides of the graphene layer. The lower SiO2 substrate was initially simplified by using additional water. Subsequent calculations were carried out with more realistic slabs comprised of water/graphene/water/SiO2 substrates. The reaction rates and mechanisms of proton transfer through the graphene were described in the framework of transition state theory and within the harmonic approximation, which is robust for systems of high densities.First-principles periodic DFT calculations were carried out to determine the lowest energy interfacial water/graphene, water/graphene/water/silica structures and the activation barriers for proton diffusion through these interfaces using the Vienna −5 eV. All of the atoms were allowed to relax in the geometry optimizations until the forces on each atom were <0.03 eV Å−1. Spin polarization was examined for all of the systems explored and applied when needed. Transition states were isolated using the nudged elastic band method44All of the calculations were carried out within the generalized gradient approximation using Perdew–Burke–Ernzerhof functionalab initio MD to validate predictions of force field in describing water/graphene systems .Supplementary Figures 1-26, Supplementary Tables 1-3, Supplementary Notes 1-13 and Supplementary References


In this way, it is possible to simultaneously obtain a regular, non-covalent paving as well as supramolecular functionalization of graphene, thus opening interesting perspectives for nanoscience applications.Two-dimensional (2D), supramolecular self-assembly at surfaces is now well-mastered with several existing examples. However, one remaining challenge to enable future applications in nanoscience is to provide potential functionalities to the physisorbed adlayer. This work reviews a recently developed strategy that addresses this key issue by taking advantage of a new concept, Janus tecton materials. This is a versatile, molecular platform based on the design of three-dimensional (3D) building blocks consisting of two faces linked by a cyclophane-type pillar. One face is designed to steer 2D self-assembly onto C(sp Graphene is of significant interest for next generation electronics  particul2 hybridized carbon atoms into sp3 hybridized. As a consequence, this disruption of the C-sp2 leads to the alteration of the characteristic electronic properties of graphene. For this reason, the non-covalent functionalization of graphene is expected to be more interesting, offering the opportunity to attach any functionality while simultaneously maintaining the integrity of the sp2-hybridized carbon network  [3 grafting strongly affects the delocalization of electrons within the graphene layer, making it incompatible for applications such as sensors [This can be achieved either by covalent or non-covalent approaches . In the perties) . This as sensors . Finally sensors .However, the controlled positioning and organization of functional molecules into self-assembled monolayers at surfaces represent a major challenge for potential applications in various fields of nanotechnology –10. Amon2-hybridized carbon supports (such as HOPG and graphene) with periodic arrays of functional 3D building blocks, known as Janus tectons [2)-based substrates, and (ii) independently exposing off-plane functionalities with controlled lateral order on demand.In this context, we recently developed a successful new strategy taking place at the liquid–solid interface at room temperature (RT) for the precise nanometer-scale 2D decoration of flat sp tectons . Here, w2)-based substrates was proposed [2)-based substrates and allowed for the first realization of a predetermined. “on demand” series of 0D, 1D or 2D topologies, based on a single rigid molecular core on HOPG. These achievements are based on the rational design of a novel functional molecular group, which turns into a non-covalent clip-like bond activated by graphite   have been designed, synthesized, and self-assembled on HOPG  at the liquid–solid interface. As expected, they form non-covalent, surface self-assembled dimers, supramolecular linear polymers, and 2D networks. The versatility of the design was then demonstrated by synthesizing bifunctional molecules bearing two functional “clips” that end-cap a central moiety consisting of, for example, a benzene ring (IV) . As showThese results demonstrate that we are now able to control the supramolecular self-assembly on HOPG. First, a new tool acting as a functional moiety for surface-specific supramolecular bonding has been designed by combining and controlling molecule–substrate epitaxial adsorption and intermolecular packing interactions. Second, the “molecular clip” concept validity was demonstrated through the good match between the various expected and experimental topologies resulting from the supramolecular self-assembly at the liquid–HOPG interface of designed building blocks.2)-based substrates. In this context, we proposed for the first time a novel and highly versatile concept, the Janus-like 3D tecton concept. This building block consists of two different faces  and a spacer linking them. Face A was designed to act as a pedestal capable of steering a 2D self-assembly onto the substrate, while B is a functional entity . The spacer acts as a pillar ensuring the decoupling of the B face from the substrate. Moreover, if the Janus tecton is laying on the substrate via the A face, the formation of a well-organized, in-plane monolayer covering the surface is expected as well as the steered positioning of the B face out of the plane. This concept was validated by designing and synthesizing the 3D tecton reported in In the second stage, the design of 3D building blocks was pursued . This stx,y,z) of a chromophore above a substrate. Moreover, due to its substantial, inherent tunability, this strategy opens up a promising novel route toward functional molecular nanostructures and new perspectives towards active surfaces and interfaces on C(sp2)-based substrates.With these last results, it was demonstrated that (i) the multi-story molecules stack perpendicular to the substrate paving HOPG with long-range ordering, and (ii) the “floor” does not disturb the self-assembly in supramolecular, linear polymeric chains, even at large scales. Thus, this approach appears to be a breakthrough given the ability to control the 3-axis positioning , exposing as an upper face different functional moieties such as triazine-4,5-diamine, 2,6-bis(2-pyridyl)pyridine and ferrocene units.a = 3.84 nm, b = 2.08 nm and α = 64°) regardless of the building block. These values are compatible with those of the lattice formed by the neat ground floor [a∙b∙sinα = 7.18 nm2) with the calculated cross-sectional areas occupied by each upper unit  can partially explain these features. All of the upper units are size-compatible with the huge footprint value.The self-assembly properties of the JAPs and JAs were investigated by STM at the liquid–HOPG interface, at room temperature . First, nd floor . Then, ind floor . Both thFinally, the self-assembly of the Janus tectons onto a graphene monolayer, grown by chemical vapor deposition onto a polycrystalline foil, was investigated. As evidenced by a typical STM image  recorded2-carbon-based substrates (including graphene) has been investigated. In contrast to other studies performed by evaporating low molecular weight molecules under UHV conditions, in our approach, the self-assembly is achieved at the liquid–solid interface, additionally allowing the physisorption of higher molecular weight molecules.These results demonstrate that for the first time a general platform for the non-covalent functionalization of flat sp2)-based substrates, the Janus tecton concept offers a versatile platform towards the non-covalent functionalization of graphene. The reported strategy is expected to be applicable for the generation of self-assembly systems exhibiting on demand functionalization, expanding the application possibilities of this functionalization method. Moreover, working at the liquid–solid interface makes this strategy easy to implement and should also provide the opportunity to control the self-assembly by tuning the molecule–solvent and solvent–substrate interactions. Finally, the successful self-assembly on graphene, together with the possibility to transfer the graphene monolayer onto various substrates, should open up new opportunities in nanoscience.Using the molecular clip concept as a tool for supramolecular bonding on C(sp


LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants.DOI:http://dx.doi.org/10.7554/eLife.13926.001 Retrotransposons, also known as jumping genes, have invaded the genomes of most living organisms. These fragments of DNA have the ability to move or copy themselves from one location of a chromosome to another. Depending on where they insert themselves, retrotransposons can modify the sequence of nearby genes, which can alter or even abolish their activity. Although these genetic modifications have contributed significantly to the evolution of different species, retrotransposons can also have detrimental effects; for example, by causing new cases of genetic diseases.Adult human cells have a number of mechanisms that work to keep the activity of retrotransposons at a very low level. However, in many types of cancers retrotransposons escape these defense mechanisms and ‘jump’ actively. This is thought to contribute to the development and spread of cancerous tumors.To understand how jumping genes are mobilized, a fundamental question must be answered: is the high jumping gene activity observed in some cell types a result of activating many copies of the retrotransposons, or only a few of them? This question has been difficult to address because there are more than one hundred copies of retrotransposons that could potentially move in humans, many of which have not even been referenced in the human genome map. Furthermore, each copy is almost identical to another one, making it difficult to discriminate between them.Philippe et al. have now developed an approach that can map where individual retrotransposons are located in the genome of normal and cancerous cells and measure how active these jumping genes are. This revealed that only a very