Bone-morphogenetic protein-7 (BMP-7) is a growth factor that plays a major role in mediating anabolism and anti-catabolism of the intervertebral disc matrix and cell homeostasis. In osteoblasts, Forkhead box protein C2 (FoxC2) is a downstream target of BMPs and promotes cell proliferation and differentiation. However, the role FoxC2 may play in degenerative human intervertebral disc tissue and the relationship between FoxC2 and BMP-7 in nucleus pulposus (NP) cells remain to be elucidated. This study aims to investigate the presence and signaling mechanisms of FoxC2 in degenerative human intervertebral disc tissue and NP cells. Western blot and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses were used to measure FoxC2 expression in the NP tissue and cells. Transfections were carried out to measure the effect of FoxC2 on BMP-7-mediated extracellular matrix upregulation. Adenoviral knock-down of Smad1 was performed to investigate the mechanism of BMP-7-induced FoxC2 expression. In degenerative NP tissue, FoxC2 was markedly upregulated and positively correlated with increased disc degeneration. Induction of NP cell proliferation was confirmed by using cell counting kit-8 assay, immunocytochemistry and real-time qRT-PCR for Ki67. FoxC2 led to decreased noggin expression and increased Smad1/5/8 phosphorylation. During combined treatment with BMP-7, FoxC2 greatly potentiated anabolism through synergistic mechanisms on ECM formation. Combination therapy using BMP-7 and FoxC2 may be beneficial to the treatment of intervertebral disc degeneration. AlthougNP cells . In addiNP cells , 12. In NP cells . To amelNP cells . FurtherNP cells .et al. found BMP-7 has a similar anabolic effect on ECM metabolism in bovine intervertebral disc (IVD) cells [et al. report that after ECM depletion in response to IL-1, BMP-7 can effectively increase the expression of IVD ECM genes by rabbit NP cells[Bone-morphogenetic proteins (BMPs), which constitute the largest subgroup of the transforming growth factor-β (TGF-β) superfamily, are often used as therapeutic factors in treating disc degeneration , 17. BMPD) cells . FurtherD) cells , 22. Tak NP cells. These rFoxC2 is a member of the forkhead box (FOX) family of transcription factors. When the abbreviation of FOXC2 contains all uppercase letters, this refers to human form. When Foxc2 has only the first letter capitalized, this refers to mouse, and when FoxC2 has the first and subclass letters capitalized, this refers to all chordates . Foxc2 het al. have shown that following treatment of skeletal precursors with BMPs, Foxc2 may play a role in the early stage of chondrogenic differentiation, because increased expression of Foxc2 is observed in relation to SOX9, noggin and aggrecan [et al. have found that Foxc2 is a downstream target of BMP-2 and promotes the survival, proliferation and differentiation of osteoblasts [Recently, Nifuji aggrecan . Furthereoblasts . Recent eoblasts . A simileoblasts . Overallth, 2014 to March 8th,2015, following approval from the Institutional Review Board of the First Hospital of Jilin University. Patients consented to the collection and use of tissue samples for research and provided written informed consent in all cases. All the human tissue samples used for this study were obtained from patients as a standard part of the patients' spinal surgeries without additional biopsies. All animal experiments were carried out in a humane manner after receiving approval from the Institutional Animal Experiment Committee of Jilin University and in accordance with the Regulation for Animal Experiments and Fundamental Guideline for Proper Conduct of Animal Experiments of our university.The study protocol was approved by the Institutional Ethics Committee of the First Hospital of Jilin University. Human lumbar and cervical IVD samples were obtained from patients undergoing spinal surgeries during March 5BMP-7 inhibitor noggin was purchased from Protect . All other chemicals and reagents were purchased from Sigma  unless otherwise specified.et al[Patient samples were collected and isolated as previously described by Yu et al. We collet al, 7 of th3 blocks, which were treated separately with 0.1% collagenase II for 4‒6 hours [2 at 37°C. Cells were then passaged and further cultured in the presence of BMP-7 (10‒100 ng/ml). On reaching approximately 80% confluence, cells were subcultured in 60-mm culture dishes (4×105 cells/well). All experiments in this study used first- or second-generation cells.Disc tissue was harvested from male Wistar rats (200–250 g). All the rats were purchased from Animal Experimental Center of Jilin University and were killed with a lethal dose of 10% chloral hydrate. Lumbar and tail IVDs were separated from the spinal column under aseptic conditions. After separation of the NP, the tissue was cut into 1-mm‒6 hours . NP cellAccording to the manufacturer’s instructions, Lenti-FoxC2, Lenti-GFP or scramble was transfected into the cells by DharmaFECT1 Reagent  at a final oligonucleotide concentration of 10 nmol/L. For each cell transfection, two replication experiments were performed.Cell and tissue lysates were prepared using modified radioimmunoprecipitation assay (RIPA) buffer as previously described . Total pPCR amplification was carried out in accordance with the previously described method. Total RNA was isolated using TRIzol reagent  following the manufacturer’s instructions. cDNA was then reverse transcribed according to the manufacturer’s instructions. Quantitative PCR was carried out using a SYBR green Jumpstart Taq ReadyMix (TaKaRa) on a Roche LightCycler 480. A threshold cycle for each PCR amplification was subjected to 35 cycles of 95°C for 15 s and 54°C for 2 min. The primer sequences and their conditions for use were summarized in 3/ well) and treated with BMP-7 for 1‒24 h. After incubation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% triton-X 100 in PBS for 10 min, blocked with PBS containing 5% FBS, and incubated with antibodies against FoxC2 (1:200), aggrecan (1:100), SOX9 (1:200), and MMP-2 (1:200) at 4°C overnight. As a negative control, cells were reacted with isotype IgG under similar conditions. After washing, the cells were incubated with DyLightTM488-conjugated goat anti-rabbit antibody and fluorescein- or DyLightTM594-conjugated goat anti-mouse antibody (Jackson Laboratory), at a dilution of 1:200 for 45 min at room temperature. Fluorescence signals were captured using an Olympus Fluoview FV1000 confocal microscope and analyzed by the FV10-ASW 1.6 Viewer program .Cells were plated in flat bottom 96-well plates  values were read at 450 nm.All measurements were performed in triplicate and data were presented as means ± standard deviation (SD). Differences between groups were analyzed by Student’s t test; *p<0.05, ** p<0.01.Of the 36 DDD patients, 19 females and 17 males, the average age of these patients was 47.36±6.651 (range 33–57 years). The clinical findings for these patients were summarized in After approximately 7‒10 days, the Primary NP cells reached complete confluence. The cells were round or polygonal with a granular cytoplasm. Collagen II and aggrecan expression in these cells were observed by immunofluorescence . As earlTo determine whether up-regulation of FoxC2 in NP cells induce survival, proliferation and differentiation of NP cells, we transfected Lenti-FoxC2 in NP cells and transfected Lenti-GFP as the control. Transfecting Lenti-FoxC2 in NP cells significantly increased the expression of Ki-67, a famous proliferative marker, in immunofluorescence staining . CompareAs mentioned above, BMPs likely played an important role during the process of FoxC2 expression . We inveTo determine whether increased FoxC2 by BMP-7 is related to the BMP signaling pathway, we quantified FoxC2 mRNA expression in the presence and absence of BMP-7, SiRNA-Smad1, Scramble and the BMP inhibitor noggin . ConcordWe next examined the effects of FoxC2 on the expression of anabolic genes. As expected, the real-time qPCR results demonstrated BMP-7 stimulated the expression of ECM genes . SpecifiDisc degeneration is a complex process caused when the delicate balance between anabolic and catabolic processes is broken. From previous researches at the molecular level, we know that genes involved in NP cell degeneration are tightly regulated and coordinated by several signaling pathways , 36–38. We demonstrated that FoxC2 was frequently upregulated in human degenerative NP tissue and significantly associated with disc degeneration grade. We cultured primary NP cells from rat discs and characterized them by assessing cell morphology and expression of Collagen I, Collagen II and aggrecan. The characteristics of NP cells in our study were consistent with those of previous studies , 44. TheBMP signals are mediated by type I and II BMP receptors and their downstream molecules Smad1, 5 and 8. Thereafter, when type I receptor kinase is activated, Smad1, 5 and 8 proteins are phosphorylated and form a complex with Smad4 to translocate into the nucleus. Within the nucleus, they then interact with other transcription factors and regulate expression of target genes. Together with other transcription factors and transcription coupling factors, the Smad complexes bind to Smad binding elements (SBEs) in the promoters of target genes whereby they regulate expression of these genes. The anaTo further understand the relationship between FoxC2 and BMP pathways, we demonstrated that overexpression of FoxC2 activated a positive regulation mechanism. Noggin was known to inhibit the BMP pathways in a negative feedback system. Overexpression of FoxC2 was found to efficiently downregulate noggin expression, thereby indirectly reducing the inhibition effect of noggin within the BMP pathways. Furthermore, overexpression of FoxC2 resulted in phosphorylation of BMP7-responsive SMAD proteins (Smad1/5/8), that modulated BMP7-SMAD1/5/8-dependent anabolic target genes in NP cells. We therefore speculated that FoxC2 played a crucial role in sustaining activation of the BMP pathways.Results from previous studies demonstrated that BMP-7 had the ability to stimulate aggrecan and collagen synthesis in human disc cells. In this study, further evidence demonstrated that both FoxC2 and BMP-7 had potent anabolic effects on rat NP cells. We found that FoxC2 enhanced the upregulation effect of BMP-7 for ECM proteins like Collagen II and aggrecan. These results not only supported our previous conclusion about the FoxC2 positive regulation effect in BMP signaling, but also provided evidence for the benefits of a novel combination treatment in disc degeneration.et al. demonstrated that Foxc2 was a common transcriptional activator of TGF-signaling [et al. have shown that forced expression of FOXC2 promoted upregulation of Wnt4 expression and activation of increased levels of BMP [Several studies have previously shown that FoxC2 can induce the BMP pathway through crosstalk with other signaling pathways. Fujita ignaling . Althougignaling . Gozo ets of BMP . It is ws of BMP , 47. ThiIn conclusion, we identified that the transcription factor FoxC2 was over expressed in human degenerative discs. The mRNA expression level of FoxC2 was positively correlated with disc degeneration grade. Furthermore, we demonstrated that overexpression of FoxC2 increased the proliferation of NP cells. Importantly, BMP-7 induced FoxC2 expression, which was a positive feedback for maintaining signal activation . Our fin


