V600E cells showed significantly higher baseline expression of PD-L1 at mRNA and protein levels compared to BRAFWT cells. MEK inhibitor treatment resulted in a decrease of PD-L1 expression across all cell lines. BRAFi treatment decreased PD-L1 expression in BRAFV600E cells, but paradoxically increased its expression in BRAFWT cells. BRAFV600E mutated patients samples had a higher level of PD-L1 mRNA compared to BRAFWT (p=0.015). Immunocompetent mice (B6129SF1/J) implanted with syngeneic 3747 BRAFV600E/WT P53−/− murine tumor cells were randomized to control, PLX4720, anti PD-L1 antibody and their combination. In this model of aggressive thyroid cancer, control tumor volume reached 782.3±174.6mm3 at two weeks. The combination dramatically reduced tumor volume to 147.3±60.8, compared to PLX4720  or PD-L1 antibody  alone. Immunohistochemistry analysis revealed intense CD8+ CTL infiltration and cytotoxicity and favorable CD8+:Treg ratio compared to each individual treatment. Our results show anti PD-L1 treatment potentiates the effect of BRAFi on tumor regression and intensifies anti tumor immune response in an immunocompetent model of ATC. Clinical trials of this therapeutic combination may be of benefit in patients with ATC.The interaction of programmed cell death-1 and its ligand is widely studied in cancer. Monoclonal antibodies blocking these molecules have had great success but little is known about them in thyroid cancer. We investigated the role of PD-L1 in thyroid cancer with respect to BRAF mutation and MAP kinase pathway activity and the effect of anti PD-L1 antibody therapy on tumor regression and intra-tumoral immune response alone or in combination with BRAF inhibitor (BRAFi). BRAF Thyroid cancer is the most common endocrine malignancy . ClinicaIn the current era of personalized medicine, strategies that exploit the mutational profile of individual tumors have come to the fore. Recent work in both preclinical studies and clinical trials has focused on the known driver mutations in thyroid cancer. Altered MAPK signaling pathway in many thyroid cancers leads to ERK phosphorylation, proliferation, and invasion hence BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) have been the subject of intense investigation. Various inhibitory molecules have been tested, including vemurafenib (PLX4032), which demonstrated a reasonable response in patients with progressive metastatic, radio-iodine refractory BRAF-positive PTC , but theAn alternative approach involving immune therapy, which previously had been rejected by most physician scientists as ineffective and cumbersome, has regained favor as a result of new knowledge about the immune checkpoint receptor, PD-1 and its ligand, PD-L1, which regulate T-cell activity in the tumor microenvironment. A role of the immune checkpoint regulators is to prevent autoimmunity and to reduce collateral tissue damage by modulating the immune response in chronic infection. Cancer cells capable of up regulating the expression of PD-L1 have the potential to become immune resistant , thereby+ T cells or NK cells in the tumor microenvironment. Indeed, human tumors show a correlation between PD-L1 levels and T cell infiltration reflecting an ongoing but unsuccessful anti-tumor immune response. PD-L1 has been investigated as a potential marker for tumor aggressiveness [ex vivo experiments. [PD-L1, also known as CD274, is a cell-surface glycoprotein of the B7 family. It is expressed on various tissues including solid tumors and can facilitate immune evasion and T cells exhaustion . The intsiveness , 16, andsiveness , 18. Theriments. –24in vivo immunocompetent murine model of anaplastic thyroid cancer. In undertaking this study, we hypothesized that PD-L1 expression in non-medullary thyroid cancer would correlate with MAP kinase signaling pathway activity, and as a result, targeted therapies that reduce MAP kinase activity, such as BRAFi and MEKi, would be found to regulate PD-L1 expression in BRAF-mutated tumors. We further proposed that blocking the interaction between PD-L1 and PD-1 with an anti-PD-L1 antibody, would have the added effect of increasing the anti-tumor activity of BRAFi-induced infiltrating T cells. In the first phase of our investigation, we studied 5 human and 4 murine thyroid cancer cell lines to determine baseline expression of PD-L1. Next, we investigated the effect of manipulating MAP kinase activity on PD-L1 expression in vitro and in vivo. Finally, we tested the effect of combining BRAFi and anti-PD-L1 antibody on tumor regression and intra-tumoral immune response in an orthotopic immunocompetent mouse model of ATC.Our study was designed to advance current understanding of the role of PD-L1 in thyroid cancer cells, thereby paving a path for future testing of PD-L1-based therapies in thyroid cancer patients. It is the first study to look at the expression profile of PD-L1 in a panel of nonmedullary thyroid cancer cells at baseline, after IFN-γ stimulation, and after treatment with MAP kinase inhibitors. It also represents the first attempt to determine the impact of PD-L1 antibodies, alone or in combination with BRAF inhibitor, on tumor volume in an V600E melanoma cell lines  and one BRAFWT (MelJuso) . In fact, the normal HTORi cell line had the lowest expression of PD-L1. Western blot analysis pointed toward higher PD-L1 protein expression in the mutant BRAF cells compared with wild type across all cell lines investigated.Given the known therapeutic implications of PD-L1 expression in melanoma, PD-L1 mRNA levels in 6 human thyroid cancer cell lines was quantified against 3 BRAFV600E-mutated tumors also expressed higher levels of PD-L1, PD-L1 mRNA expression levels were analyzed in randomly selected 28 fresh frozen PTC tumors and their matched normal thyroid tissue samples. Fifty-seven percent had BRAFV600E mutations on routine sequencing. None of the demographic or tumor characteristics were significantly different between the BRAFV600E (n=16) and the BRAFWT (n=12) groups (Table V600E-mutated tumors showed significantly higher expression of PD-L1 mRNA compared to BRAFWT tumors (log2 of fold changes: median (inter quartile range): 0.51 (−0.05 to 1.04) vs. −0.70 (−2.24 to 0.28) (P = 0.015) , whereas in the SW1736, only decreases in PD-L1 protein expression were observed. In BRAFWT cells treated with PLX4720 in the presence of IFN-γ, consistent increases were only observed in the protein levels of PD-L1. Significant increases in PD-L1 mRNA were observed only in the HTh-7 (ATC) cell line  at baseline and in response to PLX720, IFN-γ, or both. Although IFN-γ caused a dramatic increase of PD-L1 expression; 3601R (93.7-fold), 3868 (13.9- fold), 3743 (74.8-fold), 3403 (20.2-fold) as compared to untreated control, none of the four cell lines showed significant (>2-fold) change in PD-L1 expression in the presence of BRAFi Figure , regardl5 genetically engineered murine ATC; TBP 3743 BRAFV600E/WT P53−/− cells are implanted into an immunocompetent mouse thyroid, resulting in a very rapidly enlarging tumor with marked infiltration of CD8+ T lymphocytes when treated with PLX4720 [3. Treatment with anti-PD-L1 Ab alone did not significantly reduce tumor volume , whereas PLX4720 treatment resulted in significant tumor volume reduction . Using the Wald chi-square for statistical analysis showed that the combination of PLX4720 and anti-PD-L1 antibody acted synergistically, and was the most effective in reducing tumor volume compared to all other treatment groups  and impressively over 66% better than those treated with PLX4720 alone (P=0.023)  Figure .+ cell exhaustion; and Granzyme B, a marker of T-cell cytotoxicity. Tumors from animals treated with PD-L1 blockade or PLX4720 alone had visibly higher infiltration of CD8+ T cells compared to controls; in the tumors treated with the BRAF inhibitor most of the T cells appeared to have infiltrated into the peripheral aspects of the tumor. Combining both drugs, however, dramatically increased CD8+ T-cell infiltration throughout the tumor  have been shown to selectively interfere with the release of cytolytic granules by cytotoxic T lymphocytes (CTLs) in a reversible and TGFβ-dependent manner, thereby attenuating CTL-mediated cytotoxicity. Previous studies have shown that the CD8:Treg ratio is an important indicator of therapeutic benefit in melanomas, where potent synergistic effects of combination therapy using BRAFi and PD1 blockade cause increased CD8d humans . Indeed,l Figure .The baseline expression of mouse PD-L1 (mPD-L1), as seen in the IHC staining of the control group, was observed to increase dramatically upon treatment with PLX4720 alone; while treatment with anti-PD-L1 antibody alone or in combination with BRAFi reduced the IHC staining expression of mPD-L1 as compared with control and BRAFi alone, respectively.V600E, in an effort to alter thyroid cancer progression. Unfortunately, the response to these efforts to inhibit the MAP kinase pathway has been relatively short-lived as a consequence of the development of resistance to treatment. Coincident with this work, immune therapy, has regained favor as a result of new knowledge about the immune checkpoint receptor, PD-1 and its ligand, PD-L1, which regulate T-cell activity in the tumor microenvironment. In particular, studies involving the use of monoclonal antibodies directed against the PD-1 receptor and its ligand PD-L1 have shown promising response rates, leading to FDA approval of PD-1 drugs for advanced melanoma, lung and renal cancer [During the last decade, most thyroid cancer therapeutics have targeted known oncogenic mutations, such as BRAFl cancer , 32. OneIn melanoma patients, the initiation of BRAFi therapy causes rapid T-cell infiltration into tumor, but the T cells are rendered ineffective after two weeks, probably resulting from a surge of expression of PD-L1 on the tumor cells . This fiThis approach might also be the logical next step in thyroid cancer, given the deregulation of the MAP kinase pathway in many aggressive thyroid cancers, but little is known about the baseline characteristics of these immune regulators in thyroid malignancy. Furthermore, as clinical trials in other malignancies mature, it is becoming increasingly clear that toxicity is a major barrier to treatment. Additional preclinical data providing mechanistic insight into the possible beneficial synergistic and toxic effects of these combinations in the various tumor components is essential.