restricted number of them are active in any given cell type. Moreover, different subsets of jumping genes are active in different cell types, and their locations in the genome often do not overlap.Thus, whether jumping genes are activated depends on the cell type and their position in the genome. These results are in contrast to the prevalent view that retrotransposons are activated in a more widespread manner across the genome, at least in cancerous cells. Overall, Philippe et al.’s findings pave the way towards characterizing the chromosome regions in which retrotransposons are frequently activated and understanding how they contribute to cancer and other diseases.DOI:http://dx.doi.org/10.7554/eLife.13926.002 At least half of our DNA is derived from repeated and dispersed sequences called retrotransposons, a class of mobile genetic elements which proliferate via an RNA-mediated copy-and-paste mechanism termed retrotransposition  L1HS-Ta subfamily and with older copies of retrotransposition-incompetent subfamilies. Theoretically, the high L1 activity observed in particular cell types could result from global unleashing of most L1HS-Ta copies. Alternatively, it could derive from a few deregulated L1HS-Ta instances. To resolve these competing models, we mapped the location of each L1HS-Ta element dispersed in the genome of a panel of normal and transformed human cells, identified a genomic signature for the transcriptionally active copies and investigated the contribution of each of them to the bulk of L1HS-Ta transcripts. We found that individual L1 instances exhibit both locus- and cell-type-specific activation, implying that L1 mutagenic activity originates from 'hot L1' inserted in permissive loci and suggesting an unforeseen new layer of cell-type specific regulation to control endogenous retrotransposons.Human L1-derived RNA-transcripts and proteins, required for L1 retrotransposition, are detected in embryonic stem cells, in embryonal carcinoma cells, and other transformed cells or tumors, as well as in neuronal progenitor cells, but not in most primary cells, such as fibroblasts . To studAs a first estimate of L1 activity, we quantified and compared the endogenous levels of the L1-encoded ORF1p protein in distinct cell-lines. We detected ORF1p expression in whole cell extracts of half of the transformed cell lines , consistWe next asked whether the observed expression of L1HS-Ta in some cells results from the transcriptional activation of all or most L1HS-Ta genomic copies, or only of a few of them. As a first step, we mapped the genomic location of all L1HS-Ta elements in each cell line of our panel. To achieve this task, we adapted for deep sequencing an existing method termed ATLAS . In brieFull-length L1 elements contain internal sense and antisense promoters located in their 5'-untranslated region (UTR). To gain insight into the pattern of L1 expression in human somatic cells at single copy resolution, we noted that the weak L1 polyadenylation signal in the 3' UTR allows a fraction of L1 transcripts to extend into the downstream genomic sequence . This prTTC28 gene (22q12.1). Uniquely mapped RNA-seq tags are detected both in the body of the L1 sequence and in the immediate downstream genomic region. However, read mapping is performed against the reference human genome; thus, reads mapping within the L1 body could originate either from this particular reference L1 copy or from a non-reference L1 copy integrated somewhere else in the genome, and are therefore not informative. The second example is a non-reference L1HS-Ta element inserted in the NEDD4 gene (15q21.3) in opposite orientation. Again, a downstream RNA-seq peak immediately follows the insertion point. Interestingly, in both cases, we do not detect upstream antisense RNA-seq tags at the vicinity of these full-length L1HS-Ta copies, suggesting that sense and antisense L1 promoter activities - or the stability of their respective transcripts - are not necessarily coupled in a chromosomal environment, in agreement with previous results obtained from plasmid borne reporter assays . First, we observed H3K4me3 ChIP-seq peaks immediately upstream and adjacent to these L1 copies, a histone mark reflecting active or poised promoters  Bernste. AlthougTTC28 and NEDD4 loci + RNA-seq data were not available in public databases, As for MCF7, L1HS-Ta expression profiles between biological replicates of all cell lines are similar; and global shRNA-mediated ORF1 knockdown in 2102Ep, followed by RNA-seq, again confirmed the L1-derived origin of downstream transcripts in this cell line . The totvs. non-expressed elements were differentially oriented relative to the overlapping genes. Consistent with previous observations  among the 20 most highly expressed L1HS-Ta copies as deriving from the L1RP transduction family (Some L1 elements with high retrotransposition activity (‘hot’ L1) belong to well-defined lineages with distinctive 3’ transductions. To evaluate the proportion of the highly expressed elements which belong to such lineages, we screened 3’ ATLAS-seq reads supporting each L1 insertion for sequence tags characteristic of the three most characterized lineages  . We founn family . Nine adAltogether our observations support a model  where: .10.7554/L1 retrotransposon expression has been proposed both as a potential biomarker of cancer prognosis and as the starting point for L1-mediated genome instability in tumors . Hence, Furthermore, we provide here a unique resource consisting of near-complete maps of L1HS-Ta elements present in widely used normal and transformed model cell lines, for which the availability of genomic datasets is regularly increasing . These maps will be of broad utility in the future to address the impact and regulation of transposable elements in the human genome. Indeed, isolated RNA-seq and ChIP-seq signals resulting from the presence of non-reference L1 copies can only be correctly interpreted if such maps are available. Thus they can act as a platform to fully interpret and profit from the many expanding public datasets generated using the same cell lines.i.e. diminished restriction factor and/or increased cofactor activities , and 72°C for 10 min. A ~6 kb PCR product was resolved by 0.8% agarose gel electrophoresis, gel-purified using Wizard SV Gel and PCR Clean-Up System (Promega), and resuspended in 50 µL of milli-Q water. The second PCR was performed using 1 µL of the purified PCR product with primers matching the L1HS 5’ UTR (LOU1662) and the downstream flanking genomic DNA sequence of the L1 copy EXP_ID_0447 (LOU1664) with the following program: 98°C for 3 min, 40 cycles , and 72°C for 10 min. A PCR product of ~6 kb fragment was gel-purified as described above. Both primers of the second PCR contained homology in their 5’ parts to the CMV promoter and to the retrotransposition reporter cassette contained in the L1 expression vector pAD135 , allowin5 cells per well. The next day, cells were transfected with 1 µg of plasmid DNA and 3 µL of Lipofectamine 2000 (Life Technologies) diluted in 200 µL of Opti-MEM (Life Technologies), and medium was replaced with fresh medium after ~5 hr. Two days after transfection, medium was supplemented with G418 (Life Technologies) at 1 mg/mL. After 10 days of selection, colonies were fixed and stained with 1 mL of a solution containing 30% methanol (v/v), 10% acetic acid (v/v) and 0.2% Coomassie blue (m/v). The colonies obtained in three independent biological replicates were manually counted to assess the retrotransposition efficiency of pVan610 clones compared to the positive control pJM101/L1.3  and 2.8 µg of pCMV-VSV-G (Addgene #8454). Growth medium was changed 8 hr post-transfection. Two days after transfection, cells supernatants from 5 dishes were collected, pooled, filtered, and concentrated by overnight centrifugation at 4,000 rpm. Pellets were resuspended in 350 µL of growth medium, aliquoted and stored at -80°C until use. Viral stocks were titrated by colony formation assay upon puromycin selection  using serial dilution of vector stocks. For RNA-seq experiments, we infected 500,000 cells in triplicates (either MCF7 or 2102Ep cells) at M.O.I 1. Cells were collected for protein and RNA extraction after 1 week of puromycin selection (1.5 µg/mL).Annealed complementary oligonucleotides with 3' overhangs + RNA was isolated from the total RNA samples and fragmented with ultrasound (1 pulse of 15 s at 4°C). First-strand cDNA synthesis was primed with random hexamers. Then, the Illumina TruSeq sequencing adapters were ligated to the 5' and 3' ends of the cDNA. The cDNA was finally amplified with PCR using a proof reading enzyme and between 12 and 14 cycles. The TruSeq barcode sequences which are part of the 3' TruSeq sequencing adapters are included in www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-4676 and E-MTAB-3788, respectively. RNA-seq data contain biological duplicates for untreated cells (two independent RNA extractions), and triplicates for shRNA-treated cells . The genomic locations of L1 insertions in the different cell lines are provided in Raw ATLAS-seq and RNA-seq data were submitted to the ArrayExpress database (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were trimmed using sliding-window mode from Trimmomatic (v0.32)  tool ( (v0.32) . The minhttp://broadinstitute.github.io/picard), after mapping a subset of the RNA-seq samples. The other mapping parameters were set to their default values.PE trimmed reads were aligned per lane to both the hg19 reference transcriptome and genome obtained from UCSC, using TopHat2 v2.0.13 . For a bThis section is related to http://www.broadinstitute.org/igv). Only the strand of first mates and with a MAPQ of 20 were taken into account, ensuring that only one read per fragment was counted and only pairs uniquely mapped were retained for downstream analyses. The window size was set to 10 and the window function used was the median. Coverage wig files from all libraries were normalized according to the total number of mapped mates 1 of each library. After normalization, wig files were converted to bigWig files using the command wigToBigWig (v4) from UCSC bioinformatics utilities. The bigWig files were used to identify transcriptional signals