Giardia intestinalis is a parasitic unicellular eukaryote with a highly reduced genome, in which only six cis-spliced and four trans-spliced introns have been discovered. However, we anticipate that more cis- and trans-spliced introns likely remain unidentified in genes encoding hypothetical proteins that occupy ca. 2/3 of all of the open reading frames (ORFs) in the Giardia genome. Consequently, comprehensive surveys of introns in ORFs for hypothetical proteins are critical for better understanding of the intron evolution in this organism.cis-spliced introns in the draft genome data of G. intestinalis strain WB, by surveying the conserved sequence motifs shared amongst the previously known introns. G. intestinalis strains can be divided into phylogenetically distinct assemblages A–H, and all the introns identified in past studies are shared among the published genome data from strains WB, DH, GS, and P15 representing assemblages A1, A2, B, and E, respectively. Nevertheless one of the two novel introns identified in this study was found to be absent in strain P15.In this study, we identified two novel G. intestinalis assemblages A1, A2, B, and E, one of the two introns identified in this study has highly likely been lost after the divergence of the assemblages. On the basis of a sequence comparison between the intron-bearing loci in WB, DH, and GS genomes and the homologous but intron-free locus in P15 genome, we propose that the loss of this particular intron was mediated by integration of the DNA fragment reverse-transcribed from mature mRNAs.By considering the organismal relationship among  Giardia intestinalis, a unicellular eukaryotic parasite belonging to the Diplomonadida (Excavata) is known to possess a highly reduced genome of only 12 Mbp in length G. intestrains DH  as well,Giardia introns/splintrons known to date bear conserved sequence motifs at the 5′ and 3′ termini, 5′-STATG-3′ and 5′-HCTRACMCVCAG-3′ , respectively. Furthermore, the two motifs may be flanked with each other within 300 bp, since all of the known introns in the Giardia genome range from 29 to 220 bp in length. We searched for genome segments that satisfied the above criteria in the draft genome data of G. intestinalis strain WB .G. intestinalis strain WB (ATCC50803) was cultivated as described previously , although reverse transcription activity of these encoded proteins has yet to be experimentally confirmed in G. intestinalis cells [There are three major models to explain how eukaryotic genomes lost spliceosomal introns: (i) ‘de-intronization’ by mutations, (ii) non-homologous end joining (NHEJ) repair of double strand break (DSB) in an intron sequence, and (iii) homologous recombination of the cDNA e.g., -23; see is cells .cis-spliced introns and their punctate distribution in the genomes of G. intestinalis assemblies. Together with the recently found trans-spliced introns, the data presented here suggest that the intron evolution in this organism is more complex than we previously thought.In this study, we found two novel The authors declare that they have no competing interests.RK determined the sequences and analyzed the data. YI and TH provided research materials. RK, YI, and TH prepared the manuscript. All the authors read and approved the final manuscript.Giardia intestinalis strain WB and the corresponding ORFs in strains GS and P15. A. orfA. Amino acid residues and nucleotides identical among the three sequences are shaded in black background. Asterisks indicate stop codons. The inserted position and nucleotide sequences of orfA intron are presented in the balloon. ORF nos. of orfA homologues in strains WB, DH, GS, and P15 are AACB02000068-1-10039-10248, AHGT01000085-3-18630-18839, ACGJ01001903-2-4190-4396, and ACVC01000007-3-18771-18962, respectively. B. orfB. The details of this figure are the same as described in A. ORF nos. of orfB homologues in strains WB, DH, GS, and P15 are AACB02000001-6-305427-304747, AHGT01000002-2-45140-45820, ACGJ01002919-1-19018-19737, and ACVC01000101-5-40937-40224, respectively. The intron sequences, which are a part of Figure Amino acid sequences of ORFs AACB02000068-1-10039-10248 (orfA) and AACB02000001-6-305427-304747 (orfB) in Click here for fileA. Deintronization by substitutions. An intron sequence (red) is changed to an exon sequence (light blue) by nucleotide substitutions (askerisks), resulting in extension of exon sequence. B. Non-homologous end joining repair of double strand break in intron sequence. In this model, ‘microhomology’ pairing between 5′ and 3′ splice sites anchors the upstream and downstream exons, which are split by double strand break. Subsequently, the broken strands are repaired, resulting in elimination of the entire intron sequence. C. Homologous recombination between an intron-containing gDNA and the corresponding intron-free cDNA (dark blue). This model assumes that the cDNA fragment, which is reverse-transcribed from a mature mRNA bearing no intron, is recombined into the corresponding intron-containing locus in the genome, resulting in elimination of the entire intron sequence.Proposed models for intron loss. Click here for file


Nanoscale carbon-based fillers are known to significantly alter the mechanical and electrical properties of polymers even at relatively low loadings. We report results from extensive molecular-dynamics simulations of mechanical testing of model polymer (high-density polyethylene) nanocomposites reinforced by nanocarbon fillers consisting of graphene flakes and fullerenes. By systematically varying filler concentration, morphology, and size, we identify clear trends in composite stiffness with reinforcement. To within statistical error, spherical fullerenes provide a nearly size-independent level of reinforcement. In contrast, two-dimensional graphene flakes induce a strongly size-dependent response: we find that flakes with radii in the 2–4 nm range provide appreciable enhancement in stiffness, which scales linearly with flake radius. Thus, with flakes approaching typical experimental sizes (~0.1–1 μm), we expect graphene fillers to provide substantial reinforcement, which also is much greater than what could be achieved with fullerene fillers. We identify the atomic-scale features responsible for this size- and morphology-dependent response, notably, ordering and densification of polymer chains at the filler–matrix interface, thereby providing insights into avenues for further control and enhancement of the mechanical properties of polymer nanocomposites. Using MD simulations, we study the mechanical response of a model polymer , reinforced by graphene flakes. The choice of HDPE as the polymer matrix is simply one of convenience that allows us to focus on the essential physics of matrix–filler interactions without additional complications arising from the matrix itself. To understand the importance of polymer chain packing at the filler surface in controlling the mechanical response, we also study the mechanical response of polymer-matrix composites with chemically comparable but morphologically distinct fullerene fillers. By systematically varying filler concentration, morphology, and size, as well as inter-chain and chain-filler interactions, we identify certain clear trends in composite stiffness with reinforcement. We have undertaken an extensive statistical sampling over atomic configurations and trajectories relative to the parameters noted above, which establishes clear confidence levels for the observed material response. In particular, we find that, to within statistical error, spherical fullerenes provide a nearly size-independent level of reinforcement of the matrix. In contrast, composites with graphene fillers exhibit reinforcement that depends strongly on the graphene filler size: although sub-nanometer graphene flakes are found to be ineffective fillers, flake sizes in the 2–4 nm range lead to appreciable reinforcement, which scales linearly with flake size, with graphene flakes outperforming with increasing size all the fullerenes examined. We elucidate the fundamental mechanisms responsible for this size- and morphology-dependent response, thereby providing insights into processing strategies for further control and enhancement of the mechanical properties of nanocarbon-reinforced polymers.2 and CH3 groups) as a single entity. Such united-atom models are commonly described using the Dreiding potentialEbond, Eangle, and Edihedral, arising from changes in the bond length (r), bond angle (θ), and dihedral angle (ϕ), respectively, which are given by the expressionsThe interatomic potentials for both the HDPE matrix and carbon fillers are based on a united-atom model, which treats individual hydrocarbon sub-units 212291011121314et al.et al.2-CH2 non-bonded interaction for the potential by Capaldi et al. is about 20% stronger than that for the potential by Buell et al.; since CH2 groups are the dominant component of the polymer matrix, this difference in non-bonded interaction strength results in a stiffer response of the matrix according to the former potential relative to that according to the latter as shown subsequently.where Nanocarbon fillers also are described using a Dreiding potentialTable S2 in the Supplementary Information14Table S3 in the Supplementary Informationet al. as HDPE1 and to that by Capaldi et al. as HDPE2; the corresponding parameterizations for the interactions in the composite systems will be referred to as HDPE1/C and HDPE2/C.Henceforth, for convenience, we will refer to the parameterization by Buell 3-(CH2)n-CH3  chains. The carbon fillers used in our study are “zero-dimensional”, cage-like fullerenes and two-dimensional (2D), planar graphene flakes. We considered fullerenes of three different sizes, C60, C180, and C540, and graphene flakes of five different sizes, C61, C181, C541, C1087, and C2161; the three smallest graphene flakes were chosen to closely mimic the carbon content of the three fullerenes examined, thereby facilitating a direct comparison of the role of filler morphology in the composite’s elastic response. The fullerene structures were prepared using the routines by Schwerdtfeger et al.Computational models of material samples were prepared with the aim of gathering extensive statistics and identifying clear trends in the elastic response as a function of filler size, concentration, and geometry. The polyethylene matrix was modeled using unbranched CHr0) with changes in orientation at each step being equal to the equilibrium bond angle (θ0); no two non-bonded atoms were allowed to approach within a distance of one bond length of each other in the SAW. For preparing composite system supercells, we first dispersed fillers in the supercell and thereafter inserted the polymer matrix using a SAW scheme under additional constraints that prohibited collisions between the polymer chains and fillers. Table S4 in the Supplementary InformationWe used a self-avoiding-random-walk (SAW) algorithm to generate supercell models of random polymer matrices. The SAW step size was chosen as the equilibrium C-C bond length of the polymer backbone (3) and, hence, required proper thermal equilibration and relaxation. To this end, we first carried out a conjugate-gradient energy minimization of the initial configuration with a force tolerance of 0.01 eV/Å implemented in the MD simulation software LAMMPSNPT) dynamics, implemented in LAMMPS, at 500 K and equilibrated over 100 picoseconds (with a time step of 1 femtosecond) to a pressure of 1 atm using a Nosé-Hoover thermostat and barostatNPT) dynamics, the samples were quenched by 25 K every 100 ps. Tg) as indicated by