+, CD4+, FoxP3+ lymphocytes and tumor associated macrophages [+ T lymphocytes to be enriched in metastatic lymph nodes especially in recurrent disease and were markedly associated with extra nodal invasion [V600E and high level of PD-L1 in 33 PTC samples was recently reported [V600E mutations, and 3 had high expression of PD-L1, there was one patient with both [The presence of PD-L1 in thyroid tumors was first described in 2003 by Brown et al. . Subsequrophages , 20. Freinvasion . Interesinvasion . Given treported . Both BRith both .V600E -mutated tumors have higher levels of PD-L1 compared with either BRAFWT tumors or matched normal tissue. Additionally, we have validated the combination of PD-L1 blockade and BRAF-targeted therapy in a novel immunocompetent model of ATC using rapidly growing genetically engineered BRAFV600E-mutated tumors. Our model is clinically relevant on multiple fronts. First, the genetically engineered cell lines have both BRAF and p53 mutations allowing for treatment to be initiated when the tumors are large and still rapidly growing. Second, the immune competent nature of our model permits us to study the immune milieu inside the tumor. Our studies did show that PD-L1 blockade has very minimal effects on the tumor when given alone, despite fairly impressive infiltration of CD8+ T cells and high activity of Granzyme B. This finding is not entirely unexpected, however, given the aggressiveness of this specific ATC model in which more conventional agents such as BRAF or Src inhibitors were less effective [In this study, we have established that thyroid cancer cell lines and tumor samples from patients with BRAFffective In contrast, the combination of PD-L1 antibody and BRAF inhibitor produced a powerful synergistic response. The synergistic improvement in tumor shrinkage in those animals treated with a combination of BRAFi and PD-L1 antibody was also associated with an increase in tumor-infiltrating lymphocytes (TIL) and TIL function. The mechanisms underlying TIL infiltration were not studied here. It could be the result of clonal expansion or increased trafficking of T cells into the tumor. Previous studies in melanoma animal models and patient trials indicate that both mechanisms are likely at play . Clearlyin vivo studies. PD-L1 expression in lymphoid organs may suppress the initial activation of T cells or perhaps even induce Tregs. PD-L1, expressed on both tumor and non-tumor cells has the potential to suppress antitumor immunity by contributing to T-cell exhaustion.Little is known about the effects of immune checkpoint regulators in thyroid cancer. The therapeutic effect of blocking either PD-1 or PD-L1 previously has not been tested. PD-L1 is expressed by multiple cell types in tumors, thus underscoring the importance of these in vitro. Both thyroid cancer cell lines and tumor samples from patients with BRAFV600E-mutated tumors had higher levels of PD-L1 compared with either BRAFWT tumors or matched normal tissue.Treatment with MAP kinase inhibitors, such as BRAFi and MEKi, can result in complex interactions given the varied effects of these inhibitors on immune cells and tumor cells Figure . In thisV600E cells, whereas in BRAFWT tumors, BRAFi activates the RAF-MEK-ERK pathway and could enhance tumor growth in xenograft models [WT) cells were treated with PLX4720 [V600E cells with BRAFi and MEKi and we activated and blocked the same pathway in BRAFWT cells with BRAFi and MEKi, respectively. We demonstrated tight correlation between MAP kinase activity (as measured by ERK phosphorylation) and the expression of PD-L1 both in the mRNA and protein levels. IFNγ, a known and potent upregulator of PD-L1, abrogated the suppression that BRAFi imposed on PD-L1, but it was MAP kinase activity that consistently correlated with fine differences of expression as exhibited in our Western blot results.Accumulating evidence points to a relationship between MAP kinase signaling activation and the induction of PD-L1 expression , 38, 39.t models , 41. Mor PLX4720 . We bloc+ T cell infiltration in melanoma patient samples [The combination of BRAFi and immunotherapy is gaining acceptance as a rational approach to cancer treatment, and is undergoing clinical investigation in melanoma , where B samples , 43 and  samples . Atefi e samples . These i+ T cell staining for cytotoxic marker. Moreover, the blockade of PD-L1 mitigated the effect of CD8+ T cells on the number of FoxP3 T regulatory cells in the tumor microenvironment. Multiple previous studies have shown that tumor-specific CD4+FoxP3 Tregs express multiple inhibitory receptors, including PD-1 and TIM3, which likely reduce the capacity of T cells in the tumor microenvironment for proliferation and cytokine production [Our previously established mouse model served as an ideal platform to explore the effect of PD-L1 blockade in the presence of BRAFi. On the basis of the above rationale, we hypothesized that a synergistic clinical and immunologic effect would be observed by combining both approaches. Indeed, our results showed dramatic tumor shrinkage when anti-PD-L1 Ab and BRAFi were combined compared to BRAFi alone. IHC analyses demonstrated an intense and dense infiltration of CD8oduction .+ cells to over express TIM3 is not clear, but its co-expression with PD-1 on tumor specific CD8+ T cells makes it an important future candidate for combined immunotherapy [It is noteworthy that PD-L1 blockade and not BRAFi resulted in higher expression of the exhaustion marker, TIM3. This observation was not consistent with Fredrick et al., who reported increased expression of TIM3 with BRAFi treatment . Ascribiotherapy –48. NeveOur study does have a number of important limitations. Although the toxicity and tolerability profile of anti-PD-1/PD-L1 in clinical trials is promising , 49, ourAdverse events (AE) related to anti PD-1/PD-L1 antibody as a single therapy and to a greater extent when combined with anti CTLA-4 or BRAFi are well documented in patients with melanoma and lung cancer , 51. MosSince previous studies have not established whether tumors that have been growing slowly over time versus those that grow more aggressively, such as in our model, alter the native immune response to the tumor, it is not entirely clear where our model fits in the spectrum. In addition, genetically engineered tumor cell lines (such as ours) may have a smaller mutation burden compared to the equivalent native tumor. It is not entirely clear whether the immune response in the genetically engineered models is a true representation of the immune response in human tumors. Since thyroid cancers generally lie on the low end of the mutation burden, this might be less of an issue in thyroid cancer than other genetically engineered models used for preclinical studies . Future in vivo model that permits exploration of the various players of antitumor immunity in the growing tumor. More than the exciting and robust immune infiltration that occurs when BRAFi and anti-PD-L1 work synergistically; it is obvious from the IHC results of our control group that aggressive thyroid tumors potentially grow without propagating anti tumor immune response. This observation, although hardly new, is striking given the robust immune infiltration when treatment with BRAFi and combinatorial therapies are implemented. Evidence from other malignancies, along with this preclinical study, suggests promise for strategies that combine immunotherapy with targeted therapy in thyroid cancer patients. The scarce number of aggressive thyroid cancer patients treated with BRAFi and the lack of ex vivo samples from these patients is a missing piece in the puzzle. We hope that in the near future, as clinical trials with targeted therapies and immune checkpoint regulators are developed for thyroid cancer, that pre and on treatment biopsy samples will be obtained from these patients to allow detailed analysis of the immune environment inside the tumors. This will allow a better understanding of the immune landscape of those who respond versus those who do not.The fact that the study of tumor immunity has gone from a fringe effort to a viable and creative option in a wide range of tumors led us to take the first exploration in aggressive thyroid cancers. Our study is unique for its utilization of an immunocompetent 2 incubator. Cells were plated at 70-80% confluence and treated for 24 hours with either 10μM PLX4720 , 10μM PD03250901 , or 500U/ml hIFNγ for human cells and 200ng/ml mIFNγ for murine cells  either as single agents or in combination as specified in the results section. At the end of the treatment, cells were collected for RNA or protein isolation. Cell characteristics and mutational status are presented in Table Six human thyroid cancer cell lines; 8505c (Deutsche Sammlung von Mikroorganismen und Zellkulturen), BCPAP , SW1736, TPC-1 , HTh-7 and HTORi; four murine thyroid cancer cell lines; 3601R, 3868, 3743, 3403 , and fouIn preparation for these studies, a dose-response curve for hIFN-γ treatment was generated and a working concentration of 500U/ml, the minimum dose required to reach a saturated response for the 24-hour treatment, consistent with previously validated concentrations (data not shown), was selected .phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (phospho-ERK), p44/42 MAPK (ERK1/2)  and β-actin were purchased from Cell Signaling Technology.The anti-PD-L1 antibody (10F.9G2) has been described previously , had lesRNA isolation was performed using Trizol , and the cDNA was synthesized using the Superscript VILO cDNA synthesis kit (Invitrogen) according to the manufacturer's instructions. PD-L1 gene expression was measured using TaqMan Gene Expression Assays  by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with technical triplicates and repeated at least twice. Gene expression levels were normalized to GAPDH expression.At the end of treatment, cells were washed twice with PBS and lysed rapidly in RIPA lysis buffer containing protease and phosphatase inhibitor cocktails  by incubating on ice for 10 minutes. Total lysates were centrifuged at 15,000 G for 15 minutes at 4°C, and supernatants collected and quantified using Bradford protein assay kit . Western blot analysis was performed using standard procedure, and the proteins were identified using specific antibodies; total ERK (tERK), phosphorylated p42/44 ERK (pERK), β-actin and PD-L1.6 cells injected into the left thyroid gland using a 27-gauge needle. The right side of the thyroid gland was not manipulated and was used as an internal control. Mice were randomized and started treatment at two weeks for the 8505 and 4 weeks for the BCPAP-implanted groups . After 14 days of treatment, mice were euthanized and both fresh and formalin-fixed tumors were collected for RNA isolation and IHC analysis, respectively.All animal experiments were conducted at the Massachusetts General Hospital in accordance with federal, local, and institutional guidelines. To test PD-L1 expression and response to BRAFi therapy using human thyroid cancer cells: 24 ten-week-old female SCID mutant mice were injected with human thyroid cancer cells (12 with BCPAP and 12 with 8505c) as previously described . Briefly5 of the TBP  3747 BRAFV600E/WT P53−/− murine tumor cell line in 24 immunocompetent syngeneic B6129SF1/J mice as previously described [To test tumor growth and anti-tumoral immune indices in response to PD-L1 – PD1 blockade, with or without BRAFi therapy, we used the genetically engineered murine ATC cell line in an immunocompetent setting. First we implanted 10escribed . In thisescribed .4-μm sections of the formalin-fixed orthotropic thyroid tumors were stained with Hematoxylin and Eosin stain and underwent IHC analysis for CD8 (Leica Microsystems), FoxP3 (eBioscience), TIM3 (Millipore), Granzyme B (abcam), mouse PD-L1 (clone 10F.9G2) (Provided by GF), and human PD-L1 (ProSci-Iinc Poway CA) primary antibodies. The primary antibody was detected using the respective biotin-free secondary antibody.V600E and 12 BRAFWT tumors with their matched normal thyroid tissue samples were analyzed for PD-L1 mRNA expression.Using the IRB-approved MGH Endocrine Surgery Tissue Bank and the Endocrine Surgery Data Registry, we obtained clinical data, fresh frozen tumors, and matched normal tissues from randomly selected patients with PTCs >1 cm. A total of 16 BRAFStatistical analysis was carried out using Microsoft Excel  and IBM SPSS Statistics . BRAF mutant and wild type groups were compared using the Student t-test for age, Fisher's exact test for sex, tumor size (T1-2 vs. T3-4) and lymph node status, and Pearson chi-square for TNM stage. All experiments were performed in technical triplicate and repeated twice unless specified otherwise. A fold change in mRNA expression of 2 or more was considered significant. A log power of 2 for fold changes in expression was used to generate box-and-whisker plots based on the median and inter quartile range, and the nonparametric Mann-Whitney U- test was used to compare independent groups. Two-way factorial analysis of variance (ANOVA) was applied to test the effects of treatments on reduction in tumor volume with the Wald chi-square test to assess the additive vs. supra-additive (synergistic) effect of two-drug combinations. Two-tailed values of P < 0.05 were considered significant for all analysis.