from the possibly full-length L1HS individual copies: RNA-seq signals upstream or downstream individual L1HS copies were used as a proxy to identify potential transcriptional signature of specific individual L1HS copies, as these genomic flanking sequences are more-often uniquely mappable. To obtain a global view of the transcription levels detectable at each LINE-1 locus, the computeMatrix command from deeptools (v1.5.9.1) was employed  were obtained from the RNA samples using SuperScript III Reverse Transcriptase (Life Technologies) as per the manufacturer’s instructions. Briefly, 1 µg of DNase-treated RNA samples were combined in a 13-µL reaction including 1 µL of a 10 mM dNTP mix, 1 µL of a 50 µM RACE primer and milli-Q water. The samples were incubated for 5 min at 65C and placed on ice for 1 min. The reactions were then supplemented with a 7-µL cDNA Synthesis Mix containing 4 µL of 5X First-Strand buffer, 1 µL of 0.1 M DTT, 1 µL of 40 U/µL RNaseOUT and 1 µL of 200 U/µL of SuperScript III RT. For each sample, a control condition was prepared without reverse transcriptase. The tubes were incubated for 50 min at 55°C and then for 15 min at 70°C. PCR amplification of the cDNA was obtained using Platinum Taq DNA polymerase (Life Technologies) as per the manufacturer’s instructions. Briefly, 1 µL of cDNA sample was combined in a 50-µL reaction with 1X PCR Buffer, 1.5 mM MgCl (RB3PA1 ), 0.2 µMhttps://support.illumina.com/sequencing/sequencing_software/igenome.html). When an L1 copy was inserted in a region corresponding to several overlapping genes oriented in the same direction, we considered the most expressed gene. Ploidy at each L1 insertion locus and for each cell line analyzed was determined using the copy number variation (CNV) tracks obtained from ENCODE (see ‘Data’ section). They were deduced from Illumina Human 1M-Duo Infinium HD BeadChip assays and circular binary segmentation (CBS), which segments the genome into amplified, normal, heterozygous deletion, homozygous deletion or uncovered regions. As expected, no L1 were inserted in region annotated as homozygous deletion. Uncovered regions were excluded from the analysis. The list of full-length L1 insertions provided in This section is related to  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a guest Reviewing Editor, who acted as one of the reviewers, and Diethard Tautz as the Senior Editor.Thank you for submitting your work entitled "Activation of individual L1 retrotransposon instances is restricted to cell-type dependent 'hot loci'" for consideration by eLife readers. They also recommend some revisions as outlined below.The reviewers agree that your manuscript provides an important contribution to the field and will be of interest to The manuscript addresses a long-standing question in our field: Namely, how many and which of the many genomic LINE-1 loci contribute to expression at the RNA level. The authors do a very nice job of integrating different types of data: RNA-seq, epigenetic studies, and LINE-1 mapping with ATLAS, to reach conclusions about this. They take into account sense/antisense orientations and leverage that information in order to 'see' reads that span the L1 3' and 5' junctions with the adjacent genomic DNA. The attention to detail is high, and I think their conclusion – that a restricted subset of L1HS-Ta loci, a subset of which is polymorphic among individual cell lines, is responsible for the bulk of L1 expression – is sound.The following major points need to be addressed in a revision, before the paper can be accepted:More information should be provided on the positions of these active LINE-1 / contrast them to inactive loci. What are the surrounding genes and the relationship between the gene loci and the LINE-1? What is the ploidy/copy-number at these sites?More discussion is required on how the authors think their shRNA to ORF1 works. Their language  suggests they believe a shRNA silencing effect at the level of the genome is at work. One would think that shRNA is just causing degradation of the mRNA, which is what accounts for reductions in the reads.'Hot' is a colloquialism for retrotransposition active LINE-1. We would suggest the authors think of different terms, for example, expressed LINE-1 in place of hot LINE-1 when RNA levels are described, permissive loci in place of hot loci when considering epigenetic context. Keeping the narrow definition of 'hot' may avoid confusion in the literature.You should consider utilising the identification of daughter elements from their shared 3' transductions with putative progenitors as evidence of retrotransposition capability (where this was not assessed directly by cell culture assays). However you do not comment on the relationship between these transduction lineages and previously identified highly active pedigrees of elements. It is well established that common, highly active polymorphic L1 insertions, while showing significant allelic variation in retrotransposition activity, nevertheless frequently belong to lineages with distinctive 3' transductions. It would be valuable to know what proportion of the highly expressed elements analysed for their retrotransposition capability (either directly or by implication from ATLAS-seq reads) belong to previously identified lineages. It may be that the apparent dominance of small cohorts of elements is modulated by intrinsic features of these subsets of L1 elements. More information should be provided on the positions of these active LINE-1 / contrast them to inactive loci. What are the surrounding genes and the relationship between the gene loci and the LINE-1? What is the ploidy/copy-number at these sites?We have now compared the cohorts of expressed vs. non-expressed L1 copies with regards to:i) Their proportion in genes;ii) For the genic subset:– Their orientation relative to genes;– The levels of expression of their associated genes;iii) Their proportion in copy number variation regions.These analyses do not support a specific enrichment of expressed copies in genes or in amplified regions of the genome. For the genic copies, however, we found that expressed L1 copies are inserted in genes exhibiting higher levels of expression than inactive L1 loci.In the revised manuscript, these results are now shown in “To test whether the expression of L1HS-Ta copies could be influenced by their genic environment, we first compared the proportion of expressed L1 insertions in genes as compared to non-expressed copies. […] The majority of L1 copies were inserted in normal regions , whetherRelated additional information has been added in the Materials and methods section, and we appended an additional column containing gene names to More discussion is required on how the authors think their shRNA to ORF1 works. Their language  suggests they believe a shRNA silencing effect at the level of the genome is at work. One would think that shRNA is just causing degradation of the mRNA, which is what accounts for reductions in the reads.We agree that the title of 'Hot' is a colloquialism for retrotransposition active LINE-1. We would suggest the authors think of different terms, for example, expressed LINE-1 in place of hot LINE-1 when RNA levels are described, permissive loci in place of hot loci when considering epigenetic context. Keeping the narrow definition of 'hot' may avoid confusion in the literature.We agree that the use of ‘hot loci’ might be a source of confusion in the future. This was changed into ‘permissive loci’ in the title and throughout the manuscript, as appropriately proposed. Thus, the revised title is now: “Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci”. (Please see answer 9. on the choice of ‘instances’ instead of ‘insertions’ and answer 6. on the revised Discussion).You should consider utilising the identification of daughter elements from their shared 3' transductions with putative progenitors as evidence of retrotransposition capability (where this was not assessed directly by cell culture assays). However you do not comment on the relationship between these transduction lineages and previously identified highly active pedigrees of elements. It is well established that common, highly active polymorphic L1 insertions, while showing significant allelic variation in retrotransposition activity, nevertheless frequently belong to lineages with distinctive 3' transductions. It would be valuable to know what proportion of the highly expressed elements analysed for their retrotransposition capability (either directly or by implication from ATLAS-seq reads) belong to previously identified lineages. It may be that the apparent dominance of small cohorts of elements is modulated by intrinsic features of these subsets of L1 elements.We addressed this question in two different ways:i) First, we compared the list of full-length insertions, independently of their level of expression, with a published dataset of insertions deriving from the 3 main L1 lineages  . Eight out of 15 full-length and lineage-specific insertions found by MacFarlane et al. are present in the studied cell lines, but none of them belong to the top 20 highly expressed L1 .EDD4). Its affiliation to the RP lineage was confirmed by Sanger sequencing and its retrotransposition activity had already been tested by cellular retrotransposition in the initial version of the manuscript (now shown in ii) Second, we screened ATLAS-seq reads before poly(A) and L1 sequence trimming for the presence of lineage-specific sequence tags. This allowed us to identify the full-length L1 copies in our dataset belonging to these transduction lineages. Through this computational approach, we identified all 8 insertions previously found in the MacFarlane et al. dataset, as well as 2 additional insertions belonging to the RP lineage. One of these two insertions was ranked in the top 20 highly expressed L1s across all cell lines  belong to well-defined lineages with distinctive 3’ transductions. […]Thus, these findings suggest that the observed high level of expression of a small cohort of L1 insertions is not an intrinsic feature of any previously identified lineage.”Moreover, a new paragraph entitled “L1 lineage identification” has been added in the Materials and methods section.