the example constructions in Tg to lie over the range 240–265 K, which contains the typical experimentally reported value of 250 KTg to the range of 260–295 K. The root mean square (RMS) displacement of the polymer united atoms computed over the (NPT) annealing schedule exceeds the radius of gyration of the polymer chains in our computational samples, which confirms proper initial equilibration of our samples prior to mechanical testingTo ensure reasonably fast convergence of the SAW algorithm, samples were produced at a low initial density (0.5 g/cmNPT) dynamics. Fixed strain increments were applied to a chosen simulation supercell vector at intervals of 10 ps to result in an overall strain rate of 109/s, while the remaining cell vectors were relaxed to attain a pressure of 1 atm. The virial stress (σij), defined asThe mechanical response of polymers is extremely sensitive to temperature. Here, we chose to carry out all of our tests at a temperature of 150 K such that all of our samples were well within the glassy regime see . The choV is the volume of the simulation cell; mp and i-th component of the velocity of particle p; i-th component of the force and the j-th component of the separation, respectively, between particles p and q; with the summations being over all particles in the simulation cell.was calculated by averaging over the last 5 ps of every applied strain increment. In Eq. (3), 2-CH2 interactions for HDPE2 are about 20% stronger than those for HDPE1, which accounts for the observed stiffer response in the former case. Specifically, at the chosen temperature of 150 K (below Tg), the calculated Young’s modulus for the neat polymer is 1.62 ± 0.02 GPa with HDPE1 parameters and 2.02 ± 0.03 GPa with HDPE2 parameters. These values are typical of Young’s moduli of glassy amorphous polymersFirst, we consider the mechanical response of neat HDPE to establish the baseline properties of the matrix prior to insertion of fillers. 61 and C181), the role of the filler is, to within statistical error, inconsequential; it is not until the graphene flake size increases to C541 that we begin to see an appreciable improvement in the composite modulus with filler concentration. On the contrary, fullerenes act as effective fillers even at small sizes  at enhancing the composite’s modulus. However, the filler-size effect for fullerenes appears to be much less significant than that for graphene flakes, with results for C60, C180, and C540 spanning a rather narrow band of moduli (with appreciable overlap of error bars) as indicated by the grey shaded regions in 541 flakes begin to result in comparable modulus enhancement as that of the fullerene fillers, while C1087 and C2161 flakes outperform the largest fullerenes considered here.Next, we analyze systematically the influence of filler size, morphology, and concentration on the response of nanocarbon-reinforced HDPE. r or, equivalently, as depicted in the insets in Ngf, in the flake. This E/E0 ~ r or E/E0 ~ Ngf1/2 at given filler concentration is an important scaling relation for the elastic response of the HDPE-graphene composites. We note that this scaling relation is as yet based only on a limited set of flake sizes in the 1–4 nm range; larger-scale MD simulations with flakes in the 10–100 nm range will be reported in a future publication to verify the validity of this scaling relation over a couple of orders of magnitude of graphene filler size. In any case, the results here are very promising as they establish a clear trend for the enhancement of polymer–graphene composite stiffness, as experimentally relevant graphene flakes have sizes that are typically in the sub-micron range. On the contrary, the limited scope for modulus enhancement with increasing fullerene filler size that is evident from our results—in conjunction with the fundamental challenges associated with the synthesis of such large fullerenes—is indicative of their reduced utility as fillers compared to graphene flakes.The size-dependent stiffening of graphene-reinforced HDPE is elucidated further in The main computational findings thus far, namely, the linear scaling of composite stiffness with graphene concentration as well as the linear dependence of composite stiffness on the lateral dimensions of the filler are generally consistent with classical composite theory61 and C181) dispersed in HDPE: 1087 and C2161 flakes are dispersed in the HDPE matrix [1087 flake [540 fullerenes, whose surfaces tend to undergo faceting and deviation from strictly spherical geometry, we also observe some layering and densification of the matrix in the vicinity of the filler’s facets [60 and C180 fullerenes. Nevertheless, it is clear that a graphene flake provides a much larger planar area relative to a faceted fullerene of comparable carbon content for adsorption and subsequent densification of polymer chains. However, due to their low surface area, small graphene flakes cannot induce such a local ordering and densification of the polymer chains.While continuum theory provides a broad understanding of the composite response, there are additional microscopic aspects of the matrix–filler interface, which may become important for load transfer at the nanoscale. For example, consider the case of small graphene flakes , we see that the effect of randomness versus ordering is insignificant, to within statistical error; with larger fillers, the responses are more intelligible although there is still some overlap of error bars. For ordered graphene flakes, we see that the response for loading along the flakes , comparable to those of typical fullerenes, there is little to no improvement in the stiffness of the composite with increasing graphene filler concentration. With increasing flake size, however, we have found an appreciable improvement in the composite modulus, with the effect of using C541 flakes being comparable to those of the fullerenes and with larger flakes  outperforming the fullerenes as fillers. This composite stiffness enhancement likely arises from more efficient load-transfer via interfacial shear (shear-lag) as well as ordering and densification of the polymer chains at the larger planar surfaces provided by larger graphene flakes. Within the limited statistics of graphene flake sizes considered in this study, the increase in composite modulus scales linearly with the flake size (circular flake radius). Given that typical solution-processed graphene flakes are much largerIn summary, we have conducted an extensive molecular-dynamics parametric study of the elastic properties of fullerene- and graphene-reinforced HDPE composites. By systematically varying the concentration, morphology, and size of fillers, as well as the non-bonded interaction strength expressed through different interatomic potentials, we have identified the contrasting roles of spherical fullerene and layered graphene fillers. CStudies of HDPE composites with larger graphene fillers are underway with the aim of establishing scaling laws for modulus enhancement over a couple of orders of magnitude of flake size as well as elucidating the chain dynamics and organization at the graphene–PE interface. Studies of nonlinear deformation and failure of fullerene- and graphene-reinforced HDPE composites also are underway and will be reported in future publications. Finally, in the present study, we have focused on the simplest model for matrix–filler interactions governed by weak dispersive forces. To the extent that strong covalent bonding between the filler and matrix is not significant, we expect that the qualitative behavior of polymer densification at the graphene–polymer interface as well as the quantitative power-law scaling relationship for composite stiffness should hold for other modes of filler–matrix interactions including, for example, hydrogen bonding between graphene oxide flakes and the polymer matrix. The use of functionalized graphene fillers could of course result in other modes of chemical bonding with the polymer matrix, and the effects of such stronger bonding of the composite’s constituents on its mechanical properties will be explored elsewhere.How to cite this article: Lu, C.-T. et al. A Comparison of the Elastic Properties of Graphene- and Fullerene-Reinforced Polymer Composites: The Role of Filler Morphology and Size. Sci. Rep.6, 31735; doi: 10.1038/srep31735 (2016).


CD274/PD-L1 gene copy number status was assessed by fluorescence in situ hybridization analysis. PD-L1 expression was detected in 36/80 (45%) cases and concordance of PD-L1 expression in primary tumor and corresponding nodal metastasis was present in only 20/28 (72%) cases. PD-1 expression was found in tumor-infiltrating lymphocytes (TILs) but not in tumor cells. CD274/PD-L1 gene amplification was detected in 19% of cases, with high level PD-L1 amplification present in 12/80 (15%), and low level amplification in 3/80 (4%). Interestingly, CD274/PD-L1 gene amplification was associated with positive PD-L1 immunostaining in only 73% of cases. PD-L1 copy number status was concordant in primary tumor and associated metastases. Clinically, PD-L1 tumor immunopositivity was associated with a higher risk for nodal metastasis at diagnosis, overall tumor related death und recurrence. Based on our findings we propose to include PD-L1 copy number status in addition to protein status in screening programs for future clinical trials with immunotherapeutic strategies targeting the PD-1/PD-L1 axis.Immunomodulatory therapies, targeting the immune checkpoint receptor-ligand complex PD-1/PD-L1 have shown promising results in early phase clinical trials in solid malignancies, including carcinomas of the head and neck. In this context, PD-L1 protein expression has been proposed as a potentially valuable predictive marker. In the present study, expression of PD-L1 and PD-1 was evaluated by immunohistochemistry in 80 patients with predominantly HPV-negative oral squamous cell carcinomas and associated nodal metastasis. In addition,  Squamous cell carcinomas comprise over 95% of malignant tumors in the head and neck region, including the oral cavity. The disease is associated with a poor prognosis, with an overall 5-year survival rate of 60%. Two-thirds of the tumors present with locally advanced or metastatic disease (stages III and IV). Historically, oral cavity squamous cell carcinoma (OCSCC) was viewed as a tobacco- and alcohol-related cancer, but infection with human papillomaviruses (HPVs) has emerged as an important risk factor for OCSCCs (reviewed in ) TreatmeCD274/PD-L1 gene, located on chromosome 9p24.1, in close proximity to PDCD1LG2 (programmed cell death ligand 2). PD-L1 can be upregulated in tumor cells and in a number of different tissue types in response to INF-γ and other inflammatory mediators  [24 [2431]  28] wit wit28] wUp to date, there is limited data concerning changes of PD-L1 expression in tumors during metastatic progression and none of the studies up to date have evaluated SCCs of the head and neck. Results of our own study show that in only 72% of cases, the PD-L1-status was retained in nodal metastatic lesions. In two studies comparing matched tumor samples with corresponding metastatic lymph nodes involving patients with RCC and triple-negative breast cancer, respectively, the concordance rates were considerably higher , 35. TisTolerance of PD-L1 positive tumors by the immune system is mediated by interaction of PD-L1 on tumor cells with its receptor PD-1, preferentially expressed on Tregs and other immune cells. Upon activation of PD-1/PD-L1, the