Comprehensive microscopic and spectroscopic characterizations confirm the resulting hybrid material possesses a 3D crumpled few-layered graphene network structure decorated with MoS2 nanoparticles. Electrochemical characterization analysis reveals that the resulting hybrid material exhibits efficient electrocatalytic activity toward HER under acidic conditions with a low onset potential of 112 mV and a small Tafel slope of 44 mV per decade. The enhanced mechanism of electrocatalytic activity has been investigated in detail by controlling the elemental composition, electrical conductance and surface morphology of the 3D hybrid as well as Density Functional Theory (DFT) calculations. This demonstrates that the abundance of exposed active sulfur edge sites in the MoS2 and nitrogen active functional moieties in N-RGO are synergistically responsible for the catalytic activity, whilst the distinguished and coherent interface in MoS2/N-RGO facilitates the electron transfer during electrocatalysis. Our study gives insights into the physical/chemical mechanism of enhanced HER performance in MoS2/N-RGO hybrids and illustrates how to design and construct a 3D hybrid to maximize the catalytic efficiency.An efficient three-dimensional (3D) hybrid material of nitrogen-doped graphene sheets (N-RGO) supporting molybdenum disulfide (MoS Monolayer metallic MoS2 with high conductivity have been explored for HER catalyst172 chemically exfoliated via lithium intercalation from semiconducting 2H-MoS2 nanostructures grown directly on graphite was reported as a highly competitive earth-abundant catalyst due to favorable kinetics, metallic conductivity, and proliferation of active sites2 materials with conductive templates or supports to form a multidimensional structure has been recently presented as an effective approach to improve the catalytic activity2 can be implemented by incorporating MoS2 with carbon nanotube (CNTs), graphene, graphite or other mesoporous carbon to form a MoS2-based hybrid or composite20212223et al. developed an HER advanced catalyst of MoS2 nanoparticles grown on graphene via a facile solvothermal approach, the resulted highly exposed edges and excellent electrical coupling to the underlying graphene sheets endowed the hybrid catalyst with excellent HER activity233N4 nanolayers@N-graphene films by integrating porous C3N4 nanolayers with nitrogen-doped graphene sheets, displaying an unbeatable HER performance which stems from highly exposed active sites, hierarchical porous structure and 3D conductive graphene network2 nanoplatelets grown on the n-type nitrogen-doped reduced graphene oxide (N-RGO) can form p−n junctions on nanoscale and act as an outstanding catalyst in photocatalytic HERHydrogen has emerged as an effective alternative to fossil fuels because it is environment-friendly energy with water as exhaust2 nanoparticles decorated nitrogen-doped reduced graphene oxide (N-RGO), a three-dimensional (3D) hybrid structure with macro-porosity by a hydrothermal route, which can efficiently catalyze HER under acidic conditions with low overpotential and small Tafel slope. The resulting MoS2/N-RGO hybrid materials display high-performance HER catalytic activity. By controlling the elemental composition, electrical conductance and morphology of the as-prepared 3D MoS2/N-RGO hybrid, the enhanced electrocatalytic activity mechanism toward hydrogen evolution was investigated in detail.Herein, we report MoS4)2MoS4 were sonicated in N, N-dimethylformamide (DMF) to generate a highly dispersed suspension. Hydrazine and polypyrrole (PPy) were then slowly added into the suspension, followed by hydrothermal assembly at 180 °C for 12 h. Besides as a reductant, hydrazine could work as a nitrogen source as PPy during the assembly process. Regarding to PPy, it acted not only as the nitrogen source, but also was used to fabricate 3D network structure which promoted the BET specific surface and conductivity of the final product. The hydrothermal environment plays a significant role in the nitrogen doping because in the absence of hydrothermal environment, the GO could be reduced directly without the incorporation of nitrogen into the graphene network. During the hydrothermal process, (NH4)2MoS4 and GO were reduced to MoS2 nanoparticles and RGO respectively by hydrazine; and MoS2 nanoparticles were grown on the RGO accompanied by incorporation of nitrogen species into the graphene lattice4)2MoS4 is removed from the starting materials; MoS2’/N-RGO where nitrogen-doped graphene sheets decorated with half quantity of MoS2; MoS2/N’-RGO where the addition order of hydrazine and PPy is exchanged; MoS2/N-RGO’ is the sample fabricated by GO of higher oxidation degree.Firstly, graphene oxide (GO) and (NH2/N-RGO synthesized was comprehensively characterized by a number of microscopic and spectroscopic tools. Scanning electron microscopic (SEM) images revealed a 3D network structure with crumpled few-layered graphene sheets with plenty of folded edges conglutinated by PPy and decorated by numerous MoS2 nanoparticles with a lateral dimension of about 35 nm (2nanoparticle growth and created an efficient template to restrain the aggregation of particles. The information presented by the transmission electron microscopic (TEM) images was consistent with the SEM in that there were laminar structures of graphene in the catalyst and MoS2nanoparticles were evenly distributed on N-RGO sheets, suggesting successful assembly between the nanoparticles and the graphene sheets  assigned to the N-RGO can also be observed. Notably, a distinguished and coherent interface was observed in MoS2/N-RGO, which is known to facilitate the electron transfer to enhance catalytic activityThe 3D MoSut 35 nm . The lare sheets . The higinset in . The two2/N-RGO hybrid. As shown in 2/N-RGO hybrid shows the characteristic peaks of N-RGO; including a prominent graphitic C 1 s peak at 284 eV, a strong O 1 s peak at 532 eV and an N 1 s peak located at 402 eV. The peaks assigned to Mo 3d and S 2p in MoS2 were also present in the survey spectrum, which confirmed the successful assembly between the nanoparticles and the graphene sheets. The detailed information of elemental composition is presented in 2. It is worthy mentioning that the N/C atomic ratio of the 3D MoS2/N-RGO hybrid was calculated up to be 5.9%, much higher than that of N-doped graphene from previous reports31322/N-RGO hybrid could lead to a more efficient catalytic activityX-ray photoelectron spectroscopy (XPS) measurements were performed to investigate the components of the prepared 3D MoS2/N-RGO hybrid. The binding energy of C-C/C=C (284.5 eV) in the 3D MoS2/N-RGO hybrid was downshifted by ∼0.5 eV compared to the graphitized carbon peak of pristine GO (285.0 eV)2, which can improve both the current density and catalytic activity of the catalyst. The N 1 s region shown in 352H4In the high resolution XPS of C 1 s , a promi4+ 3d5/2, Mo4+ 3d3/2, MoO3 or MoO42−, Mo5+ and S 2 s, respectively, are displayed in the high resolution XPS of Mo 3d5/2 and Mo 3d3/2 peaks are close to the meta-stable 1 T phase of MoS2 (associating with better HER performance)406+ results from the oxidation of the catalyst in air to form MoO3 or MoO42−6+ results in the formation of Mo5+3/2, S 2p1/2, S22− (or S2−) ligands and the S4+ of sulfate groups, respectively404322− and/or S2− is likely to be caused by the doping of S in the graphene sheet or formation of an S-rich MoS2 structure. This facilitates the development of high HER activity at the active sites for HER on the edge of MoS2where the unsaturated S atoms are exposed to adsorb H74043As shown in 2/N-RGO hybrid were characterized using the nitrogen isotherm (2 adsorption-desorption isotherm of 3D MoS2/N-RGO hybrid showed an intermediate shape between types II and IV (in the IUPAC classification) with a small H3 hysteresis loop extending from P/P0 = 0.45 to 0.95, demonstrating the presence of mesoporous structure and slit-shaped pores2 adsorption-desorption measurement revealed the as-prepared 3D MoS2/N-RGO hybrid had a Brunauer-Emmett-Teller (BET) specific surface area of 1066.6 m2 g−1, which was significantly larger than many reported porous carbon nanomaterials4647482/N-RGO hybrid materials and formation of numerous porous structures during the sintering and elimination process in the presence of PPy. The pore-size distribution of the 3D MoS2/N-RGO hybrid calculated by the DFT method exhibited one dominant