Computational studies were performedto understand the interfacial behaviour of MoS2 and graphene, whichproves high stability of the composite with high interfacial binding energy(−2.02 eV) among them. Further, the lithium and sodiumstorage properties have been tested and reveal excellent cyclic stability over 250and 500 cycles, respectively, with the highest initial capacity values of1300 mAh g−1 and 640 mAhg−1 at 0.1 Ag−1.Developing nano/micro-structures which can effectively upgrade the intriguingproperties of electrode materials for energy storage devices is always a keyresearch topic. Ultrathin nanosheets were proved to be one of the potentialnanostructures due to their high specific surface area, good active contact areasand porous channels. Herein, we report a unique hierarchical micro-sphericalmorphology of well-stacked and completely miscible molybdenum disulfide(MoS In recent years, room temperature sodium-ion batteries (SIBs) have been the object ofsignificant interest for their potential application in large-scale energy storagesystems. This is mainly caused by concerns about insufficient lithium ores to satisfythe increasing demands for lithium-ion batteries (LIBs), as well as because sodium is arelatively cheaper option compared to lithium, which could be significant in large-scaleapplications such as grid storage. In addition, the electrochemical principles of SIBsare identical to those of LIBs2+ between the MoS2 layers456782 has been greatly improved , large volume changesoccur during charge-discharge cycling, which results in poor cycling stability. Severalmethods have been reported to successfully improve the cycling stability, such asexfoliation and restacking of MoS2 layers2 layers,1113215161718192021+ per MoS2. Considering the advantages of graphene as ahighly conductive and stable material, Wang et al.2—graphene nanocomposite with an improved electrochemicalperformance when compared to pristine MoS2, however, there is no clearevidence of well-ordered stacking of the MoS2 and graphene nanosheets amongthemselves. Three-dimensional (3D) hierarchical micro-spherical architectures withnanostructures as building blocks are considered to be electrochemically andstructurally stable morphology that could lead to improved practical application of suchactive materials in battery systemsAmongst various anode materials, molybdenum disulfide is one of the earliest compoundsstudied for rechargeable LIBs due to its layered structure, which can intercalateLi2-graphene composites with a unique micro-spherical morphologysynthesized via the spray-drying technique, which is an industrial-scale synthesisprocedure for a large-scale production of composite powders with a controllable narrowparticle size distribution and nano/micro-spherical morphology2 and graphene nanosheets could be an efficient structure to enhancethe interfacial effect between graphene-like MoS2 and graphene, thus takingfull advantages from both MoS2 and graphene components and achieving highcapacity, excellent cycle life and high rate capability as electrode materials for LIBsand SIBs.In this work, we are reporting structurally and electrochemically optimizedMoS2and graphene oxide, two proportions of MoS2: graphene oxide i.e., 80:20and 60:40 were optimized based on structural and electrochemical aspects. Thesynthesis of the MoS2-graphene composites with two different grapheneratios is briefly described in 2 andgraphene oxide were mixed in 80:20 (MoS2-G1) and 60:40(MoS2-G2) ratios to form a homogenous mixture. The graphene oxide sheetsand MoS2 sheets were well dispersed among each other and no aggregationwere observed after leaving the solution undisturbed overnight. The solutions forthe samples were then pumped through a nozzle using a peristaltic pump into acustom-made spray-drying reactor at 350 °C. The resultantblack fluffy powders were collected using a cyclone collector attached to the spraydrying reactor. The products were then annealed at 800 °C inH2/Ar gas flow for 2 h to fully reduce the grapheneoxide. The samples were then characterized using X-ray diffraction (XRD) todetermine the phase of the material as shown in 2, MoS2-G1, and MoS2-G2,show peaks which can be indexed to hexagonal MoS2 (ICDD# 37-1492) and noimpurity peaks can be observed. In order to determine the amount of graphene presentin the samples after annealing, thermo-gravimetric analysis (TGA) was performed onthe samples. The samples were loaded into alumina crucibles and heated in flowingair at the rate of 5 °C/min up to700 °C. Based on the assumption that all the MoS2is converted into MoO3 at 700 °C, the carboncontent estimated for MoS2-G1 and MoS2-G2 is13 wt% and 26 wt%, respectively, as shown in 2 sample can be attributed to adsorbed CO2 on thesurface of the samples. The ratio of C to O is roughly 1:2. The atomic percentages(%) of C in MoS2-G1 and MoS2-G2 agree well with the resultsfrom TGA, where the later has a higher amount of graphene sheets present. For allthree samples, the ratio of Mo to S is roughly 1:2, which suggest there is minimalor no oxidation of MoS2. This is further confirmed by Raman spectroscopy,as shown in −1 can be attributed tohexagonal phase MoS2, which is in well agreement with the literatureelsewhere−1 and1597 cm−1, respectively, can be observedon the spectra of MoS2-G1 and MoS2-G2.After preparation of composite material with various proportions of MoS2, MoS2-G1 and MoS2-G2samples was investigated using field emission scanning electron microscopy (FESEM),and the images are shown in 2-G2 are shown in 2 nanosheets. In addition, the spherical morphology ofthe sample enables the sheets to naturally stack on top of each other rather than ina random stacking. When the SAED pattern was collected from a larger area coveringthe sides of the sphere, rings were observed instead of the bright dots. Both of theSAED patterns can be indexed to the hexagonal MoS2 phase consistent withthe XRD results. High resolution TEM (HRTEM) was also used to study the distributionof the MoS2 and graphene nanosheets. 