anti-tumor response of the immune system is attenuated as T-cell functions are suppressed by the mechanisms named above. In HNSCC, co-receptor signals on T-cells mediated by PD-1 can inhibit antitumor response in a pre-clinical model , 36, 37.In our OCSCC patient cohort, PD-L1 positive carcinomas had a significantly higher risk for nodal metastasis at the time of diagnosis . Moreover, the risk of disease-and overall tumor-related death was significantly higher in PD-L1 positive OCSCC. In comparison, results of previous studies have been contradicting in this regard, probably reflecting differences in methodologies used to assess PD-L1 status and differences in patient characteristics, including percentage of HPV-related SCC tumor subgroups  28]. It. It28]. CD274/PD-L1, PD-L2 and JAK2 genes [Expression of PD-L1 in tumors cells is mediated by different mechanisms. It can be induced by autocrine or paracrine factors within the tumor microenvironment, especially INF-γ or HIF1α. In this case, PD-L1 expression is dynamic, with variable, time-dependent expression. On the other hand, expression can be driven by gene amplification events involving the 9p24.1 locus. The 9p24.1 chromosomal locus contains the K2 genes , 23. SelK2 genes  and has K2 genes , 23. OthK2 genes , 38-40.CD274/PD-L1 gene locus, in 19% of OCSCC cases. The amplification driven PD-L1 expression in a subgroup of OCSCC may identify a new subgroup of HNSCC with a disease-specific genetic alteration. Further studies are needed to evaluate the impact of PD-L1 amplification on pathogenesis and disease progression and on prognosis of this newly identified subgroup of HNSCC.Using FISH analysis, we found amplification of 9p24.1, including the PD-L1 amplification [CD274/PD-L1 gene copy number gain as a potential mechanism for PD-L1 overexpression in the present study may provide a rationale for treatment of HNSCC patients, especially in a subgroup of OCSCC with PD-L1 amplification. Future studies have to investigate whether PD-L1 copy number status, which is stable in the primary tumor and corresponding lymph node metastasis, might be a better predictive marker for tumor response to blockage of PD-1/PD-L1 pathway [CD274/PD-L1 gene amplification and the PD-L1 immunohistochemical status is surprisingly poor, with 4 amplified cases being PD-L1 immunonegative. The reason for this discrepancy is currently not clear, but may involve technical problems with the antibody clone used or posttranscriptional or posttranslational modifications.Blocking the PD-1/PD-L1 axis is a promising treatment option in multiple tumor types. Up to date, several clinical studies have been conducted, including especially patients with melanoma and non-small cell lung cancer (NSCLC), but also HNSCC (reviewed in ). These fication . Responsfication , 42-45. fication , 46, 47. pathway  than PD-CD274/PD-L1 amplification is a novel finding in OCSCC and is a frequent event, often  associated with high PD-L1 expression levels and stable during metastatic progression. Based on these data, we propose to include CD274/PD-L1 amplification analyses into studies recruiting patients for immune checkpoint protocols.In summary, we show that PD-L1 is expressed in 45% of OCSCC, when a 5% cut-off of positive cells is applied. PD-L1 positive cases have a significantly higher risk for nodal metastasis at diagnosis. PD-L1 expression is associated with higher risk for overall tumor-related death and recurrence. Although PD-L1 expression status in corresponding nodal metastasis is equivalent to expression in primary tumors in the majority of cases, a considerable number of cases (32%) were discordant. th edition). Complete clinicopathologic data including age, gender, localization of primary tumor, local, nodal and distant recurrences and overall and disease-free survival were available for all patients . Grading and staging was undertaken according to the current World Health Organization Classification of Tumors of Head and Neck (2004) and the AJCC tumor, node, metastasis (TNM) classification  using a Tissue Microarrayer  with a core size of 0,6 mm. A minimum of 2 and (where feasible) up to 4 tumor cores from tumor invasion front and tumor center were taken from the primary tumors in areas previously marked by two pathologists . Corresponding regional nodal metastasis from 28 patients were also included in the TMA, selecting 1 or (where feasible) 2 different areas from the metastatic deposits, depending on size of metastasis.IHC was performed on 2 μm sections from each TMA using a PD-L1 primary antibody  at a dilution of 1:100 and a PD-1 primary antibody  at a dilution of 1:50. Stainings were run on an automated immunostainer with an iVIEW DAB detection kit . Immunohistochemical expression of PD-L1 in tumor cells was assessed by counting the percentage of positive tumor cells and quantifying staining intensity in a 4-tiered grading system including “no staining” (0), “weak staining” (1+), “intermediate staining” (2+), “strong staining” (3+). PD-L1 positivity was arbitrarily defined as at least 5% tumor cells displaying membranous PD-L1 staining of any intensity. The cut-off value of 5% was chosen, because many clinical trials involving immunomodulatory therapeutics have chosen this value. Immunohistochemical expression of PD-1 in TILs was assessed by counting the percentage of PD-1 positive TILs. PD-1 positivity was counted as at least 1% positive TILs.in situ hybridization, the SPEC CD274, PDCD1LG2/CEN9 Dual Color Probe containing a mixture of fluorochrome direct labelled SPEC CD274, PDCD1LG2 probe specific for the CDC274/PD-L1 and PDCD1LG2/PD-L2 genes located at 9p24.1 and an orange fluorochrome labeled CEN 9 probe specific for the classical satellite III region of chromosome 9 (D9Z3) at 9q12  was used according to the manufacturer´s instructions. At least 20 nuclei per sample were counted. PD-L1 amplification was defined as PD-L1/CEP9 ratio ≥ 2.0, with high level amplification defined as ≥ 4.0 and low level amplification defined as ≥ 2.0 and ≤ 4.0. Polysomy 9 was defined as average PD-L1 copy number > 3 signals/cell.Dual-color FISH analysis was performed on 2μm sections from formalin-fixed paraffin-embedded tissue. Sections were deparaffinized, dehydrated in 100% ethanol and air-dried. For DNA was isolated from formalin-fixed, paraffin-embedded tissue of primary tumor samples using the QIAamp DNA FFPE Tissue Kit  in combination with the QiaCube  according to the supplier's instructions. HPV analysis and determination of HPV subtypes was performed with the HPV LCD 3.5 Array - Kit  according to the manufacturer's recommendations.2-test or Fisher's exact test, depending on the cell counts of corresponding contingency tables. Likewise, quantitative data was compared between groups using the non-parametric Mann-Whitney U test. Cumulative incidence functions were estimated for censored outcomes to account for competing risks such as death. All statistical tests were performed on exploratory two-sided 5% significance levels.Descriptive and exploratory statistical analyses were performed using SPSS 21  and R 3.2.0 . The distribution of qualitative data was compared between groups using χ


Immunotherapy has become a key strategy for cancer treatment, and two immune checkpoints, namely, programmed cell death 1 (PD-1) and its ligand (PD-L1), have recently emerged as important targets. The interaction blockade of PD-1 and PD-L1 demonstrated promising activity and antitumor efficacy in early phase clinical trials for advanced solid tumors such as non-small cell lung cancer (NSCLC). Many cell types in multiple tissues express PD-L1 as well as several tumor types, thereby suggesting that the ligand may play important roles in inhibiting immune responses throughout the body. Therefore, PD-L1 is a critical immunomodulating component within the lung microenvironment, but the correlation between PD-L1 expression and prognosis is controversial. More evidence is required to support the use of PD-L1 as a potential predictive biomarker. Clinical trials have measured PD-L1 in tumor tissues by immunohistochemistry (IHC) with different antibodies, but the assessment of PD-L1 is not yet standardized. Some commercial antibodies lack specificity and their reproducibility has not been fully evaluated. Further studies are required to clarify the optimal IHC assay as well as to predict and monitor the immune responses of the PD-1/PD-L1 pathway. Among the many molecularly defined checkpoint proteins,PD-1 is a type 1 transmembrane protein of the immunoglobulin superfamily,,PD-1 binds two B7 family ligands, namely, PD-L1 and PD-L2 (B7-DC or CD273),Cancer cells can activate PD-L1 expression through various oncogenic signaling pathways, such as phosphoinositide 3-kinase/protein kinase B (PI3K/PKB)Antibodies that target either PD-1 or PD-L1 are being developed to block ligand-receptor interaction and to improve antitumor immune response by allowing T cells to attack the tumor. To date, these antibodies have demonstrated exciting clinical responses against many cancer types.,-PD-L1 is expressed in several tumor types, such as melanoma, glioblastoma, and cancers in lung, kidney, head and neck, stomach, colon, pancreas, breast, cervix, cervical, and ovarian cancer. This protein has also been observed in hematologic malignancies, such as multiple myeloma, lymphoma, and various leukemia types,,PD-L1-positive cancers may indicate immune active tumors that could be sensitive to anti-PD-1 and/or PD-L1 therapies because of their correlation with poor prognosis in many of these malignancies, including lung adenocarcinomaPD-L1 has dynamic expression, and its evaluation by IHC is not well standardized. Previous studies have used a range of various antibodies, treatments, tumor types, and criteria to determine the positivity of samples. Therefore, a coherent definition of PD-L1 positivity must be established to facilitate further study of PD-L1 as a potential biomarker for the PD-1/PD-L1 pathway blockade. Given the intrinsic heterogeneity of PD-L1 expression in many tumors, the present results must be interpreted with caution. However, a biomarker of the response to a specific immunotherapy treatment is yet to be found.,,In contrast, although testing the biopsied tumor tissue remains a recommended method for mutation analysis, challenges associated with serial tumor biopsy, particularly in NSCLC, have spurred the search for non-invasive blood-based assays that allow the frequent assessment of biomarkers as a part of routine clinical carevs. investigator’s choice of chemotherapy  as first-line therapy for stage IV or recurrent PD-L1-positive NSCLC, and a phase I study of nivolumab in combination with gemcitabine/cisplatin, pemetrexed/cisplatin, carboplatin/paclitaxel, bevacizumab maintenance, erlotinib, ipilimumab, or as monotherapy in patients with stage IIIb/IV NSCLC.Immunotherapy has shown promising results in early NSCLC clinical trials involving PD-1 or PD-L1 antibodieset al.vs. docetaxel for patients previously treated with PD-L1 positive NSCLC (NCT01905657), and combination studies with ipilimumab or chemotherapy for NSCLC patients (NCT02039674).As another anti-PD-1 mAb, pembrolizumab received FDA approval in October 2014 and can be used for treating epidermal growth factor receptor (EGFR) mutation-negative and ALK rearrangement-negative NSCLC that has progressed on or after platinum-based chemotherapy. Approval was granted based on the results of a phase I trial by Garon BMS-936559 and MPDL3280A are anti-PD-L1 mAbs. BMS-936559 showed modest