peak at 5.30 nm and another two weak peaks at ∼1.56 and 3.79 nm, respectively , (103) and (110) of MoS2 could be observed in MoS2/N-RGO, indicating the MoS2 nanoparticles have been successfully grown on the N-RGO surface. The disappearance of the typical (002) peak of MoS2located at 14.1° resulted from the small size of the MoS2 nanoparticles preferring to attach to the surface of the silicon supports5051The porosity structures of the 3D MoSisotherm . The N2 Inset of , suggest2/N-RGO hybrid was further confirmed by Raman spectra analysis. As shown in −1 associated with the D and G bands of graphene can be observed in GO, N-RGO and MoS2/N-RGO. The reduction of GO induces the recovery of the graphene structure, which leads to the increase of 2D bands in N-RGO and MoS2/N-RGO. Notably, compared to the GO with a G band at 1599 cm−1, the G band of N-RGO (1586 cm−1) and MoS2/N-RGO (1593 cm−1) showed a downshift due to the incorporation of N heteroatoms2 located at 375.5 and 403.5 cm−1  were observed in the Raman spectrum of MoS2/N-RGO ranging from 300 to 450 cm−1  suggested that the interaction between the Mo precursors and graphene could efficiently avoid the aggregation of MoS2The successful fabrication of the 3D MoS450 cm−1 53. In co2/N-RGO hybrid toward the HER was investigated in 0.5 M H2SO4 solution with commercial Pt catalyst (10 wt % Pt on carbon black) as the reference. N-RGO, MoS2’/N-RGO, MoS2/N-RGO, MoS2/N’-RGO and MoS2/N-RGO’ were also prepared to investigate in detail the influence of composition and structure to the HER catalytic activity. XPS , MoS2/N’-RGO (155 mV) and MoS2/N-RGO’ (200 mV) showed more negative onset potential, indicating lower HER activity. The current intensity of the 3D MoS2/N-RGO hybrid was higher than the other catalysts along the whole potential region. To further investigate the HER activity, the linear portions of the Tafel plots were fitted to the Tafel equation (η = a + b log |j|), where j is the current density and b is the Tafel slope. In 2/N-RGO hybrid showed a Tafel slope of 44 mV per decade, which outperformed MoS2’/N-RGO of 114 mV per decade, MoS2/N’-RGO of 71 mV per decade and MoS2/N-RGO’ of 57 mV per decade. The slight higher Tafel slope compared to MoS2 nanoparticle on graphene with a value of 41 mV2 nanosheets on graphite with a value of 43 mV2/N-RGO hybrid. Electrochemical impedance spectroscopy (EIS) was measured to evaluate the HER catalytic activity of these catalysts. The MoS2/N-RGO displayed the lowest faradaic impedance among these prepared catalysts , which was larger than MoS2’/N-RGO and MoS2/N’-RGO, which lead to better reactant accessibility and superior efficiency of build of the triple-phase boundaries (gas–electrode–electrolyte)582 582  and MoS2ted MoS2  displaye2 and nitrogen impurities in RGO boost the activity of the catalyst. The relative content (RC) of S22− and/or apical S2−related to active HER catalytic activity, where RC is mean that sulfur elemental compositions (wt %)  calculations showed that graphite N shows the smallest Gibbs free-energy (|∆GH*|) value of 0.76 eV, followed by pyrrole N of 1.10 eV and pyridinic N of 1.73 eV, indicating graphite N was the best electrocatalytic activity from the viewpoint of thermodynamics and pyrrolic N also possessed the enhanced electrocatalytic activity than pyridinic N toward HER   multiplys (wt %) , in MoS22’/N-RGO , MoS2/N-2/N-RGO’ , while t2/N’-RGO  was not ydrazine . The MoS2/N-RGO’ , it was ward HER . Additioward HER , larger ward HER , which l2/N-RGO hybrid were characterized by comprehensively microscopic and spectroscopic methods including SEM, TEM, HRTEM, BET, XRD, XPS and Raman spectroscopy. Electrochemical characterization data including low onset potential, small Tafel slope, low charge transfer resistance and high electrochemical stability revealed the high performance of the MoS2/N-RGO hybrid toward HER catalytic activity compared to analogous composites. Detailed experimental analysis implies that the abundant active S22− and/or S2− ligand species and pyrrolic nitrogen and graphene N of the 3D MoS2/N-RGO hybrid and its good conductance were all beneficial to its enhanced HER catalytic activity. The exploration of the enhanced HER mechanism of the MoS2/N-RGO hybrid provides guidelines to design and construct 3D hybrids to maximize their catalytic efficiency.In summary, we have developed an efficient hydrothermal route to successfully synthesize a 3D hybrid material of nitrogen-doped graphene oxide sheets-supported molybdenum disulfide nanoparticles with high capability of hydrogen evolution. The morphology and element of the as-generated 3D MoS2H4·H2O, 80%) was purchased from Guangdong Guanghua Sci-Tech Co., Ltd (JHD). (NH4)2MoS4, Polypyrrole  and Pt/C (10% Pt) were obtained from Sigma-Aldrich. N, N-dimethylformamide  and KOH were purchased from Sinopharm Chemical Reagent Co., Ltd. Sulfuric acid  and ethanol (99.9%) was acquired from Beijing Chemical Works. All aqueous solutions were prepared with doubly distilled water.Graphene oxide  was obtained from XF NANO, INC and graphene supermarket, respectively. Hydrazine monohydrate (N4)2MoS4 and 10 mg of GO was dispersed in 10 ml of DMF by 10 min of sonication at room temperature. Then 0.1 ml of N2H4•H2O was added to the above solution followed by 30 min of sonication. 100 mg PPy was then added, followed by sonication for 10 min to form a stable complex solution. The mixture solution was transferred to a 40 mL Teflon-lined autoclave and heated in an oven at 180 °C for 12 h with no intentional control of ramping or cooling rate. The product was collected by centrifugation at 10000 rpm for 5 min and washed with DI water several times to remove most of the DMF. Subsequently, the product was redispersed in 3 ml of DI water and freeze-dried overnight, followed by thermal treatment at 600 °C for 3 h in N2 gas with 400 standard cubic centimeters per minute (sccm) to remove the organic species (DMF) and improving crystallinity. The obtained product was denoted as 3D MoS2/N-RGO. Other samples were also fabricated in the same way for comparison: N-RGO where (NH4)2MoS4 is removed from the starting materials; MoS2’/N-RGO where Nitrogen-doped graphene sheets decorated with half quantity of MoS2; MoS2/N’-RGO where the addition order of hydrazine and PPy is exchanged; MoS2/N-RGO’ is the sample fabricated by GO of higher oxidation degree.22 mg of (NHDLD instrument equipped with an Al Kα X-ray source. Raman spectrum of powder samples were recorded on an InVia-Reflex Raman microscope with a laser excitation wavelength of 532 nm.X-ray diffraction (XRD) was performed with a Rigaku X-ray diffractometer with Cu KR target. The porosity was measured with a nitrogen adsorption-desorption isotherm using a surface area analyzer . The structure of products was observed under a field-emission scanning electron microscope  and a transmission electron microscope . X-ray photoelectron spectroscopy (XPS) spectra were obtained using an AXIS ULTRA2). After the catalyst ink dried, 5 μl of 1 wt% Nafion solution was dropped onto the GCE and the working electrode was prepared. Liner sweep voltammetry (LSV) was conducted in nitrogen-purged 0.5 M H2SO4 at a scan rate of 3 mV s−1 at 1400 rpm. Electrochemical impedance spectroscopy (EIS) was carried out in the same configuration in nitrogen-purged 0.5 M H2SO4 from 10−2 to 106 Hz with a modulation amplitude of 5 mV. SCE was calibrated with respect to reversible hydrogen electrode (RHE). The calibration was performed in the high purity H2 saturated electrolyte with a Pt wire as the working electrode and the counter electrode. LSV were run at a scan rate of 0.1 mV s−1, and the potential at which the current crossed zero was taken to be the thermodynamic potential for the hydrogen electrode reactions. In 0.5 M H2SO4, E (RHE) = E (SCE) + 0.314 V. All the potentials reported in our manuscript are against RHE.All electrochemical studies were performed using a CHI 852C electrochemical workstation  in a standard three-electrode setup. A three-electrode configuration consisting of a saturated calomel electrode (SCE) as the reference electrode, a graphite rod as the counter electrode and a glass carbon RDE after loading the catalyst as the working electrode was employed. Typically, 0.5 mg of catalyst was dispersed in 500 μl of DI water by sonication to form a homogeneous ink. Then, 20 μl of the catalyst ink  was loaded onto a glassy carbon electrode (GCE) of 3 mm in diameter .