2nanosheets are stacked on top of each other, forming a sandwich-like structure. Sucha unique spherical microstructure with inter-stacked graphene and MoS2nanosheets is due to a combination of several factors. The miscibility and stabilityof the graphene oxide and the MoS2 nanosheets in aqueous solution is veryimportant to provide the sandwich-like stacking, while the spray-drying process iscrucial in providing the spherical morphology of the end product. The thickness ofthe stacks of graphene and MoS2 nanosheets was determined to be from3–15 layers by studying 10 random areas using HRTEM. d-spacing of the MoS2 and graphene nanosheets, whichwere measured to be 0.63 nm and 0.34 nm, respectively. TEManalysis of MoS2 and MoS2-G1 was also carried out, yieldingsimilar results. The images and diffraction patterns are presented in 2, MoS2-G1 and MoS2-G2 samples andrepresented in The morphology of the MoS2 and graphene. A (5 × 5)single graphene layer containing 50 carbon atoms was used to match a(4 × 4) MoS2 monolayer containing 16Mo and 32 S atoms. The lattice mismatch between the graphene and the MoS2monolayer is only 1.3%. Plane-wave basis VASP code was used to perform all thecalculations26302 geometry. The equilibriumdistance between the graphene layer and the top of the MoS2 monolayer iscalculated to be 3.34 Ǻ. The interface adhesion energy,Ead, was obtained according to the followingequation,Computational studies were performed to further understand the interfacial behaviourof MoSEcomb, Egraphene, andEMoS2 represent the total energy of the relaxed hybridgraphene-MoS2 complex, the pure graphene sheet, and the MoS2monolayer, respectively. The interface binding energy is as high as−2.02 eV for the whole model interface, which indicates veryhigh stability. To characterize the electron coupling at thegraphene-MoS2 interface, three-dimensional (3D) charge densitydifference plots were calculated by subtracting the electronic charge of the hybridgraphene-MoS2 nanocomposite from those of the separate graphene layerand the MoS2 monolayer, as shown in 2 surface in the ground electronic state.Where −1 over 50 cycles. In the firstdischarge, MoS2-G2 shows the highest capacity at 1300 mAhg−1, while MoS2-G1 and MoS2show 800 mAh g−1 and 630 mAhg−1, respectively. Large irreversible capacity isobserved for all three samples, as the first charge capacities are 945, 660, and480 mAh g−1 for MoS2-G2,MoS2-G1, and MoS2, respectively as shown in −1 for MoS2-G2, MoS2-G1, andMoS2, respectively, at the end of cycling. The samples were furthertested for their rate performances up to the current density of 5 Ag−1. The MoS2-G2 sample showed the bestrate performance, retaining 590 mAh g−1 at5 A g−1. The MoS2-G1 andMoS2 managed to retain 435 and 387 mAhg−1, respectively, at 5 Ag−1. When the rate was recovered to 0.5 Ag−1, all the samples showed capacity recovery, whereMoS2-G2, MoS2-G1, and MoS2, recovered 820, 680,and 560 mAh g−1, respectively. It should benoted that the rate performances improved with increasing graphene content in thesamples. This can be justified by the increased conductivity provided by thegraphene nanosheets in the samples. In order to investigate the long-term cyclingstability, the samples were tested at 50 mAg−1 for the initial 5 cycles, then at 1 Ag−1 up to 250 cycles. The MoS2 samplesrecorded a stable capacity of 500 mAh g−1 upto the 75th cycle, and then the capacity gradually faded over 50cycles. The capacity after the 125th cycle is negligible. This couldbe attributed to huge volume expansions of pristine MoS2 duringcharge-discharge process and gradually results in pulverization of electrodes overcycle life. The spherical shape of the MoS2 sample yielded improvedcycling stability and rate performances when compared to the bulk MoS2,which has been reported by us previously2-G2, and MoS2-G1 samples exhibit stable cycling over 250cycles, retaining 780 and 700 mAh g−1,respectively. From the cycling tests in lithium half-cells, it can be noted thatgraphene nanosheets in the samples are crucial for improving both the capacity andthe cycling performance.The samples were studied for their lithium storage properties and the results areplotted in xMoS2, remaining Na1-xions reacting with MoS2 and reduction of Mo4+ to metallicMo along with formation of Na2S particulates, respectively. Theseobservations are in consistent with the reports elsewhere1617−1, all three samples showstable cycling behavior over 50 cycles. The initial discharge and charge capacitiesare 640 and 400, 620 and 420, and 430 and 280 mAhg−1, for MoS2-G2, MoS2-G1, andMoS2, respectively. The irreversible capacities of about 35% .These excellent electrochemical properties in both LIBs and SIBs could be attributedto various reasons such as: (i) the well-ordered stacking and excellent miscibilityof the MoS2 layers and graphene sheets, which leads to improvedconductivity, and thereby, improved cycling performance and rate capability; (ii)suppression of volume changes by the structurally stable nanosheets in microspheresduring cycling; and (iii) good penetration of electrolyte into and among theMoS2 and graphene nanosheets. In addition, these microspheres withhierarchical nanostructures benefit from both micro-materials (high tap density) andnanomaterials (short Li/Na diffusion pathways), leading to densely packed electrodesand improved battery life in practical applications333435Furthermore, the samples were studied for their sodium storage properties inroom-temperature sodium half-cells using similar testing conditions to those forlithium cells see . In the  35% see can be dth cycle 0 mAh g−12)-graphene composite with uniquehierarchical microsphere morphology was prepared by the spray-drying technique. Thecomposite microspheres consist of well-ordered stacks with MoS2 andgraphene nanosheets with a high interfacial binding energy(−2.02 eV). Under testing for their lithium and sodiumstorage properties, MoS2-graphene (26 wt.%) microspherespresented excellent cycling stability and rate capability, with initial dischargecapacities of 1300 mAh g−1 and640 mAh g−1 at 0.1 Ag−1 in LIBs and SIBs, respectively. Notably, in SIBsat 1 A g−1, MoS2-G2 showed 93%capacity retention after 500 cycles. These enhanced electrochemical features areattributable to the unique hierarchical composite microspheres with uniformdistribution of graphene nanosheets among MoS2 layers.In summary, a molybdenum disulfide .