activity (ORR of 6%-17%) among patients with advanced cancers, including NSCLC, in a phase I multicenter trial (NCT00729664). Objective response  was observed in 5 of 49 evaluable NSCLC patients,,PD-L1 is up-regulated in cancer and is expressed in tumor cells in 40%-50% of NSCLCs independent of tumor histology,Several new immune-based treatments for small cell lung cancer (SCLC) are currently in clinical development. These treatments include the mAb-targeting Delta-like ligand 4 (DLL4) demcizumab (NCT01859741) and nivolumab with or without ipilimumab (a mAb antibody against CTLA-4) (NCT01928394),Several companies have developed different primary antibodies for analyzing both PD-1 and PD-L1 proteins by IHC. Some studies suggest that tumor PD-L1 expression that is detected by IHC may predict clinical responses to anti-PD-1/PD-L1 therapyet al.The similarities among the PD-L1 antibodies that are used in trials, as well as the staining localization, threshold for signal detection, and test conditions, need to be investigated to obtain a robust protocol. Gadiot et al.The phase I study of Topalian A recently published patent describes antibodies with specific sequences that bind to human PD-L1 as well as reveals the benefit of detecting PD-L1 expression in FFPE human tissue samples by IHC (WO/2014/100079)Roche/Genentech and Bristol–Myers Squibb have developed different companion assays for PD-L1 expression, each with its own experimental PD-L1 antibody. These antibodies include Spring Biosciences clone SP142 , Spring Biosciences clone SP263 , and Dako clone 28-8 . To date, the properties and concordance between these IHC antibodies have not been reported, and only clone SP142 is commercially available. The characteristics of all antibodies that are discussed in this section are listed in ,,et al.For an antibody to be considered as a favorable diagnostic tool, it must show sensitivity, specificity, reproducibility, and robustness in detecting the target by IHC. Some of the numerous available assays for detecting PD-L1 expression by IHC can only stain cancer cells, whereas the other assays can stain tumor-infiltrating immune cells. Therefore, patients with tumor cell staining and/or immune cell staining were examined in some studies, whereas the other studies only included patients with PD-L1 expression in tumor cellset al.Two patterns of cellular distribution of PD-L1, namely, membranous (cell surface) and cytoplasmic, have been described in tumor cells to indicate PD-L1 positivity. Membranous PD-L1 expression is present in tumors and inflammatory cells. PD-L1 staining pattern also differs between the assays. While some assays only evaluated membranous staining, others considered both membranous and cytoplasmic staining. PD-L1 is a type 1 transmembrane protein, and its cytoplasmic localization can represent intracellular stores of ligand that may relocate to the cell surface depending on cell stimulation. Interestingly, Brahmer ,,,The literature provides four definitions of PD-L1 sample positivity that are independent of the sample location and the staining of cells, i.e., whether ≥1, ≥5, ≥10, or ≥50 of cells per area are stained positive for PD-L1Immunotherapy for lung cancer is a new and exciting therapeutic modality. Multiple mAb candidates that target the PD-1/PD-L1 immune checkpoint have demonstrated reliable responses in tumors, including lung cancer, whereas some mAb candidates have shown remarkable antitumor effects in different clinical trials. Unfortunately, the lack of a reliable biomarker of response obscures such a scenario. Data on the correlation between PD-L1 positivity and patient responses to the different PD-1/PD-L1 blocking agents are also conflicting. PD-L1 is up-regulated in many cells and cancer types and contributes to the malignancy of these cancers by interacting with PD-1 and inhibiting T cell activation, thereby limiting the detection and destruction of tumor cells by the immune system. This ligand may play important roles in the inhibition of immune responses in both lymphoid and non-lymphoid organs. If PD-L1 is up-regulated in a tumor without an appropriate immune infiltrate, the blockade may have no effect because the tumor lacks the effector cells that fight the cancer.Several immunohistochemical antibodies have been developed for detecting PD-L1 expression in FFPE tissue. Characterizing tumors and immune cells via PD-L1 protein expression by IHC may help identify those patients who can benefit from the mAb candidate anti-PD-1 and anti-PD-L1 agents. As a result, the ability of PD-L1 protein expression to become a favorable predictive marker of response has been measured in various ways in several clinical trials. The conflicting results of PD-L1 staining may be attributed to the small sample sizes that are tested in different assays and/or to the variability of the used antibodies. No precise cut-off has also been established for determining PD-L1 positivity by IHC. The limitations in the specificity and reproducibility of some antibodies may explain the contradictory relationships between assays.Confirming PD-L1 as a predictive biomarker presents a promising new therapeutic opportunity to administer those agents that prevent PD-1/PD-L1 pathway interaction in advanced or metastatic lung cancers and other tumors. Further studies must be conducted to clarify the optimal IHC assay, validate and standardize the definition of PD-L1 positivity, and explore the relationship among various expression levels of the PD-L1 protein, as well as the effect of such levels on the prognosis of lung cancer patients with PD-1/PD-L1-directed therapies.


The cases were divided into two groups: those without pTDP-43-positive neuronal cytoplasmic inclusions in the hippocampal dentate granule cells , and those with such inclusions . Furthermore, the Type 2 cases were divided into two subgroups based on semi-quantitative estimation of pTDP-43-positive dystrophic neurites (DNs) in the temporal neocortex: Type 2a  and Type 2b . Clinico-pathologic analysis revealed that cognitive impairment was a feature in patients with Type 2a and Type 2b, but not in those with Type 1, and that importantly, Type 2b is a distinct subtype characterized by a poor prognosis despite the less severe loss of lower motor neurons, the unusual subcortical dendrospinal pTDP-43 pathology, and more prominent glial involvement in cortical pTDP-43 pathology than other two groups. Considering the patient survival time and severity of motor neuron loss in each group, transition from Type 1 to Type 2, or from Type 2a to Type 2b during the disease course appeared unlikely. Therefore, each of these three groups was regarded as an independent subtype.Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are types of major TDP-43 (43-kDa TAR DNA-binding protein) proteinopathy. Cortical TDP-43 pathology has been analyzed in detail in cases of FTLD-TDP, but is still unclear in cases of ALS. We attempted to clarify the cortical and subcortical TDP-43 pathology in Japanese cases of sporadic ALS (The online version of this article (doi:10.1186/s40478-016-0335-2) contains supplementary material, which is available to authorized users. Amyotrophic lateral sclerosis (ALS), an adult-onset, fatal neurodegenerative disorder, is the most common type of motor neuron disease (MND). Most cases 90-95 %) appear to occur randomly without a family history (sporadic ALS) , 2. The  % appearSince the identification of a nuclear protein, 43-kDa TAR DNA-binding protein , as the major component of the ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive, tau-and α-synuclein-negative inclusions (FTLD-U) and ALS , 12, manTypes A, B, C and D) of FTLD-TDP pathology [Type D being associated with mutations of the VCP  gene [Type B, being characterized by moderate numbers of NCIs and very few DNs throughout all cortical layers. However, one of the above studies demonstrated that 7 of 10 cases that were sporadic and exhibited MND in addition to dementia had cortical ubiquitin pathology characterized by a mixture of numerous NCIs and frequent small DNs [Type A of the new classification system mentioned above [Cases of FTLD-U were originally divided into three types by two independent groups using their own classification systems based on the morphology and anatomical distribution of cortical ubiquitin neuronal lesions, including NCIs and DNs , 22. Aftathology , the newin) gene , 25. In ed above ; this fiUsing a phosphorylation-independent anti-TDP-43 antibody, we have previously demonstrated that immunopositive NCIs and glial cytoplasmic inclusions (GCIs) can occur in many brain regions in ALS, and that cases can be classified into two types – type 1 and type 2–based on the distribution pattern of NCIs in the CNS and hierarchical cluster analysis of the pattern . Type 2 In the present study, we attempted to reevaluate the cortical and subcortical TDP-43 pathology in cases of sporadic ALS using the above monoclonal antibody, which never recognizes endogenous non-phosphorylated TDP-43 in nuclei, thus allowing unambiguous identification of pathologic structures. The results obtained eventually allowed us to classify the examined cases into three pathologic groups, whose clinical, pathologic and biochemical features were then analyzed.The present study was conducted within the framework of a project, “Neuropathologic and Molecular-Genetic Investigation of CNS Degenerative Diseases”, approved by the Institutional Review Board of Niigata University. Informed consent was obtained from the patients’ families prior to genetic analyses.We retrieved all cases of pathologically confirmed ALS from our institutional autopsy files covering the period between 1975 and 2013, reviewed the medical records and identified 128 cases of clinically sporadic ALS without any family histories of similar neurological disorders. All of the patients were of Japanese ancestry, and their clinical information was obtained retrospectively by reviewing their medical records.Among these 128 cases, the tissue samples were of poor quality due to complications of infarction, etc. and/or sampling in 26 cases, pathologic features indicative of complications arising from other major neurodegenerative diseases affecting the cerebral cortex and basal ganglia were evident in 4 cases , and no TDP-43-positive inclusions were detected in the CNS, including the lower motor neurons, in 2 cases. Accordingly, a total of 32 cases were excluded, leaving 96 cases  for analysis. Seven cases were found to have only a few Lewy bodies, with α-synuclein-positive NCIs and DNs confined to the brainstem. These cases were considered to be incidental Parkinson’s disease and were included in the present study. All of the studied cases showed loss of upper and lower motor neurons as well as ubiquitin-positive skein-like inclusions in the remaining lower motor neurons, and Bunina bodies were evident in the remaining lower motor neurons in 91 of the 96 cases.Multiple formalin-fixed, paraffin-embedded CNS tissue blocks for all cases were available for the present study. For the motor cortex, frontal cortex , temporal cortex (including the hippocampus), basal ganglia, hypoglossal nucleus, and cervical and lumbar anterior horns, 4-μm-thick sections stained with hematoxylin-eosin (H-E) were used for semi-quantitative analysis employing a 4-point scale  of neuronal cell loss , frontal and motor cortices and basal ganglia for immunohistochemical studies. The sections were autoclaved at 120 °C in 10 mM citrate buffer, pH 6.0, for 10 min, and then