Resistive random access memory (ReRAM) devices have been extensively investigated resulting in significant enhancement of switching properties. However fluctuations in switching parameters are still critical weak points which cause serious failures during ‘reading’ and ‘writing’ operations of ReRAM devices. It is believed that such fluctuations may be originated by random creation and rupture of conducting filaments inside ReRAM oxides. Here, we introduce defective monolayer graphene between an oxide film and an electrode to induce confined current path distribution inside the oxide film, and thus control the creation and rupture of conducting filaments. The ReRAM device with an atomically thin interlayer of defective monolayer graphene reveals much reduced fluctuations in switching parameters compared to a conventional one. Our results demonstrate that defective monolayer graphene paves the way to reliable ReRAM devices operating under confined current path distribution. Nowadays one of the most widely used nonvolatile memories is flash memory. It has been employed to numerous mobile devices and becomes a representative product supplied by the Si based semiconductor industries. The flash memory is expected to reach limitations in operating speed, power consumption, and density of memory in near future because it is a charge-storage type memory based on a Si transistor. To overcome the limitations, many researchers have tried to develop next generation nonvolatile memories (NG-NVM) with high performances, which do not rely on stored charges and Si transistors123ReRAM whose resistive change is induced by applied external electrical stress is considered as one of these NG-NVMs. In addition to the advantageous properties of oxide-based ReRAM such as simple composition, facile fabrication process, and compatibility with conventional semiconductor processes, this resistance-change memory has presented promising nonvolatile memory effects including fast operation speed, low power consumption, and high scalability5678Resistive switching can be classified into two categories: Uni-polar and bi-polar resistive switching caused by conducting filament formation in bulk and oxygen migration at interface, respectively. Especially, uni-polar resistive switching, which is usually observed in binary oxide, has been explained by conducting filament (CF) mechanism in switching materials. The creation and rupture of CFs presumably result from Joule heating910111213161920+ ion-assisted reaction (IAR) system, which is monitored by Raman spectroscopy24In this paper, to reduce fluctuation in switching parameters, we suggest the insertion of a highly defective graphene (d-graphene) monolayer between an oxide film and an electrode. Because localized electronic structure of graphene can be modified by ionic treatments2223242/Si substrate using chemical vapor deposition (CVD) method . In Raman spectroscopy data from honeycomb lattice of graphene, three most considerable features are the G, 2D, and D peaks, which appear around 1580 cm−1, 2700 cm−1, and 1340 cm−1, respectively. Our Raman data only shows G and 2D peaks with G/2D peak ratio of about 0.5, which are very similar with those of intrinsic MLG2427282925+ ion beam was generated by a cold hollow cathode-type ion source. Generated number of Ar+ ions is measured by Faraday cup placed at a distance of 50 cm from the ion source. Working pressure during Ar+ ion bombardment is kept at 0.1 mTorr with Ar flow rate of 5 sccm.Before we fabricate a MGIM structure, we need to confirm quality of our graphene. Raman spectroscopy analysis in + ions at kinetic energies of 240 eV, 250 eV, 260 eV, and 270 eV, respectively, with number of bombarded Ar+ ions about 5 × 1014 per unit area (cm2) and time (second). The 240 eV of applied kinetic energie is minimum value of stably controllable area in our experimental system. Relative defect concentrations in the pristine and defective graphenes can be compared using Raman spectroscopy data, as shown in + ion bombardment induces the breaking of carbon-carbon bonds in graphene honeycomb structure31242633+ ions. To identify the location of defects, we obtained Raman scanning microscopy images of D/G peak ratio in 5 μm × 5 μm scanning areas of two d-graphenes irradiated with Ar+ ions at kinetic energies of 240 eV and 270 eV, as shown in the upper and lower panels of + ions at kinetic energies of 240 eV and 270 eV, respectively. The observed bright spots designate the positions of conducting paths where higher local current (~50 pA) passes through d-graphenes than that (~1 pA) of the other regions. + ion irradiation with lower kinetic energy induces less conducting paths on d-graphene than that with higher kinetic energy. As Jafri et al. mentioned, the higher concentration of conducting paths on d-graphene with more defects can be attributed to the defect induced mid-gap states, which create a region exhibiting metallic behavior around the vacancy defects on grapheneWith attempt to provide confined current path distribution, we introduce defects on graphene by bombarding it with Ar2/Si substrate using dc reactive sputtering method with the same condition of our previous works 3637383940414243To fabricate a MGIM structure, d-graphene introduced in forming), measured current abruptly increases and the structure reaches a low resistance state (LRS). During the following voltage sweep with step voltage of 0.05 V from 0 V, switching from LRS to HRS and switching from HRS to LRS occur at Vreset and at Vset, respectively. Compliance current of 1 mA is considered as a minimum value which can allow forming and reproducible set process in our NiO capacitor systems. The MGIM structure initially shows higher Vset value and lower current level of HRS than those of the MIM structure owing to the additional resistance from the inserted d-graphene interlayer. + ions at kinetic energies of 240 eV (MGIM240), 250 eV (MGIM250), 260 eV (MGIM260), and 270 eV (MGIM270) as well as a MIM structure. The cumulative probability for each structure was obtained after over 200 switching cycles. The distributions of the switching voltages, Vset and Vreset, are important parameters indicating memory device performances. According to the cumulative probability data, we can confirm that the switching voltage distributions of MGIM structures are narrower than that of a conventional MIM structure. Especially, MGIM240 shows much narrower distributions of Vset  of 0.32 V) and Vreset  than those of Vset  and Vreset  in the MIM structure. Due to such wider distribution, Vset of the MIM occasionally becomes higher than that of the MGIM. MGIM240 also shows the narrowest distributions in the cumulative probability plots of resistance values in HRS and LRS states  and after (less-residue MGIM240) transfer of the MLGs on NiO films. Less-residue MGIM240 shows narrower distribution of Vset  than that for MGIM240 due to the removal of residues on the MLG during Ar+ ion bombardment although distribution of Vreset  for less-residue MGIM240 is comparable to that for MGIM240. Each resistance state for less-residue MGIM240 is stable similarly to that for MGIM240, as shown in In previous reports, graphene devices have shown suppressed performances than those predicted by theory owing to residues, ripples, vacancies, etc4546+ ions bombarded on MLG/NiO may induce defects in the NiO layer as well as on the MLG. According to a previous study about ReRAM treated using IAR+ bombardment. It was found that the forming voltage could be controlled by modification of surface roughness and oxygen vacancy concentration of an oxide layer, which depended on kinetic energy of the bombarded Ar+ ion. However, in our case, the existence of MLG interlayer might reduce the effect of Ar+ bombardment on the underlying NiO layer. To demonstrate the protection role of graphene, we fabricated MIM structures with defective NiO layers directly bombarded by Ar+ ions at kinetic energies of 240 eV (DMIM240), 250 eV (DMIM250), and 260 eV (DMIM260).During the modified fabrication process, Ar+ ion increases, they are comparable to those of MIM. In contrast, DMIM240 reveals larger fluctuations in switching voltages and currents than those of less-residue MGIM240 with d-graphene, as shown in + ion bombardment.+ ions at kinetic energy of 240 eV has lower conducting path concentration than that of 270 eV leading to more reduced randomness during formation and rupture of CFs in an oxide layer underneath a d-graphene. To vary the number of defects on graphene, we fabricated two MGIM240 structures in which graphenes are bombareded with different numbers of Ar+ ions: 3 × 1014/cm2 and 8 × 1014/cm2  simply modified by insertion of a defective graphene layer between a top electrode and on oxide layer. This atomically thin interlayer inserted into an MIM device can reduce randomness during formation and rupture of conducting filaments by confined current path distribution in the modified system. MGIM structures show much narrower range of V2/Si substrate. During the growth process, the substrate is heated to 650 °C within 10 min under ~10−7 torr and then treated with H2 plasma. After purging with Ar gas for a couple of minutes, C2H2 is added (C2H2:Ar = 1:40) for graphene growth at the same temperature. A 35 nm thick polycrystalline NiO thin film was prepared by dc reactive sputtering method on Pt/Ti/SiO2/Si substrate .Monolayer graphene was synthesized by inductively coupled plasma enhanced chemical vapor deposition (ICP-CVD) on a Cu/Ni/SiO2/Si was spin-coated with polymethyl methacrylate (PMMA) 950 C4 on which we attached a pressure sensitive adhesive ultraviolet tape. Peeling the tape against the Si wafer physically separated the tape/PMMA/graphene/Cu/Ni layer due to poor adhesion of the metal films to SiO2. After etching of the underlying Cu/Ni by soaking in FeCl3 and cleaning in water, the tape/PMMA/graphene layer was pressed onto the NiO/Pt/Ti/SiO2/Si. The successive removal of the tape and PMMA in ethanol and acetone, respectively, left only the graphene layer on the NiO/Pt/Ti/SiO2/Si.For the monolayer graphene transfer, graphene/Cu/Ni/SiOMost electrical properties of the MGIM device (such as resistive switching characteristics and fluctuations of the switching parameters) were measured using a Keithly 2400 sourcemeter at room temperature and atmospheric pressure, but all retention measurements were performed additionally at 85 °C and 1 mTorr. The electrical data were obtained on a single device for each kind of structure, which showed the best performance among several ten devices. The Raman spectroscopy measurements were performed at room temperature with a 532 nm diode-pumped solid state laser and a microscope setup with a laser spot diameter of 200 nm.How to cite this article: Lee, K. et al. Enhancement of resistive switching under confined current path distribution enabled by insertion of atomically thin defective monolayer graphene. Sci. Rep.5, 11279; doi: 10.1038/srep11279 (2015).