The goal of the current study is to clarify the relationship between social information processing  and aggressive tendencies. Thirty adults were recruited in the eye-tracking study that measured various components in social information processing. Baseline aggressive tendencies were measured using the Buss-Perry Aggression Questionnaire (AQ). Visual attention towards hostile objects was measured as the proportion of eye gaze fixation duration on cues of hostility. Hostility attribution bias was measured with the rating results for emotions of characters in the images. The results show that the eye gaze duration on hostile characters was significantly inversely correlated with the AQ score and less eye contact with an angry face. The eye gaze duration on hostile object was not significantly associated with hostility attribution bias, although hostility attribution bias was significantly positively associated with the AQ score. Our findings suggest that eye gaze fixation time towards non-hostile cues may predict aggressive tendencies. Aggressive tendencies not only predict violent behaviors, but also self-injurious or suicidal behaviors ,2. UnravCognitive distortions have been thought to play a role in hostility attribution bias . Mood dyA total of 36 young adults (age range: 18–30 years) were recruited for this study. The exclusion criteria include (1) a history of psychotic disorders, (2) a history of depressive or bipolar disorder, and (3) a history of substance use disorders. We used the self-reported questionnaire to exclude the individuals with these four types of mental disturbances.Twenty-five of the 36 participants were recruited through on-line advertisement, and provided their consents by signing in the on-line spreadsheet. Another 11 participants were enrolled through the biorepository at Cincinnati Children’s Hospital Medical Center (CCHMC). This biorepository project has been conducted based on the protocol approved by Institutional Review Board (IRB) of CCHMC. The individual in this manuscript has given written informed consent (as outlined in PLOS consent form) to publish these case details.We used Buss-Perry Aggression Questionnaire (AQ) to assess the baseline aggressive tendencies . The queStimuli: We have designed a series of pictures to present embedded ambiguous signs of hostility in the context of social interactions to each participant. These social interactions were classified into two categories: “confrontational” and “non-confrontational.” During the confrontational session, a series of 10 pictures of facial expressions retrieved from MMI Facial Expression Database [Database  were preEye tracking experiment: Eye gaze patterns were recorded using the Eyelink II and analyzed using Eye Link® Data Viewer  according to the user’s manual. The area of interest (AOI) was determined by the research team to concentrate on the regions that were hypothesized to represent the target of social information processing. For images related to social interactions, the AOIs were defined as regions including the face of each character and other body parts that intuitively referred to the body language . For images of individual facial expressions, the AOIs included eyes, noses, and mouths, which were found to more frequently viewed than the other parts of the face [the face .2 = 0.2, and alpha-value = 0.05, we estimated that we would achieve the power of 0.71 with the sample size equal to 36. Therefore, our sample size might be adequate given that we aimed to obtain a moderate statistical power. The primary predictor variable was the eye gaze fixation time at each AOI. All key continuous variables  were normalized with inverse rank-based transformation before the further analysis. We used the step-wise regression model to select significant independent variables to partially remedy under-estimated coefficients due to multicollinearity. The relationship between aggressive tendencies (AQ scores) and eye gaze fixation times was examined using the general linear model with PSS scores, gender, and age being adjusted as covariates. The area of AOI was adjusted in the regression model. Two-sided alpha value = 0.05 was used as the cut-off to screen for significant association findings. We later performed single-value permutation tests  to generate empirical p-values in order to alleviate inflated type-I errors due to multiple tests. We also test spatial homogeneity for eye gaze fixation points in the AOI with Ripley’s K functions. The measure of the degree of spatial homogeneity was used to examine whether the degree of “attention dispersion” correlated with the AQ score. Attention dispersion might be regarded as another aspect of visual attention.Under the assumption of effect size fOur findings suggest that selective visual attention towards objects with hostility may be associated with aggressive tendencies in young adults. Specifically, we found that individuals with greater aggressive tendencies seemed to pay more visual attention to the non-facial parts of the hostile character in the context of social interactions, compared to individuals with lower aggressive tendencies. This may lend little support to the hypothesis that selective visual attention to hostile objects can contribute to aggressive tendencies. Similarly, we found that individuals with greater aggressive tendencies seemed to pay more visual attention to non-eye regions of the angry face, compared to their peers with lower aggressive tendencies. This may imply that individuals with greater aggressive tendencies may show less eye contact when being faced with possible hostility, compared to their peers with lower aggressive tendencies. Taken together, aggressive tendencies appear to be inversely correlated with eye contact in response to the object of hostility. The association appears to be more remarkable when the individual interacts with another hostile individual than serving as a by-stander in the scene of social conflict.Eye contact indicates several different kinds of social communications. Previous studies have suggested that eye contact aversion often occurs in an aggressive encounter . In primTo evaluate if the data based on early short time-windows were different from the current results, we re-examined the eye gaze fixation points during the first 5 seconds. The results show that there was no statistically significant correlation between aggressive tendencies and region-specific fixation time. Our explanation is that the visual stimuli in the first 5 seconds might be primarily driven by immediate emotional response, which might not be able to sufficiently distinguish individuals with higher aggressive tendencies from individuals with lower aggressive tendencies. When the viewing time is extended, how visual attention is sustained towards certain regions might be able to distinguish individuals with higher aggressive tendencies from individuals with lower aggressive tendencies.The primary limitation of the current study is the sample size. The age range of our sample  and characteristics of participants (volunteer college students) may also discourage the generalizability of our findings. In addition, we did not perform stringent multi-testing corrections, such as Bonferroni method, and hence inflated type-I errors may remain to be a problem. However, we did perform single-value permutations to generate empirical p-values to identify significant associations, so inflated type-I errors may be partially alleviated. Finally, we relied on self-reported information to exclude subjects with mental disorders. The impact of under-reported cases of mental disorders may lead to uncertain directions of bias on our findings.Several recent studies also report neural correlates or eye gaze patterns in individuals viewing violent conflicts. Briefly, how an individual reacts to stressful stimuli  may be affected by several different factors, such as affective information in the background ,24. FurtIn conclusion, we have found that selective visual attention associated with hostile emotions might serve as a physiological/behavioral marker for aggressive tendencies in non-mentally ill individuals. Since aggressive tendencies may predict aggressive and violent behaviors, selective visual attention towards objects of hostility may also predict aggressive or violent behaviors. Replication of these findings in a larger population is needed to evaluate the clinical utility of eye gaze fixation time  in at-risk individuals. The findings may inform early detection and prevention modalities of aggressive/violent behaviors in the future. Previous evidence suggests that the patterns of visual attention bias towards hostility in healthy individuals seem to depend on the diagnosis of mental disorders. Further research is warranted to compare the cognitive and behavioral mechanisms underlying aggressive tendencies across different diagnoses.S1 Dataset(DOCX)Click here for additional data file.