immunostained with a mouse monoclonal antibody against phosphorylated TDP-43  . Selected sections were also immunostained with a rabbit polyclonal phosphorylation-independent anti-TDP-43 antibody . Immunolabeling was detected using the peroxidase-polymer-based method using a Histofine Simple Stain MAX-PO kit  with diaminobenzidine (DAB) as the chromogen. To estimate the neuropathological staging of changes associated with Alzheimer’s disease, we performed Gallyas-Braak silver impregnation, and immunohistochemistry using mouse monoclonal antibodies against hyperphosphorylated tau protein  and β-amyloid . Then, we evaluated the Braak stages of neurofibrillary tangles and amyloid deposits , 27, andn = 63) and the other with such inclusions   and Type 2 accompanied by abundant DNs   Fig. . We then11) Fig. .Fig. 1PrA double-labeling immunofluorescence study was performed to assess the anatomical localization of pTDP-43 deposits forming granular and dot-like DNs. Sections of the temporal lobe and basal ganglia, including the neostriatum and globus pallidus from three representative cases of Type 2b were examined using rabbit polyclonal anti-pTDP-43 (phospho Ser409/410)  and mouse monoclonal anti-neurofilament H (non-phosphorylated) , as well as rabbit polyclonal anti-pTDP-43 (phospho Ser409/410) and mouse monoclonal anti-synaptophysin . The second antibodies used were Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555 goat anti-mouse IgG . The sections were treated with an Autofluorescence Eliminator Reagent , mounted under glass coverslips using VectaShield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) nuclear stain , and analyzed using a Carl Zeiss confocal laser scanning microscope (LSM700).n = 22), Type 2a (n = 12) and Type 2b (n = 7) among cases logged after 1990. In the Type 1 cases, a significant number of pTDP-43-positive NCIs and GCIs were seen in the motor cortex. Therefore, from the Type 2a and Type 2b cases, we selected cases in which a larger number of pTDP-43-positive NCIs and GCIs were evident in the motor cortex than in the temporal or frontal cortex.The numbers of neurons and glial cells possessing cytoplasmic pTDP-43-positive inclusions (NCIs and GCIs) were assessed in the motor cortex using a double-labeling method with in situ hybridization (ISH) and immunohistochemistry, and compared between the three groups. For this study, we selected representative cases of Type 1 (neurofilament 3 (hNF: 150-kDa medium) (GenBank accession number: BC002421) was used. As a result, the sections from 11 cases were found to be inadequate for ISH, leaving 30 cases  logged between 1990 and 2012 for the subsequent immunohistochemical study.ISH was performed in these three groups using newly prepared paraffin-embedded 10-μm-thick sections from the motor cortex, as described previously , with mi2, covering the cortical layers II-VI. The numbers of hNF-positive cells with nuclei, hNF- and pTDP-43-positive cells with nuclei, and hNF-negative and pTDP-43-positive cells with nuclei were counted manually.The hybridized sections were immunostained using mouse monoclonal anti-pTDP-43 , and then counterstained with Nuclear Fast Red solution . In each case, 10 sequential images of the motor cortex were taken through a × 20 objective lens using a single ISH-labeled and immunostained section. The total area taken from a section from each case was 1.5 mmhttp://www.r-project.org/). Differences were considered statistically significant at P <0.05.To compare clinical and pathologic features between the three groups, we used Kruskal-Wallis test with post-hoc Steel-Dwass test for non-parametric analysis of independent samples, multiple regression analysis to determine whether independent variables can predict the value of the dependent variable, Kaplan-Meier plots and a log-rank test to compare survival distributions, and Fisher’s exact test with Bonferroni-corrected multiple comparisons or Ryan’s multiple comparison test for comparison of categorical data. These statistical analyses were performed using GraphPad Prism version 5.0 , SPSS Statistics version 12.0  and R version 3.1.2 (n = 4) cases and from the temporal cortex of Type 2a (n = 4) and Type 2b (n = 4) cases, as well as from the frontal cortex of FTLD-TDP Type A, B and C (two each), as described previously [Fractionation of frozen brain tissues and TDP-43 immunoblotting were performed on selected cases. Protein lysates were generated from the motor cortex of Type 1 . We amplified all the exons of the TARDBP (NM_007375.3) gene using a series of primers, followed by sequence reaction [C9ORF72 (NM_018325.2), repeat-primed PCR was performed on an ABI 3130xl genetic analyzer  using Peak Scanner software v1.0 (Applied Biosystems), as described previously [The presence or absence of reaction . For scrBP NM_0075.3 gene eviously .P = 0.024), and that the survival time from disease onset was shorter for Type 2b than for Type 1 (P <0.0001) and Type 2a (P = 0.003). Multiple regression analysis using age at onset and ALS subtype as independent covariates and the survival time from disease onset as a dependent variable, a significant correlation was found between ALS subtype and the survival time from disease onset (P = 0.039), whereas the correlation for age at onset was not statistically significant (P = 0.106). Comparison of initial symptoms between the three groups showed that limb weakness was more frequent whereas bulbar and other symptoms were significantly less frequent in Type 1 than that in Type 2a (P <0.0001) and Type 2b (P <0.01). Cognitive impairment was present in 15 (16 %) of the 96 patients, the incidence being similar to that (10-15 %) of overt cognitive impairment meeting the criteria for frontotemporal dementia reported previously in patients with ALS [P <0.001) and Type 2b (P <0.00001). There were no evident differences in the age at death between cases with (n = 15) and those without (n = 81) cognitive impairment .The demographics and clinical features of the studied patients are summarized in Table t P = 0.0, whereasP <0.001 and P = 0.001, respectively) or in Type 2a . No neuronal loss in the basal ganglia was evident in any of the cases. None of the present cases showed hippocampal sclerosis characterized by neuronal loss and gliosis in the hippocampal formation, which is found often in FTLD-TDP but rarely in ALS [The pathologic features of the studied cases are summarized in Table y in ALS , 41. They in ALS , 93 and P <0.001) or in Type 2b . In around half of the cases in both the Type 2a and Type 2b groups, pTDP-43-positive NCIs showed a temporal cortex-predominant distribution pattern, in comparison with the motor cortex. No pTDP-43-positive neuronal intranuclear inclusions were evident in any of the cases examined.In each group, a proportion of cases showed FTLD manifested by frontotemporal atrophy and frontotemporal cortical neuronal loss with gliosis; the rate of occurrence of FTLD was only 6 % in Type 1, being much lower than in Type 2a  and Type 2a (P <0.0001), respectively, than in Type 2b. These inclusions were also detectable with the phosphorylation-independent anti-TDP-43 antibody  Table . The ratody Fig. .Fig. 2DeSMI-32 and synaptophysin are known to be dendritic and presynaptic markers, respectively. In the temporal cortex and globus pallidus, pTDP-43-positive granular and dot-like structures were observed along the SMI-32-positive dendrites Fig. . In the hNF (mRNA) and immunohistochemistry using an antibody for pTDP-43 (protein). Brain cells positive for hNF were recognized as neurons   for Type 1, 573.7 (516.7-619.7) for Type 2a, and 575.4 (533.3-650.3) for Type 2b, P = 0.316. The density of GCI-bearing glial cells was higher in Type 2b than that in Type 1 (P = 0.027) and Type 2a (P = 0.018)    Fig.  i, altho18) Fig.  ii. The 04) Fig.  iii, alt14) Fig.  i.Type B, and different from those in FTLD-TDP Type A, showing no evident differences between the three ALS groups.The data obtained by immunoblot analysis of sarkosyl-insoluble fractions are shown in Fig. TARDBP) gene in the 83 cases examined, there were no mutations in the coding regions except for one case with a known synonymous mutation in exon 6 [C9ORF72 repeat expansion was observed.With regard to the sequencing of the TDP-43  [Type A in the classification system mentioned above . Furthermore, immunoblot analysis of sarkosyl-insoluble pTDP-43 revealed that the band patterns of Type 2b and Type 1 were indistinguishable and differed from those of FTLD-TDP Type A  [Comparison of the ubiquitin- or TDP-43-related cortical pathology of FTLD described by many investigators suggested that Type 2a apparently corresponded to . (2011) , 40, 42,e A Fig. . By contis (PLS) . TherefoFrom a clinico-pathologic viewpoint, cognitive impairment was much less frequent in patients with Type 1 as we suggested previously ; in otheIn the present study, clinical information was limited to that obtained retrospectively from the medical records of the patients for whom details of some clinical symptoms were unavailable. This was one of the limitations of the present study. It has been shown that classification of patients with ALS based on clinical assessment provides information for better understanding the wide spectrum of this motor and cognitive disorder . MoreoveThe results of double-labeling immunofluorescence using anti-pTDP-43 antibody and dendritic SMI-32) or presynaptic (synaptophysin) markers in the temporal cortex and basal ganglia of patients with Type 2b suggested that deposition of pTDP-43 might occur in neuronal dendritic spines  repeat expansion in a non-coding region of C9ORF72 is the major genetic cause of FTD and ALS (c9FTD/ALS) in the Caucasian population [None of the patients in the present study had mutations in pulation , but extpulation , 50, 51.pulation , being mpulation .In conclusion, we have demonstrated heterogeneity of cortical pTDP-43 pathology in sporadic ALS, allowing the disease to be classified into three groups: Type 1, and Type 2a and Type 2b. Several significant clinical and pathologic correlations among these groups were evident. It was re-confirmed that dementia is not a feature of Type 1. Furthermore, the distinct subtype, Type 2b, accompanied by dendrospinal accumulation of pTDP-43, was shown to be characterized by a poor prognosis despite the less severe loss of lower motor neurons, the unusual subcortical dendritic pTDP-43 pathology, and more prominent glial involvement in cortical pTDP-43 pathology than other two groups. Further studies will be needed in order to establish the character of this subtype, Type 2b. Recognition of the heterogeneity of the anatomical and cellular localization of pTDP-43 will be important for clarifying the pathomechanisms involved in pTDP-43 aggregation and propagation in sporadic ALS, and eventually for developing future strategies of treatment and prevention.ALS, amyotrophic lateral sclerosis; DN, dystrophic neurite; FTLD, frontotemporal lobar degeneration; GCI, glial cytoplasmic inclusion; MND, motor neuron disease; NCI, neuronal cytoplasmic inclusion; pTDP-43, phosphorylated 43-kDa TAR DNA-binding protein