Cultured cells were characterized by immunophenotyping. Before and after administration of the agents, the cultures were observed by inverted and environmental scanning electron microscopy (ESEM). The number of live cells and the proliferation rate were monitored by MTT assay.Rituximab and adalimumab were the least toxic agents to chondrocytes, whereas adalimumab and etanercept were to osteocytes.During periods of intense active inflammation, the concentration of the preferred BAs after inhibition of inflammation needs to be emphasized when their effects on cartilage and bone tissue are considered at the cellular level if the clinical practice is to continue. Rheumatic diseases are systemic inflammatory conditions that may cause severe joint degeneration and are associated with high morbidity and mortality rates . In receDisease-modifying anti-rheumatic drugs are declared as the traditional treatment of rheumatic diseases and, even if these drugs reduce the symptoms of the disease in a long-term, they have limited effects to terminate the disease completely . Involvein vitro). Using these methods, we aim to evaluate the toxicity and effects of the BAs on the viability and proliferation of the cultured cells.In the present study, we aim to observe the effects of three TNF inhibitors, etanercept (ETA) (soluble TNF receptor fusion protein (p75-IgG fusion protein)), infliximab (INF) , and adalimumab (ADA) , and two IL-1 antagonists, abatacept (ABA) (CTLA4-IgG1 fusion protein) and rituximab (RIT)  was approved by the Local Scientific Research Ethics Committee of Gulhane Military Medical Academy (GMMA) on February 27, 2014, with the authorization number 1491–29–14/1539. To perform molecular analyses on cellular material, written informed consent was obtained from patients. Specific BAs were numbered and researchers were blinded to each specific BA added to each culture medium. All experiments were carried out at least three times.n = 6, mean age 65 years; stage 4 gonarthrosis according to the Kellgren-Lawrence protocol ). RIT allowed chondrocyte proliferation up to 36 h (p < 0.01 [p = 0.0000]). In the same period of time, the viability rate of chondrocytes in the ADA group was similar to the RIT group (p < 0.01 [p = 0.0000]). In the ETA, ABA, and INF groups, the number of live chondrocytes was lower than that of the RIT, ADA, and control groups, suggesting that ETA, ABA, and INF inhibited chondrocyte proliferation (p < 0.01 [p = 0.0000]). Proliferation analysis performed 48 h after BA application showed no living cells or proliferation in all groups, other than the control group (p < 0.01 [p = 0.0000]).At 24 h, in the RIT group, more viable chondrocyte cells were present, compared to the other groups (p < 0.01 [p = 0.0000]). From the results of the proliferation test, which was made at the 24th hour, it was clear that proliferation occurred in ADA and ETA groups at 36th hour (p < 0.01 [p = 0.0000]).In the osteocyte cultures, the number of live cells was the greatest in the ADA group, followed by the ETA group, 24 h after BA application, compared to the control and all other groups (p < 0.01 [p = 0.0000]).The viability rate of osteocytes in the ABA, INF, and RIT groups was found to be very low compared to that in the control, ADA, and ETA groups, suggesting that these BAs inhibited osteocyte proliferation (p < 0.01 [p = 0.0000]).The results of proliferation test at the 60th hour showed no proliferation in all BA groups, while proliferation in the control group was continued , INF , and ADA , and two IL-1 antagonists, ABA (CTLA4-IgG1 fusion protein) and RIT  on human primary chondrocyte and osteocyte cultures. An additional aim was to evaluate the toxicity and effects of these BAs on the viability and proliferation of the cells in question.Owing to difficulty in isolating osteocytes from the bone tissue, characterization of osteocytes and chondrocytes via immunoflow cytometry was performed before their use in the experiments. Immunophenotypic characterization of these cells is usually performed on expanded cells rather than primary cell cultures. Although no perfect marker has been defined for cells grown in culture, it is known that hematopoietic markers, such as CD34, CD14, and CD45, are not expressed. CD44, a receptor for ligands such as hyaluronan and osteopontin, is a marker of osteocytes, but there is no such a specific receptor for chondrocytes , 34. ForPharmacological alternatives of BAs used in this study, including bevacizumab, ranibizumab, aflibercept, and Ziv-aflibercept were studied on different tissues, and mild mitochondrial toxicity was reported . FurtherThe cases included in the study were those which confirm that the toxicity in osteocytes and chondrocytes was a result of BA therapy and not a patient-specific cellular response; we ensured that the patients from whom the tissue samples were provided were not exposed to methotrexate, fludarabine, cyclophosphamide, high-dose steroids, and/or an anti-pneumococcal vaccination, and that they did not have protein allergies or a history of RA.p < 0.01 [p = 0.0000]).In the present study, the data obtained at the 24 h showed that the number of live cells and the rate of chondrocyte proliferation were highest in the RIT group, and that ABA and INF were the most toxic to chondrocytes. However, at the 48 h, neither viable cells nor proliferation were observed in all groups, except the control (p < 0.01 [p = 0.0000]).At the 24 h, the highest number of viable osteocytes was in the ADA group, followed by the ETA group. But, viability rates in the ABA, INF, and RIT groups were lower. Based on the proliferation analyses at the 48 h, similar to the chondrocyte cultures, there were no viable osteocytes and proliferation in all groups, except for the control. Osteotoxicity was highest in the RIT group, followed by the INF group . Tested doses are determined based on the previous studies and systemic peak levels of patients. Our data indicate that chondrotoxicity and osteotoxicity of BAs should be taken into consideration on the treatment regimen during active inflammation periods.RIT and ADA were found to be the least toxic biologics for cartilage, whereas ADA and ETA were the least toxic for bone cells (


Researchers seeking to develop complex statistical applications for mobile devices face a common set of difficult implementation issues. In this work, we discuss general solutions to the design challenges. We demonstrate the utility of the solutions for a free mobile application designed to provide power and sample size calculations for univariate, one-way analysis of variance (ANOVA), GLIMMPSE Lite. Our design decisions provide a guide for other scientists seeking to produce statistical software for mobile platforms. An increasing number of scientists and statisticians use mobile devices, such as smart phones and tablets. Moving statistical power and sample size software to mobile devices allows designing studies anywhere, anytime, even when away from the office.Developers of mobile statistical applications face a common set of design challenges. In this paper, we list the challenges and provide general solutions. In addition, we provide detailed example solutions from our experience developing GLIMMPSE Lite. GLIMMPSE Lite calculates power and sample size for studies that use analysis of variance (ANOVA). Our design decisions should be useful for other developers of statistical software aimed at mobile platforms.We present a list of challenges we faced in designing statistical power software for a mobile platform. We encountered each one of the challenges listed below while designing our mobile power and sample size application, GLIMMPSE Lite.Power calculations for ANOVA in particular, and the general linear model in general, involve complex matrix operations such as inversions and multiplication of large matrices. In addition, sample size calculations do not have a closed form, and require numerical search algorithms. Given that the calculations are both memory and processor intensive, statistical power applications may run poorly on mobile devices with limited random access memory (RAM) or slow processors.To perform a power or sample size calculation, a user must input several pieces of information about the design. Inputs include the Type I error rate, the list of independent and dependent variables, the hypothesis of interest, and choices for means and variances. Developing an intuitive user interface to accept these inputs is a challenge. For mobile devices, developers have an additional challenge of tailoring the interface to the small screen format.Users of power and sample size software have differing levels of statistical training. Some users may need additional assistance to understand inputs to the power or sample size calculation. The limited screen size of a mobile device forces designers to present concise help pages that do not clutter the view.Using a client-server architecture is a better option than performing power and sample size calculations directly on the mobile device. In the client-server architecture, the mobile device elicits inputs from the user and sends the data to a server with greater computing resources. In turn, the server performs the actual calculation. The client-server architecture solves the problem of limited computing resources on the mobile device, but introduces a potential bottleneck in communication with the server. Application performance may be optimal over wi-fi or 4G cellular networks, but performance may degrade on slower cellular data networks such as 2G and 3G. To maintain adequate performance at slower data speeds, the communication protocol must be both lightweight and optimally utilize available bandwidth.With an increasing number of mobile device users and the potential for wide distribution from online application stores, the number of users for a popular application can increase very rapidly. Thus, the client-server architecture supporting the mobile application must scale well. This means that the application must handle an increasing number of users in a short time frame without failure.Every mobile platform has its own software development kit (SDK). Applications developed for one mobile platform are not compatible with other mobile platforms. Thus, development time increases proportionally to the number of devices supported.Due to frequent updates, developers must ensure that any application is compatible with current versions of the mobile operating system and a reasonable subset of previous versions. The problem is compounded when supporting multiple brands of mobile devices.Most mobile devices are capable of running multiple applications simultaneously. Therefore, a power and sample size application must manage shared resources such as file storage or network connections appropriately, and gracefully handle errors generated when interacting with other applications.To increase the usability of power and sample size calculations on a mobile platform, users must be able to save outputs and use them later.To facilitate reproducible research, scientists and statisticians need an accurate record of the steps involved in a power or data analysis. Therefore, users must be able to save any design, and recall it for reuse later.http://glimmpse.samplesizeshop.org), our free and web application that calculates power and sample size for study designs with normally distributed outcomes. GLIMMPSE provides researchers with a user-friendly interface for calculating power and sample for the general linear multivariate model and the general linear mixed model with and without a Gaussian covariate. A full description of GLIMMPSE appears in GLIMMPSE Lite is a smartphone application that calculates power and sample size for ANOVA study designs on two mobile platforms: iOS from Apple GLIMMPSE Lite is a prototype mobile application that implements a subset of the functionality of GLIMMPSE. Limiting GLIMMPSE Lite to ANOVA designs enabled us to solve the general design challenges of porting statistical power software to mobile platforms with minimum development time. Using the experience we gained from developing GLIMMPSE Lite, we plan to expand the functionality of the mobile application to include multilevel and longitudinal designs in future releases.The remainder of the manuscript is organized as follows. The next section presents a review of statistical power software packages available for standard web browsers, personal computers, and mobile platforms. In the Challenges section, we describe general solutions to the computing challenges listed above, and then the specific solutions we used to implement GLIMMPSE Lite. Finally, in the Conclusions section we present conclusions and discuss directions for further research in this area.The credibility of a statistical software package primarily depends on three factors: 1) if the user can clearly understand what statistical methods were chosen to perform the power and sample size calculations, 2) if the user can understand what computational technologies were used, and 3) if experiments were used to validate the power and sample size results. SPSS Statistical power software packages that are distributed as stand-alone desktop installations can cost upwards of $500, even for an academic version. These packages include SPSS Usability differs across these software packages. Specifically, POWERLIB requires knowledge of SAS/IML programming statements. UnifyPow must be installed on the user's workstation, has no graphical user interface, and was last updated in 2008. GLIMMPSE, our free, web-based application, can be accessed via a standard web browser and is easy to use with no prior hands-on training.Several other statistical power software packages that are available for free are listed in As part of our design process, we surveyed all statistical packages for mobile devices available on the App Store As outlined in the Introduction section, migrating a statistical