This review article aims to provide an overview of chemically modified graphene, and graphene oxide (GO), and their impact on toxicology when present in biological systems. Graphene is one of the most promising nanomaterials due to unique physicochemical properties including enhanced optical, thermal, and electrically conductive behavior in addition to mechanical strength and high surface-to-volume ratio. Graphene-based nanomaterials have received much attention over the last 5 years in the biomedical field ranging from their use as polymeric conduits for nerve regeneration, carriers for targeted drug delivery and in the treatment of cancer via photo-thermal therapy. Both in vitro and in vivo biological studies of graphene-based nanomaterials help understand their relative toxicity and biocompatibility when used for biomedical applications. Several studies investigating important material properties such as surface charge, concentration, shape, size, structural defects, and chemical functional groups relate to their safety profile and influence cyto- and geno-toxicology. In this review, we highlight the most recent studies of graphene-based nanomaterials and outline their unique properties, which determine their interactions under a range of environmental conditions. The advent of graphene technology has led to many promising new opportunities for future applications in the field of electronics, biotechnology, and nanomedicine to aid in the diagnosis and treatment of a variety of debilitating diseases. There is only a relatively small contribution regarding the safety profile and toxicology data in the literature on graphene-based materials outlining their interactions in biological systems with cells and tissues. Over the last 5 years alone, over 424 publications and cited articles relate to graphene toxicology, which has increased to 1,015 publication by 2009 to approximately 3,753 in 2013, whereby the vast majority focus on the physical and material properties of graphene and is a subject of intensive research , rolled into 1D structures as a single-sheet CNTs, or stacked into 3D-layered structures such as graphite , mechanical, optical transmittance (~97.7 %), structural, and thermal behavior, and has shown great promise for many application areas relating to electronics, semiconductor fabrication, and the biomedical industry   at high temperature  using gasses such as methane –CH4 (g) as a carbon source as highlighted in Fig. 4 forms graphene on top of the Cu or nickel (Ni) foil . Further exfoliation with ultrasonication, thermal or energetic conditions help to oxidize stacked layers of hexagonally arranged carbon atoms that are bonded together with an inter-planar force to obtain graphene layers. The use of this method led to the formation and production of single-layer-reduced GO  bottom-up and 2) top-down fabrication techniques. Bottom-up fabrication is achieved using several methods to prepare high-quality graphene such as chemical vapor deposition (CVD). These methods produce highly crystalline graphene, but are not suitable for mass production  of graphite has been reported as 2 g/per kilo  is reacted with polyethylenimine-modified GO (PEI-GO) acting as a nanocarrier attached to the nanocrystals to yield PEI-GO–nanocrystal  converts light or optical energy to heat by absorption of a range of nanomaterial , leading to the thermal ablation resulting in the death of cancer cells. In recent years, PTT as a minimally invasive, controllable, and highly efficient treatment method has drawn widespread attention in the treatment of cancer. A large number of research groups have developed various light-absorbing nanomaterials as PTT agents  implants used in the peripheral and central nervous system (CNS). Nanoparticles incorporated into polymeric conduits, acting as fillers, such as, graphene, CNTs, and fullerene, can become one possible solution in the production of conducting materials, which are necessary for stimulating cell growth, and delivery of therapeutic agents. Identification of neural stem cell differentiation is an essential stage for the practical application of stem cell technology in regenerative medicine. Cell differentiation and monitoring is incredibly important for the application of neural stem cells (NSCs) in the treatment of neurodegenerative disease such as Alzheimer’s , morphology, geometry and aggregation behavior and solubility will influence the particle distribution within the surrounding environment. Nanoparticles when present in physiological fluids such as plasma or serum redistribute themselves throughout the host tissues and are transported to the liver, lungs, spleen, heart, kidney, and bone marrow due to their material and surface properties, and how they interact as a consequence of proteins that adsorb on to the surface can cause nanoparticle aggregation and cell uptake , as a result of environment toxicity or in vitro or in vivo medical treatments using graphene either as an implantable device or as drug delivery carrier. Animal experiments are performed according to policy guidelines standardized by the Organization for Economic Co-operation and Development (OECD) in the UK, and are known to determine common toxic characteristics of a broad range of materials and chemicals. This includes eco-toxicity or animal and human toxicity.Toxicology studies are now becoming more advanced in small- and large-scale animal studies in vivo and human cell lines in vitro. Direct hazard assessments and current methodology for studying nanomaterials help to reveal gaps in the knowledge and deficiency of current assessments. For example, the lethal dose (LD50) of graphite, CNTs, and fullerenes reported as 2 g/Kg , epoxy (–C–O–C–), and hydroxyl (–OH) functional groups. Moreover, limitation of GO use in a variety of biomedical applications is due to the absence of stable dispersions. In the following sections, we review current nanotoxicity studies carried out with GO over the last 5 years. Moreover, Tables GO is water-soluble nanomaterial and has been investigated extensively as a material for industrial applications for electronics and use in biomedical engineering. This is due to the large vacancy of planar surface area for efficient filling of aromatic drug molecules through 2) groups, whereas linear PEIs contain all secondary amines. The production of linear PEI-grafted GO (LP-GO) conjugates, and their efficacy to transfer nucleic acids into the mammalian cells is investigated  with the loading ratio more than 100 %  (GO/TiO2) hybrid (GOT) was studied by using Ti (OC4H9)4 and GO as reactants. The result presented no toxicity of GO in vitro as an electron sink in GOT efficiently increased the photodynamic therapy (PDT) activity  cells based on current data has shown that at low concentrations, GO does not enter into the cells and shows no signs of cytotoxicity. However, GO is known to be cytotoxic and is dose-dependent and known to cause oxidative stress in A549 cells, and induce a loss in cell viability at high concentrations , ZnO, TiO2, tin (Sn), and CNTs at concentration higher than (100 μg/mL). Graphene concentrations higher than 1 μg/mL induced DNA damage at a significantly lower concentrations  and TRC105 (an antibody that binds to CD105)  loaded onto PEG-functionalized graphene oxide (GO) via supramolecular π–π stacking investigated and obtained GO–PEG–HPPH complex, shows high HPPH loading efficiency. The in vivo distribution and delivery were tracked by fluorescence imaging as well as positron emission tomography (PET) after radiolabeling of HPPH with 64Cu. Compared with free HPPH, GO–PEG–HPPH offers dramatically improved photodynamic cancer cell killing efficacy due to the increased tumor delivery of HPPH  system including liver and spleen were observed post-injection (i.p.) and are highlighted in Fig. Functionalized GO toxicity and their distribution have been studied in mice using radiolabeling techniques ]) was investigated  that are able to distinguish intact and damaged DNA and catalytic activity of hemin through graphene π–π interactions have successfully synthesized. Based on this, for detection of single-stranded DNA (ss-DNA) and damage DNA induced by chemicals such as styrene oxide (SO), NaAsOo-methacrylate (FMA) to yield multi-functional graphene (MFG) with water dispersibility based on 2.5 mg/mL concentration  for linking the fluorescein Graphene-based nanomaterials have great potential for large number of future technologies ranging from biotechnological and biomedical applications including, drug delivery, PTT, and cancer targeting and therapy. Graphene materials are known to vary widely in terms of their physical and chemical properties such as their dimensions and chemical functional groups. Moreover, they possess unique physical and chemical material properties such as optical, electrical and thermal conductivity, and high surface-to-volume ratio. In addition, they can be easily linked to macromolecules through covalent or non-covalent attachment. The types of graphene material and their chemical modifications to produce novel graphene compounds provide different levels of dispersibility and impurities within the nanomaterials. Based on the previous toxicity investigations of graphene, graphene oxide and their derivatives all exhibit in vitro toxicity. However, it showed that cells are very sensitive to size, shape, solubility, and concentration of graphene nanomaterials. GO is considered more biocompatible than graphene due to its greater solubility/dispersibility, which results in less damage and toxicity in human cell types such as skin fibroblasts and red blood cells, and bacteria. Unfortunately, there are no current or sufficient in vivo studies outlining their nanotoxicity. The results indicate that upon initial exposure of the materials, the most prominent route into the human body lies within the respiratory system. However, they are less effective in liver, kidney, and spleen. Furthermore, all cells exhibit time and dosage dependency depending upon protein adsorption and reactions governed by aggregation. As this review represents only a few studies in relation to graphene-based materials, their toxicology profile remains at the very early stages of development for a range of biomedical applications. However, before such materials reach the clinic, their toxicology profile and safety efficacies are essential steps in their evolution. Such great potential will offer a variety of new and powerful tools based on graphene materials for use in the areas of advanced imaging, disease diagnosis, and targeted therapies for the treatment of a range of severely debilitating diseases.