Monte Carlo simulations and tight-binding calculations shed light on the properties of three-dimensional nanographene, a material composed of interlinked, covalently-bonded nanoplatelet graphene units. By constructing realistic model networks of nanographene, we study its structure, mechanical stability, and optoelectronic properties. We find that the material is nanoporous with high specific surface area, in agreement with experimental reports. Its structure is characterized by randomly oriented and curved nanoplatelet units which retain a high degree of graphene order. The material exhibits good mechanical stability with a formation energy of only ∼0.3 eV/atom compared to two-dimensional graphene. It has high electrical conductivity and optical absorption, with values approaching those of graphene. Recent efforts in graphene research include the realization of three-dimensional (3D) bulk materials with graphene nanoplatelets (nanoribbons) as their building blocks –4. These2 geometries of the graphene plane, which may possibly alter the anticipated electronic conduction and optical properties of these materials.The reported up-to-date 3D graphene structures, produced by a variety of experimental techniques, such as chemical vapor deposition (CVD) , assemblThis inherent disorder, therefore, raises a number of questions about the stability and the microstructure of these porous networks, and how this disorder influences the optoelectronic properties of 3D nanographene. Some key issues to be tackled include the rigidity and robustness of the networks, their high specific surface area, whether and in what degree they preserve graphene order and bonding percolation, and if conduction and optical absorption are strongly influenced by the deformations of the nanoplatelets.Here, we report what is presumably the first attempt to simulate and study theoretically the structural, mechanical, and optoelectronic properties of realistic 3D nanographene (3D-NG) materials. We achieve this through a combination of atomistic Monte Carlo simulations, to generate the networks in a random unbiased way, and tight-binding calculations to relax them properly and extract their basic properties. The outstanding finding of our studies is that 3D-NG exhibits good mechanical stability and high electrical conductivity and optical absorption, approaching those of 2D graphene.We construct and characterize the 3D-NG networks in two steps, following a procedure we used earlier to construct carbon nanofoam networks . In the s and p atomic-like orbitals and distance- and environment-dependent parameters for transferability between different structures. The successful description of various carbon phases by this model was previously demonstrated [Γ point for the calculation of energies and optoelectronic quantities.In the second step of our procedure, the 3D-NG networks generated by the MC method are fully relaxed using tight-binding molecular dynamics (TBMD) simulations in the  canonical ensemble. We first anneal extensively the networks at 2000 K in order to bring the structures out of any local energy minima, and then we relax the volume/density at 300 K, where the structural, mechanical, and electronic properties are inferred. The calculations are carried out within the TB framework developed at the Naval Research Laboratory (NRL) . This isnstrated , 13. DueThe model also describes nicely the well-known , 15 inst2 atoms and the formation of new, both large and small, atomic rings. These have a profound effect on the electronic properties, as shown below. The density of the network is 0.5 g/cm 3, making the material mechanically very robust. This should be considered as an upper limit to the density of 3D-NG, in view of the rather small size of the computational box used here and the possibility of larger pore sizes that inevitably lower the density. Biener et al. [3 for 3D-NG prepared by pyrolysis and etching of sol-gel organic polymers. Ultra-low density (of the order of mg/cm 3) foamy structures might also be possible to achieve, but these should be less stable.We first begin with the microstructure of 3D-NG. Panel  of sp 3 sites, some of them located at the junctions between the units as linking geometries, while the rest (9 %) are sp 1 sites, found both at the junctions and decorating the internal pore surfaces. The curved nature of the nanoplatelets is reflected in the appearance of both large and small atomic rings, other than the dominant (70 %) six-membered (6 m) rings forming the ideal graphene layer. We find, for example, a large fraction of 9-m rings (9 %), 7-m rings (5 %), and 8-m rings (4 %). There are also small 3-m rings (5 %) and 5-m rings (8 %). Non-6-m rings and non-sp 2 sites put their signature on the electronic density of states to be discussed below.As expected, the main bonding hybridization in our model in Fig. 22/g, compared to 2636 m 2/g for ideal monolayer graphene. Our values are in accord with the experimental value of 3000 m 2/g reported by Biener et al. [We calculate the specific surface area (SSA) of our networks to be in the range of 3000–3200 m r et al. . Most li3) amorphous carbon (a-C) network (shown in the inset), which was generated with the same TBMD methodology. There are some striking differences between the G(r) of 3D-NG and a-C. One comes from the feature between the second and third peak, at ∼2.8 Å. This extra peak corresponds to the third-nearest-neighbor distance in the graphene hexagon, and is absent in a-C. Another notable difference is that peaks beyond remain intense in 3D-NG, while they vanish in a-C. These differences show that not only short-range order but also medium-range order correlations, and thus graphene order, are highly preserved in 3D-NG despite its distorted nature. It is evident that the sharp peaks in the G(r) of graphene are broadened into the peaks of 3D-NG at exactly the same radial distance. Overall, we may characterize 3D-NG as a continuous random network (CRN) at the microlevel, in the sense that the units percolate throughout but they are randomly oriented, while high order prevails at the nanolevel.Figure 2B0 of our networks by fitting their energy versus volume curves to the Murnaghan’s equation of state [B0 of 60 GPa. All networks studied have values of B0 in the range of 55–65 GPa despite their porous nature. This is lower than values for typical 3D carbon materials with low density, such as graphite-like a-C (B0∼200–250 GPa), but still high enough to make 3D-NG durable for potential applications. Again, our values should be an upper limit since for networks with larger pores and smaller density we expect lower moduli. Work is in progress to estimate the hardness of the material.As a stringest test of the mechanical stability of 3D-NG, we calculated the bulk modulus of state . Such a Further evidence for the stability of 3D-NG networks is provided by calculating their formation energies relative to the undulated graphene plane at 300 K. We find values ranging from 0.2 to 0.5 eV/atom. For example, the total energy of the network shown in Fig. 2εF arising from the disorder due to the ripples. The EDOS of 3D-NG, on the other hand, follows more or less the shape of the graphene EDOS, which is in accord with the experimental work by Biener et al. [εF, it is completely gapless. As these states around εF are π orbital states, and given the deformations induced by crosslinking, one wonders whether their π orbitals are properly aligned so as to keep their delocalized nature, as in graphene, and give a metallic character to the material.We now proceed to the study of the electronic structure of 3D-NG. Figure 2r et al. , but exhci are the coefficients in the expansion of the eigenstates in terms of the local orbitals, and which is a measure of the localization of the electronic states in the system. The localized states and their IPR are denoted by vertical spikes in Fig. εF region is practically free of localized states. This is in sharp contrast to a-C where the π and π∗ states near εF are localized, due to misalignment of the π orbitals, causing the low conductivity of the material [1-bonded atoms at the internal surfaces, while sp 2 and sp 3 sites contribute minimally.We investigate this issue by calculating the inverse participation ratio (IPR) defined by material . The IPRπ orbitals in the presence of deformations can be explained by noting that the large-membered rings in the network induce locally a planar geometry, causing the proper alignment of π orbitals. This is significant because many of the atoms contributing significantly at εF are sp 2-bonded atoms located at large 8- and 9-m rings. Otherwise, such atoms would contribute localized states at εF, as defect atoms do. We observed a similar phenomenon of large-ring-induced local planarity in our previous studies [The proper alignment of π states in 3D-NG are delocalized at εF is expected to have a strong effect on the conductivity. In its standard frequency-dependent form, this is given by We now proceed to the calculation of the optoelectronic properties of the material. We first examine the electrical conductivity. Our finding that the Ei and Ef are the energies of the initial occupied valence eigenstates |i〉 and the final unoccupied conduction eigenstates |f〉, respectively. The rigorous relation i〉 and |f〉 eigenstates into atomic states  |a〉 and |b〉 where )〈f|r|i〉  has beenj and all atomic orbitals in the basis set. The basis sets of the atomic orbitals |a〉 and |b〉 are in principle different. Considering the LCAO expansions in Eq.  [ω (or E) → 0.where the index |r|l〉=0) . Using tσDC of the graphene layer is ∼ 2×10−4μΩ−1cm−1. This agrees with several experimental measurements in graphene materials [−3μΩ−1cm−1. This agreement provides us with a benchmark and shows the reliability of our approach for the calculation of the transition matrix elements. For 3D-NG, our calculations predict that σDC is of the order of ∼ 5×10−5μΩ−1cm−1, which underlines that 3D nanographene retains in a large extent the conductivity of 2D graphene. For comparison, the experimental work of Worsley et al. [−6μΩ−1cm−1, while Chen et al. [−5μΩ−1cm−1, which is closer to ours. Obviously, the conductivity of covalently bonded 3D-NG will depend on the connectivity and percolation of units, the pore size, and the density.The results of our calculations of the conductivity as a function of the energy are given in Fig. 6aterials –21. For y et al.  on 3D-NGσDC of graphite-like a-C, which is highly sp 2-rich, and more than eight orders of magnitude higher than sp 3-rich tetrahedral a-C [−8μΩ−1cm−1 for the former and practically zero for the latter. As discussed above, this is due to the strong localization of π states in the a-C phase due to the misalignment of π orbitals.Note that, as shown in Fig. 6dral a-C . Our comε2(ω) is the imaginary part of the dielectric function and n is the refractive index of the material [The 3D-NG materials are also highly absorbtive, as demonstrated in Fig. 6material , revealsIn conclusion, we reported here the first simulational and theoretical study of the structural, mechanical, and optoelectronic properties of realistic 3D nanographene materials, through Monte Carlo simulations and tight-binding calculations. We found that 3D-NG is characterized by nanoporosity and high specific surface area. It is a random network at the macrolevel but with high graphene order at the nanolevel, within the nanoplatelet units. It exhibits good mechanical stability and high electrical conductivity and optical absorption, approaching those of 2D graphene. These characteristics and the calculated relevant quantities are in good agreement with experimental measurements. Actually, some of them, such as the electrical conductivity, are even higher than what is reported experimentally. This may indicate that there is room for improvement of these materials at the lab through better unit connectivity and percolation, and pore size and density control.