power software package to a mobile platform raises several challenges. In this section, we describe each challenge, discuss possible solutions, and then present the specific solutions we adopted to develop GLIMMPSE Lite.n is the largest dimension of the matrices. Thus, calculating power and sample size becomes increasingly slow as the size of the experiment increases, even on a powerful desktop machine. http://samplesizeshop.org/documentation/glimmpse/validation-results/). Total CPU time to calculate power using GLIMMPSE was as much as 101 seconds per calculation for complex designs. The timing experiments were performed on an Intel i7-2600 quad core, 3.40 GHz processor with 8GB of RAM, running 64-bit Windows 7.Power and sample size calculations require computationally intensive algorithms including matrix multiplication, matrix inversion, numerical integration, and search. To give an idea of the difficulty of the problem, the complexity of matrix multiplication is By contrast, a typical fast mobile platform comes equipped with 1GB of RAM and a processor speed of 1100 MHz. The lower RAM and slower processors on a mobile device would lead to unacceptably slow calculation times, if the calculation were performed on the mobile device. There are two approaches to solving the computational speed issue. One approach is to invest strong efforts to optimize the algorithm. In the case of power calculations, this is impossible, as the inherent complexity is irreducible. The other option is to export the calculation to another more powerful machine, as is done in the client-server model. The mobile device accesses a web service to perform the power and sample size calculations. Because the web server has fewer limitations in RAM and processing power, the approach is faster. The mobile device needs only to elicit inputs to the power calculation from the user, format the request to the server, and display the results.When designing GLIMMPSE Lite, we decided to use a client-server approach based on the Java web services architecture Power Service calculates power and sample size. The Chart Service produces power curves. The File Service provides upload and save functionality. For full details on the GLIMMPSE software architecture, see http://samplesizeshop.com/documentation/glimmpse/. GLIMMPSE Lite currently interacts only with the Power Service. Thus, the ANOVA power and sample size calculations are performed on the server side, rather than overburdening the mobile device.The architecture contains three web services. The Statistical power software involves a complex set of inputs. The user must choose the Type I error rate, list the dependent and independent variables, and specify hypotheses, means, variances, relative group sizes, correlations, repeated measures, covariates, and the type of statistical test. To assist users in creating a study design, a simple and easy to understand user interface was needed. To address the challenge, we took the following approach: 1) We divided the input requests so that the user only had to understand and input one item on each screen; 2) We added logic checks to prevent the user from entering nonsensical input; and 3) We tested the resulting user interface in a focus group.We implemented GLIMMPSE Lite on two smartphone mobile platforms: iOS and Android. Our choice of these two platforms is based on their current share of the worldwide mobile phone market . Creating a usable interface required different design decisions for each platform. To illustrate the design decisions for each platform, we describe how to specify an ANOVA study design using GLIMMPSE Lite on each mobile platform.The GLIMMPSE Lite user interface first presents the “Design” screen, a top-level list of the required inputs for an ANOVA design, in a tabular format, as shown in While the GLIMMPSE Lite user interface is similar for both platforms (iOS and Android), a few design solutions are specific to the SDK available for each platform, as discussed below.To start the ANOVA study design, the user first selects whether to solve for power or sample size. To facilitate the selection, we use the “Segmented Control” widget from the iOS SDK, as shown in Each time the user selects power or sample size, the “Design” screen in The Type I error rate is the probability of rejecting a null hypothesis when in fact the null hypothesis is true. The Type I error rate is a number between 0 and 1. For the iPhone, we use the “Rotating Wheel” widget available on iOS, shown in A one-way ANOVA design compares means among two or more groups. Currently, GLIMMPSE Lite supports up to 10 groups. As shown in GLIMMPSE Lite supports power and sample size for ANOVA designs with equal or unequal group sizes. The default study design assumes equal group sizes. For unequal group sizes, GLIMMPSE Lite sliding bars allow the user to select the relative size of the groups see Fig. 7. If solving for power, the user must specify the smallest group size. Currently, GLIMMPSE Lite allows users to enter up to 4 smallest group sizes. A separate power calculation is performed for each group size. Since a user may enter any integer value for this input, we provide text boxes on both iOS and Android platforms, as shown in If solving for sample size, the user must input the desired power level for the ANOVA study design. Currently, GLIMMPSE Lite allows users to select up to four commonly used power levels on each platform, as shown in GLIMMPSE Lite assumes equal variance across all groups. The user enters a mean value for each group and a single standard deviation value. Since the user can enter any real value for means and any positive number for the standard deviation, we use text boxes on both iOS and Android platforms, as shown in The GLIMMPSE Lite user interface allows the user to select the “Relative Group Size” and “Means and Variances” screens after the “Number of Groups” input is complete. If a user taps on the cell titled “Relative Group Size” or “Means and Variances” without completing the “Number of Groups” input, the GLIMMPSE Lite interface alerts the user with an error message shown in To evaluate the GLIMMPSE Lite user interface, we conducted a focus group study with nine participants. To match our target market, the focus group included five PhD level biostatisticians, three PhD level epidemiologists, and a Master's level student in biostatistics. The goal of the focus group was to assess the usability, look and feel, and text readability of the GLIMMPSE Lite user interface. Participants used a Likert scale The look and feel of the GLIMMPSE Lite application was appealing.I could easily read the text even on the small screen.My calculation was successful.Many users of our software have limited experience with power or sample size calculations. They may not be trained in statistical concepts such as the Type I error rate or power. Adding help pages is the obvious solution. However, on a mobile platform, the amount of help is limited by the mobile device screen size.To address this challenge, we implemented a separate help page for each input screen. Separating the help pages from the input screens allows the GLIMMPSE Lite user interface to access information only when needed, while maintaining a clean design. The help page is available for all users upon request. The text was designed to be understandable for individuals with limited statistical training, while still providing adequate theoretical details for trained scientists and statisticians. Sophisticated or experienced users who need no help can proceed directly to the input screen without looking at the help pages.Users can navigate to the help pages in one of two ways. They can go directly to the help page from the first screen. They can also view the help screens from anywhere within the input sequence, without losing any study design inputs. The start screen of the GLIMMPSE Lite user interface is shown in Users who need assistance during the input sequence can always access the help pages by tapping one of three tabs displayed at the bottom of the screen as shown in Tutorial tab leads to a table showing each available help page.The Design tab summarizes the study design created so far, lists finished inputs, and indicates which screens remain to be filled in.The About Us tab includes funding information and details on our development team. GLIMMPSE Lite users can submit a query or report a bug via the “Contact Us” button provided at the bottom of the “About Us” screen  As discussed in the Introduction section, each mobile platform has its own software development kit. In other words, native applications developed for one mobile platform will not compile on other mobile platforms. Therefore, the development time for a mobile application increases proportionally to the number of supported mobile platforms.With limited resources on our development team, we chose to develop GLIMMPSE Lite only for the iOS and Android mobile platforms. http://www.samplesizeshop.org/. The Android version can be installed directly from Google Play. The iOS version is available from the App Store.The GLIMMPSE Lite application for both iOS and Android platforms is available at Mobile devices have a rapid development cycle, and mobile operating system change frequently. New operating systems may be unstable, and manufacturers may not fix problems on a regular schedule. Therefore, computer scientists must decide what subset of mobile platforms they can afford to support. The problem is further complicated by the fact different variants of the operating system may only run on specific phone hardware. Even after the target operating systems and devices are chosen, developers must test the mobile application on all combinations of the supported operating systems, mobile devices, and software development kits.We felt that supporting iOS and Android would cover the largest number of mobile users while not overburdening the development team. We have released our GLIMMPSE Lite application for iOS versions 5 and 6 for iPhone and iPod touch devices. iOS 5 is considered a stable iOS release by Apple. The iPhone 5, the latest mobile phone released by Apple, runs iOS 6. We have tested our GLIMMPSE Lite application on both iOS 5 and 6 versions via two iPhone simulators (5.0 and 6.0), four physical iPhones, and two iPod touch devices.The GLIMMPSE Lite application for the Android platform has been released for application programming interface (API) levels 9  onwards. We have tested our GLIMMPSE Lite application via Android Virtual Devices for Gingerbread with API levels 9 and 10 Resources on a mobile device, such as random access memory (RAM), file storage, and internet bandwidth, are typically shared among multiple running applications. Therefore, a statistical power application must efficiently use the shared resources and gracefully handle errors caused by interaction with other applications installed on the mobile device.The GLIMMPSE Lite product includes extensive error checking when interacting with other applications. For example, GLIMMPSE Lite allows users to save power results either by sending an email or by producing an image in the camera folder on the device. When a valid email cannot be found, GLIMMPSE Lite will alert the user with an appropriate error message, similar to that shown in Upon completion of a power or sample size calculation, GLIMMPSE Lite allows the user to save the results via the following methods:Many users will want the ability to re-create a power analysis, as well as looking at the results of the power analysis in the future. We allow both activities. In case an internet connection is available (wi-fi or 3G/4G), the power and sample size results can be emailed via the default email client registered on user's mobile device, as shown in In case the mobile device loses an internet connection, the calculated power and sample size results can still be saved locally on the mobile device via the “Save As Image” option, as shown in For credible studies, scientists and statisticians need to be able to save study designs and reproduce the power and sample size results. The GLIMMPSE software allows users to save their study design anytime during the design input process. The saved study design can be used later to reproduce the power and sample size results without following all the steps involved in the input process. The GLIMMPSE Lite user interface saves the ANOVA study design as a JSON file that can be used on the GLIMMPSE web application via the “Upload a study design” feature.This paper described the challenges that occur when porting statistical power software to a mobile platform. We suggested specific solutions for each challenge, and gave examples from the development of GLIMMPSE Lite, our mobile ANOVA power and sample size application.Our next effort in this direction is to implement GLIMMPSE Mobile. GLIMMPSE Mobile will allow users to calculate power and sample size for multilevel and longitudinal studies. Currently, GLIMMPSE Lite is available on only iOS and Android mobile platforms. To increase the user base for GLIMMPSE Mobile and make power and sample size calculations more accessible to users, we will target the development of GLIMMPSE Mobile to multiple mobile platforms. For this purpose, we will use Phonegap Chart Service can generate a power curve image in response to an HTTP GET request.While GLIMMPSE Lite does not currently provide power curves, the underlying architecture will allow us to easily implement these capabilities for GLIMMPSE Mobile in the future. If a future GLIMMPSE Mobile release requests a power curve, the existing With the release of GLIMMPSE Lite, we have provided a free, open-source, validated power and sample size tool for users seeking to design ANOVA studies. In addition to being a useful tool for scientists, we hope that the explanations of our design decisions provide a guide for other designers seeking to produce statistical software for mobile platforms.