GMC cultures were split into four groups treated for 24 h: Group N (blank control group), Group A (10−7 mol/L AngII), Group LT (10−7 mol/L AngII and 10−5 mol/L losartan), and Group Pred (10−7 mol/L AngII and 10−5 mol/L prednisone). GMCs proliferation was measured by the MTT and trypan blue assays. The distribution of TRPC6 was monitored by immunofluorescence, the expression of TRPC6 was detected by RT-PCR and Western blotting, and [Ca2+]i was measured by laser scanning confocal microscopy. The results showed that the maximal proliferation of GMCs was induced by treatment with 10−7 mol/L AngII for 24 h. In Group A, the distribution of TRPC6 was not uniform in the cell membrane, there was increased accumulation of this protein within the cytoplasm, and the increased expression of TRPC6 and [Ca2+]i was consistent with the proliferation of cells. In Group LT, losartan inhibited the proliferation of GMCs significantly, the levels of TRPC6 and [Ca2+]i were diminished, and the distribution of TRPC6 was improved. Prednisone also significantly inhibited the proliferation of GMCs and had no effects on the expression of TRPC6 and [Ca2+]i in Group Pred. These findings suggested that AngII could enhance the expression of TRPC6, increase [Ca2+]i, and demonstrate a time–dose–response relationship with the proliferation of GMCs, while losartan reversed the effect of AngII on GMC proliferation.This study investigated the changes in intracellular [Ca In the past 20 years, epidemiological studies have suggested an increase in the incidence of chronic kidney disease in children . Glomeru2+. TRPC6 is the core member of TRPC superfamily [2+]i  and is involved in a number of kidney diseases i and the involvement of ion channels in cell proliferation have become hot topics. In this study, GMC proliferation induced by AngII was observed. The expression of TRPC6 at the gene and protein level, as well as the relationship between cell proliferation and [Ca2+]i in the GMC proliferation process, was further investigated. Furthermore, the blocking effects of losartan were studied, and the mechanism of cell proliferation induced by AngII was explored from the perspective of ion channels.Transient receptor potential channel (TRPC) is a transmembrane protein that allows for transmembrane movement of Caerfamily , which mdiseases , 5.Changfibrosis –9. Previby AngII . Therefo2 at 37 °C and grew adherently in 20 % fetal bovine serum medium . Morphology was observed with an inverted microscope. GMCs at logarithmic growth stage were inoculated into six-well plates, fixed with paraformaldehyde for 30 min, and then blocked by goat serum  for 1 h. Afterward, GMCs were incubated with mouse monoclonal antibodies , including anti-desmin protein, anti-α-actin protein, anti-myosin protein, anti-vimentin protein, anti-keratin protein, and anti-factor VIII at 4 °C overnight. This was followed by inoculation with FITC-labeled goat anti-mouse IgG  at 37 °C for 1 h. GMCs were monitored and visualized by fluorescence microscopy. GMCs of 3–8 generations were used for further experiments.Glomerular mesangial cells  were cultured in incubators containing 5 % CO−8 and 10−7, 10−6 and 10−5 mol/L, were used to stimulate the GMCs for 0, 24, 48, and 72 h, respectively. Next, 10 μL of MTT  was added to each well and incubated at 37 °C for 4 h; afterward, 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The optical density (OD) at 490 nm was measured by a microplate reader. After mixing the cells with a 0.4 % trypan blue solution for 3 min, the number of living cells and dead cells were counted with a hemocytometer. Proliferation rate (%) was calculated with the following formula: /OD value in blank control × 100 %. Cell viability (%) was calculated with the following formula: number of living cells/(number of living cells + number of dead cells) × 100 %.Cells were inoculated into 96-well plates. In the serum-free medium, cells adhered to the surface, and different concentrations of AngII , including 0 (blank control group), 10−7 mol/L of AngII to induce GMC proliferation for 24 h), the experiments were grouped as follows: Group N (blank control group), Group A (10−7 mol/L AngII), Group LT (10−7 mol/L AngII and 10−5 mol/L losartan), and Group Pred (10−7 mol/L AngII and 10−5 mol/L prednisone). Intervention groups (Group LT and Group Pred) were pre-treated by losartan or prednisone, respectively, for 1 h prior to the addition of AngII. Inhibition rate (%) was calculated with the following formula: /OD value in group A × 100 %.After determining the optimal concentration and time of AngII treatment , and mixed with rabbit anti-mouse TRPC6 polyclonal antibody . After overnight incubation, FITC-labeled goat anti-rabbit IgG  was added, and the distribution of TRPC6 was observed by immunofluorescence microscopy.Total RNA was extracted with the addition of Trizol reagent  into all groups. cDNA was synthesized using 1–2 μg of total RNA. The primers were designed using the Primer Express software (Primer Premier 5.0) based on sequences obtained with the GenBank accession numbers. The sequences of the primers and amplicon sizes are shown in Table Cell lysis solution was added into the cells of each group. The protein concentration was determined by the bicinchoninic acid (BCA) method. Thirty five micrograms of total protein was denatured at 100 °C for 5 min, run on a 120 g/L sodium dodecyl sulfate polyacrylamide gel  for 1.5 h, and then wet transferred to a polyvinylidene difluoride  membrane. The membrane was blocked with 5 % non-fat milk at room temperature for 30 min, followed by incubation with rabbit anti-mouse TRPC6 polyclonal antibody  at 4 °C overnight. The membrane was subsequently incubated with HRP-labeled goat anti-rabbit IgG  at room temperature for 1 h. After the final rinse, the membrane was visualized with an enhanced chemiluminescence reagent and exposed to X-ray films. Band intensity was estimated using Gel-Pro image analysis software. The relative expression levels of target proteins were calculated as the ratio of the target protein band intensity to that of the internal control GAPDH.−6 mol/L of Fluo-3AM  at 37 °C for 45 min. Following this step, cells were inoculated into special culturing dishes for laser scanning confocal microscope (LSCM) and washed with D-Hanks three times. Then, the measurements were taken by LSCM with the excitation wavelength set at 488 nm. The [Ca2+]i was scanned per 5 s for 10 min, with each scan covering 20 cells. The results were analyzed by Zeiss image 4.2 software. The [Ca2+]i was calculated with the following formula: F/Fa .Cells from each group were re-suspended with 5 × 10P value <0.05 was considered to be statistically significant.All values were expressed as mean ± SD, and analysis of variance was calculated using SPSS 11.5 statistical analysis software. Fisher’s LSD test was used for the pairwise comparison. A GMCs grew adherently and showed larger soma with fusiform, irregular star-shaped, or elongated shapes. There were several dendrites of various lengths on the cell, which connected the cell groups into a network with a round- or egg-shaped cell nuclei in the middle. The GMCs stained positive for desmin, α-Actin, myosin, and vimentin and negative for keratin and factor VIII, indicating that the cultured cells were in fact GMCs Fig. .Fig. 1Im−8 mol/L AngII, while addition of 10−7, 10−6, and 10−5 mol/L of AngII caused increased cell proliferation at 24, 48, and 72 h; the proliferation at 24 h was much more significant than that at 48 h and 72 h . In addition, at 24 h, the proliferation with AngII at 10−7 mol/L was of greater statistical significance than that observed with AngII at 10−8, 10−6, and 10−5 mol/L (P < 0.01) . Furthermore, compared with Group A, the GMC proliferation was significantly inhibited in Group LT and Group Pred  ; when compared with Group A, the expression of TRPC6 mRNA in Group LT was diminished (P < 0.01), and there existed no significant difference in Group Pred (P > 0.05) P < 0.01); as compared with Group A, the expression of TRPC6 protein in Group LT declined (P < 0.01), and no significant difference in Group Pred was observed (P > 0.05) 2+]i increased significantly after the addition of AngII. The fluorescence intensity began to increase at the third min and peaked at the fourth minute. The fluorescence began to decrease 1 min later and returned to the starting level at the eighth minute ; as compared with Group A, [Ca2+]i decreased in Group LT (P < 0.05), and there was no significant difference in Group Pred (P > 0.05).After incubation with Fluo-3AM, a calcium indicator, green fluorescence was uniformly distributed in cells Fig. . The i can promote the proliferation of GMCs; for example, AngII can increase [Ca2+]i by signal transduction pathways, which in turn regulates cell proliferation [2+]i and the proliferation of GMCs are significantly inhibited i reached the maximum level at the forth minute after incubation with AngII. These observations suggested that TRPC6 and Ca2+ played important roles in the process of GMC proliferation. After incubation with losartan, TRPC6 distribution was improved, and [Ca2+]i was consistent with the cell proliferation, indicating that AngII affected TRPC6 and Ca2+ through AT1R, and further regulated cell proliferation. Prednisone inhibited cell proliferation, but had no effect on the expression of TRPC6 and [Ca2+]i, indicating that cell proliferation was not necessarily caused by changes in [Ca2+]i. It had been reported that glucocorticoid does not cause the changes in [Ca2+]i, and that its effects on cell proliferation could be achieved by inhibiting nuclei factor NF-κB or increasing P21 protein to arrest the cell cycle of GMCs at G0/G1 i in GMCs, which in turn induced its proliferation. The mechanism by which this occurs begins with the binding of AngII to AT1R on the surface of GMCs, which causes phospholipase c (PLC) to catalyze the cleavage of 4,5-phosphatidyl inositol diphosphate (PIP2), producing 1,4,5-inositol triphosphate (IP3). IP3 can then activate Ca2+ channels, leading the flow of extracellular Ca2+ into the cell. This influx results in increased [Ca2+]i in the sarcoplasm, which in turn regulates transcription and causes the proliferation of GMCs. There exist target sites for AngII on the TRPC6 channel. Through TRPC6, [Ca2+]i can be increased by AngII stimulation. And it is possible that TRPC6 and AngII are relevant in terms of regulating GMC proliferation via controlling [Ca2+]i i and induced GMC proliferation shared similarities with the mechanism by which TRPC6 regulated [Ca2+]i, as both were associated with an IP3-regulated calcium channel, and TRPC6 mediated Ca2+ to flow inward to promote the proliferation of GMCs.AngII promoted the increase of [Ca [Ca2+]i . In the f a SOCC . These s