Graphene is composed of a single atomic layer of carbon which has excellent mechanical, electrical and optical properties. It has the potential to be widely used in the fields of physics, chemistry, information, energy and device manufacturing. In this paper, we briefly review the concept, structure, properties, preparation methods of graphene and its application in lithium ion batteries. A continuous 3D conductive network formed by graphene can effectively improve the electron and ion transportation of the electrode materials, so the addition of graphene can greatly enhance lithium ion battery’s properties and provide better chemical stability, higher electrical conductivity and higher capacity. In this review, some recent advances in the graphene-containing materials used in lithium ion batteries are summarized and future prospects are highlighted. However, these materials have some drawbacks that limit their use. For example, carbon materials have good cycle performance but low initial charge and discharge efficiency; tin-based oxides have good cycle but high irreversible capacity loss in the first cycle ), outstanding thermal conductivity (3000 W/(m · K)) 3. These materials have electronic conductivities of 10-4 S/cm  are LiCoOExisting studies show that pure graphene can’t become a direct substitute for current carbon-based commercial electrode materials in lithium ion batteries due to its low coulombic efficiency, high charge–discharge platform and poor cycle stability  and poor lithium ion diffusion (10- 14 -10- 16 cm2/S) lead to capacity fade quickly under high rate charge and discharge  nanoparticles, mixed them with graphite oxide in solution. The mixed solution was then underwent a spray-dried and sintered process to obtain the LiFePO4/graphene composite (LFP/G) shown in Figure 4, with a thickness of approximately 2 nm (about 3–5 layers of graphene) and formed a continuous lamellar structure; LiFePO4 nanoparticles had uniform size at 2–5 nm. The obtained material displays a more regular morphology and structure, which can potentially lead to a large enhancement of conductivity. In addition, this material can be further carbon coated to obtain carbon coated LiFePO4/graphene (LFP/(G + C)), which presents superior rate and cycle performances.When used as an electrode material, LiFePO4, Li3V2(PO4)3 is an attractive cathode material for LIBs, because its average extraction/reinsertion voltage is about 4.0 V, and its theoretical capacity is 197 mAh/g 3 forms a monoclinic structure and has a high operating voltage and shows a good performance at high discharge currents. However, its intrinsic low electronic conductivity (240 nS/cm at 25°C) limits its rate capacity, so graphene is added to improve its electrochemical performance. Li3V2(PO4)3/graphene cathode material has been prepared by sol–gel, solid state and spray-drying synthesis methods , high surface-to-volume ratio, ultra-thin thickness which can shorten the diffusion distance of ions, structural flexibility that paves the way for constructing flexible electrodes, thermal and chemical stability which guarantee its durability in harsh environments.Graphene has opened new possibilities in the field of lithium ion battery materials due to its light weight, high electrical conductivity, superior mechanical flexibility, and chemical stability  can cause large volume changes. This leads to the pulverization of the particles and the electrical disconnection of the electrode. In order to circumvent this, new anode materials with graphene have been examined in many recent studies. For example, the performance of anode electrode was improved when tin nanoparticles embedded in grapheme was used , the actual capacity with Co doped can only reach a maximum of 400 mAh/g. When the composite with graphene formed through a two-step liquid method, followed by hydrothermal synthesis, the delithiated ratio capacity of this compound is about 900 mAh/g at low current density (40 mA/g), closing to the theoretical capacity. When current density reaches 1600 mA/g, the specific capacity of this compound maintains 390 mAh/g .MnCuO has low band gap energy and high catalytic activity. However, as an anode material, it has a low conductive performance and large volume expansion effect. These shortcomings can be improved by forming CuO/graphene composites  and low price (Choucair et al.2O3/graphene composites by a hydrothermal method. The addition of graphene prevented Fe2O3 from aggregation and also buffered material’s volume expansion. At l160 mA/g current density, the reversible capacity of the composite remains 660 mAh/ g after 100 cycles.Fe2 with graphene is another nanocomposite that can be synthesized by a facile gas/liquid interface reaction (Cai et al.-1 at a current density of 100 mAg-1. This specific charge capacity drops to 150 mAhg-1at a high current density of 3000 mAg-1. The advantage of this nanocomposite is that the oxygen-containing groups on the graphene sheets can be reduced after the heat treatment and residual functional groups including the OH and COOH on the surface of graphene sheets can strongly interact with the metal ions during the synthesis process .The random hybridization of TiO2 nanoparticles and ultrathin graphene sheets form a three-dimensional porous structure of the TiO2-graphene nanocomposite. The small nanoparticles provide a short mean-free-path for electron and lithium ion to travel during the lithium ion insertion/extraction process, resulting in excellent rate capability.TiO4-graphene, Tin-based, Si-based and transition metal based electrode materials with graphene have been extensively studied in this paper. The composite materials’ advantages can be summarized as following. Firstly, graphene’s flexibility makes it an ideal material to buffer metal electrode’s volume expansion and contraction during the charge–discharge process. This improves the electrode material’s cycle life performance. Further, the excellent electrical properties of graphene can enhance the conductivity of metal electrode material. Moreover, the addition of graphene can control the growth of metal oxide particles. Smaller particles means the diffusion distance of lithium ions and electrons is reduced, this improves the material’s rate performance. Finally, the lithium storage capacity for most metal oxide composite materials with graphene has improved greatly.Compared to traditional carbon electrode materials, graphene, due to its low initial coulombic efficiency, high charge and discharge platform and other shortcomings, cannot directly replace traditional graphite as an anode material. However, graphene does demonstrate high electrical conductivity, good mechanical strength, excellent flexibility, great chemical stability and high specific surface area. This is especially noticeable when graphene is chemically converted with a greater proportion of functional groups, proving that it is suited for use as a base composite electrode material. When used as electrode material, graphene can effectively reduce the size of the active material, prevent agglomeration of nanoparticles, improve electrons and ions transmission capacity, as well as enhancing the electrode’s mechanical stability. As a result, graphene-containing electrode materials have high capacity and good rate performance. Lithium metal oxide-graphene, LiMPOIn addition, various approaches are taken to prepare graphene. These vary from simple mechanical mixing to the well controlled in situ reactions and interfacial reactions, which result in better graphene morphology and structure. Developing easily replicable methods for producing graphene is the key to future application of graphene in lithium ion batteries.


The size-controllable and ordered Au nanostructures were achieved by applying the self-assembled monolayer of polystyrene microspheres. Few-layer graphene was transferred directly on top of Au nanostructures, and the coupling between graphene and the localized surface plasmons (LSPs) of Au was investigated. We found that the LSP resonance spectra of ordered Au exhibited a redshift of ~20 nm and broadening simultaneously by the presence of graphene. Meanwhile, the surface-enhanced Raman spectroscopy (SERS) of graphene was distinctly observed; both the graphene G and 2D peaks increased induced by local electric fields of plasmonic Au nanostructures, and the enhancement factor of graphene increased with the particle size, which can be ascribed to the plasmonic coupling between the ordered Au LSPs and graphene. Graphene is the first two-dimensional carbon atomic crystal which is constructed by several layers of honeycomb-arrayed carbon atoms. This promising material is highly attractive for the fabrication of high-frequency nanoelectronic and optoelectronic devices due to its exceptional optical and electrical properties, such as extreme mechanical strength, ultrahigh electrical carrier mobility, and very high light transmittance –3. Unfor2O3 spacer layer inserted between the graphene and nanoparticles Im(α). The calculated results of Qabs for a single 30-nm Au nanosphere with and without graphene coating show that the absorption peak of the Au nanosphere exhibits a slight redshift (about 20 nm) after graphene coating, which is in good agreement with the experimental data. The discrepancy of the absorption results between calculation and experimental results can be ascribed to the simplified assumptions during the calculation such as the semi-infinite substrate, the dipole approximation, the homogeneous particle size, and the crystal perfection of the Au nanosphere [Theoretical calculation based on the dipole approximation (results not shown here) has been conducted to understand the redshift of Au LSPs after graphene coverage. In the calculation, the Au nanosphere is utilized as a representative of the Au nanostructures and is placed above a transparent substrate. The thickness of the substrate is assumed to be semi-infinite, and absorption of the substrate is completely omitted. The dielectric constant of graphene is based on an assumption that the optical response of every graphene layer is given by optical sheet conductivity , 9, and itten as 1\documenectively . The absnosphere , 10.−1), G peak (1590 cm−1), and 2D peak (2690 cm−1). The G peak originates from the first-order Raman scattering process, while the 2D peak is due to a double-resonance intervalley Raman scattering process [2/Si substrate. It is apparently revealed that the intensities of graphene G and 2D peaks are significantly enhanced with increasing size of the ordered Au nanostructures from 100 to 140 nm. With increasing size of the ordered Au nanostructures to 140 nm, the Raman peak intensities of graphene on top of Au display an enhancement factor of ~3-fold for the G peak and 2.5 for the 2D peak. Therefore, the SERS of graphene caused by the ordered Au nanostructures was distinctly observed, and the underlying mechanism will be discussed in detail later.To shed light on the effects of plasmonic ordered Au nanostructures on the Raman properties of graphene, the Stokes Raman spectra of pristine graphene and graphene transferred on differently shaped and sized Au nanostructures were measured and the corresponding results are shown in Fig.  process , 18. TheISERS is the increase in Raman intensity with respect to its original intensity I0, σ is the relative cross-sectional area of the nanoparticle, Q(ω) is the plasmonic enhancement from the Mie theory with ωs representing the Stokes Raman frequency, α is the particle radius, and h represents the separation between the particle center and the surface of the graphene sheet. From Eq. Q(ω) of the nanoparticle. The shapes of the particles vary with different initial thicknesses of the Au film, although majority of Au nanostructures can be treated as spheres or triangle prisms. Experiments of the absorption spectra have shown that the plasmonic resonance positions for the ordered Au nanostructures with various size ranges from 100 to 140 nm lie around the excitation laser wavelength  in Eq. ΔISERS will be distinctly increased which can be simply estimated from Eq. Generally, both the LSP and charge transfer can play a role in the SERS of graphene modes, since the SERS electromagnetic enhancement results from the Raman excitation and coupling with the LSP of nanostructures, while the metal-induced charge transfer can lead to the chemical resonance enhancement . In our mentclasspt{minima−1 for graphene that was transferred on the surface of the ordered Au nanostructures while the 2D peak position almost remains constant. It has been reported that the G peak of graphene is blueshifted for both electron and hole doping, while the 2D peak is redshifted for electron doping and blueshifted for hole doping [Now we turn towards the nature of the chemical interaction between the ordered Au nanostructures and graphene, which is another physical mechanism for the SERS especially for the graphene-nanoparticle hybrid system , 19. Froe doping , 21. In In summary, the coupling between graphene and LSPs of ordered and size-controllable Au nanostructures has been investigated systematically by directly transferring graphene on the surface of the Au nanostructures. The absorption spectra of Au exhibit a redshift of ~20 nm after graphene coverage, which can be ascribed to the plasmonic coupling between the Au LSPs and graphene. On the other hand, the graphene SERS is significantly observed, and intensities of the G and 2D peaks increase with increasing size of the ordered Au nanostructures. The electromagnetic plasmonic effect rather than the charge transfer mechanism is considered to be the dominant mechanism for the SERS effect of graphene. We believe the results are beneficial not only for further understanding the coupling mechanism between graphene and the ordered metallic nanostructures, but also for developing plasmonic graphene-based optoelectronic devices.