Controlling the propagation properties of the terahertz waves in graphene holds great promise in enabling novel technologies for the convergence of electronics and photonics. A diode is a fundamental electronic device that allows the passage of current in just one direction based on the polarity of the applied voltage. With simultaneous optical and electrical excitations, we experimentally demonstrate an active diode for the terahertz waves consisting of a graphene–silicon hybrid film. The diode transmits terahertz waves when biased with a positive voltage while attenuates the wave under a low negative voltage, which can be seen as an analogue of an electronic semiconductor diode. Here, we obtain a large transmission modulation of 83% in the graphene–silicon hybrid film, which exhibits tremendous potential for applications in designing broadband terahertz modulators and switchable terahertz plasmonic and metamaterial devices. Graphene has demonstrated the ability to modulate terahertz (THz) waves by optical or electrical excitation, but modulation depths have been low. Here, Li et al. demonstrate enhanced modulation and polarity-dependent THz attenuation using external voltage bias and photoexcitation on a graphene–silicon film. Over the past few years, graphene, as a two-dimensional version of graphite, has attracted a lot of attention from physicists, engineers and material scientists. Graphene also holds great promise for photonic and optoelectronic device applications1245678In the terahertz regime, graphene is also one of the most prosperous materials in modulating the propagation properties of the terahertz wave. One way to realize such modulation is to apply external voltage on the graphene–insulator–semiconductor structures, so that the Fermi level in graphene could be tuned accordingly and thus the optical conductivity due to the intraband transition process or free carrier absorption101112131411121617In this study, we investigate the impact of dual excitation behaviour of a graphene–silicon hybrid film where we achieve enhanced modulation of the terahertz waves at low voltage bias, that is, a few volts and low fluence photoexcitation. We discover that the transmission modulation of the terahertz waves could be achieved under different polarities of the voltage bias and simultaneous photoexcitation with a continuous wave (CW) green laser (532 nm). Moreover, on photoexcitation, the graphene–silicon hybrid film transmits the terahertz waves when biased with a positive voltage, whereas it blocks the wave when biased with a negative voltage, resembling the characteristic of a typical semiconductor-based electronic diode that allows passage of current only for positive bias and blocks the current when negatively biased. Thus, the proposed graphene–silicon hybrid film could function as ‘a diode' for the terahertz waves, which we would address from here on as graphene–silicon terahertz diode (GSTD).N-type, resistivity ρ>8,000 Ω cm) in sequence, by using wet etching method to remove the copper and poly-(methyl methacrylate), respectively2022242. The double-layer graphene was chosen due to enabled higher modulation depth and improved efficiency in terms of diode performance when compared with the monolayer counterpart  led to a negligible change in the transmission, whereas the increase in the photoexcitation power (with fixed positive bias) caused a visible change, corresponding to a modulation depth of <12%. The modulation depth is defined as: P=140 mW, modulation depth of up to 51% was achieved when the bias was changed from 0 to −3 V. With further increase in the illumination power P to 420 mW, the modulation depth was increased to a maximum value of 83% when −4 V bias was applied. The terahertz transmission reached a saturation point at a negative bias voltage of approximately −3 V for the three different levels of photoexcitation. Besides, the dynamic electrical behaviour of the GSTD device was characterized. By applying an AC rectangular voltage at various frequencies, the modulation speed was observed to be around 1 kHz  on the transmitted terahertz waves1213In such a graphene–silicon hybrid film, we attribute the observed ‘diode' characteristics to the variation in carrier density in the graphene layer due to the hybrid graphene–silicon interface. As a control experiment, we also prepared another sample in which the double-layer graphene was transferred onto a silicon substrate with a 300-nm-thick top SiOTo acquire further understanding of the ‘diode' characteristics, we now discuss the carrier properties of the graphene layer under optical illumination and electrical biasing. Based on the terahertz time-domain spectroscopy measurements, the frequency-dependent amplitude transmission was obtained by N=2 is the number of graphene layers, Z0 is the vacuum impedance, ns is the effective refractive index of the substrate (see Methods) and (ω) is the complex sheet conductivity of graphene, which can be approximately described by the Drude model930where, Γ being the carrier-scattering rate and D being the Drude weight, and D can be further expressed aswith VF (=1.1 × 106 m s−1) is the Fermi velocity, e is the electron charge, ℏ is the reduced Planck constant and n is the carrier concentration.where, D and Γ. The solid black curves in D and Γ are shown in D increased, whereas the scattering rate Γ decreased with an increase in the negative bias voltage. According to D=18 × 103 × G0 cm−1 and Г=22 cm−1, respectively, where G0=2e2/h is the quantum of conductance. By applying n1=3.07 × 1013 cm−2. In contrast, the carrier density at 280 mW, −3 V is estimated out to be n2=6.90 × 1013 cm−2, which is more than twice of n1. The enhanced carrier density implied that a slight increase in the negative bias voltage (0 to −3 V) would inject a significant number of carriers into the graphene layer. It is also found that a clear enhancement in Drude weight appears with the reinforcement of the photoexcitation power under the same negative voltage due to an increase in the number of carriers excited in the silicon substrate. Whereas for the carrier-scattering rate, it indicates an inverse variation trend compared with the Drude weight, which is very interesting, as the larger carrier density normally reveals a higher scattering rate due to stronger carrier–carrier interactions. This behaviour could have been a result of the trap-filling effect due to impurities in the graphene layer, which inhibits the carrier recombinationD and Γ when there is no applied bias voltage. This implies that the carrier diffusion from the photoexcited silicon only played a minor role in the carrier accumulation in graphene. The level of the fitted Drude weights and carrier-scattering rates also agrees well with the recently reported results930EF in graphene extracted from By fitting the amplitude transmission based on N-type semiconductor, the graphene would act as a ‘P-type material' in this configuration. When the GSTD sample was photoexcited by the green laser, a photo-conductive silicon layer was generated beneath the graphene layer where the electron density was enhanced due to carrier generation. However, the amount of photoexcited carriers generated in the graphene layer was much smaller due to small optical absorptionTo further elucidate the mechanism, we could interpret the behaviour of the GSTD sample as that of an electronic ‘PN' junction. As the silicon substrate used here is an In the above analysis, we attribute the transmission modulation to the conductivity change in graphene; however, the conductivity of the photo-conductive silicon layer was also modified under the external bias, which could also contribute to the modulation of terahertz transmission at the same time. To validate the calculated results, transmission spectra of the GSTD were simulated using CST Microwave Studio, where the photo-conductive silicon layer was also taken into account (see Methods). It could be seen from the simulation results shown in In summary, we have experimentally demonstrated a graphene–silicon hybrid film that behaves as an efficient ‘diode' for the terahertz waves under simultaneous CW photoexcitation and DC bias voltage. Compared with the existing electronically controlled devices, the GSTD achieves a large modulation depth of up to 83% at a small negative gate bias voltage of −4 V. Our work also highlights that graphene is compatible with silicon-based electronics and photonics. The active tuning behaviour of the graphene–silicon hybrid film would enable promising applications in terahertz technology, such as communications, plasmonic engineering and graphene-based metamaterials.ns=3.45 when we fit the Drude weight D and scattering rate Г using The thickness (∼1 μm) of the photo-conductive silicon layer, which was generated below the graphene under photoexcitation, was determined by the penetration depth of the 532-nm laser in silicon−1, respectively, based on experimental measurements (ɛr+iɛi (as shown in ɛr=−σi/(ωdɛ0) and ɛi=σr/(ωdɛ0)14σr+iσi was extracted from Finite-element frequency-domain method was carried out to simulate the transmission spectra using CST Microwave Studio. In the simulation, conductivity variation of the photo-conductive silicon layer under various photoexcitation power was taken into consideration. The conductivity of the photo-conductive silicon layer under 140, 280 and 420 mW photoexcitation was set to be 600, 1,200 and 1,800 S murements . When biQ.L., Z.T., J.H. and W.Z. proposed the active graphene–silicon hybrid metamaterial. Q.L. and X.Z. completed numerical simulations. Q. L. and J.G. fabricated the graphene–silicon hybrid diode sample. Q.L. and Z.T. performed all the measurements. Q.L., X.Z., L.D. and Z.T. discussed the comparisons between simulations and experiments. Q.L., X.Z., L.D., Z.T., J.G. and R.S. analysed the measured data. J.H., R.S. and W.Z. supervised the theory and the measurements. All the authors discussed the results and contributed to the writing of the manuscript.How to cite this article: Li, Q. et al. A graphene–silicon hybrid diode for terahertz waves. Nat. Commun. 6:7082 doi: 10.1038/ncomms8082 (2015).Supplementary Figures 1-4 and Supplementary References


The levels of soluble intercellular adhesion molecule-1 (sICAM-1) have been reported to increase in patients with idiopathic pulmonary fibrosis. However, the utility of sICAM-1 has not been reported in detail. The aim of this study was to investigate whether sICAM-1 was a useful biomarker for stable idiopathic pulmonary fibrosis (IPF) and early phase of acute exacerbation of IPF. The patients who were diagnosed with IPF between 2013 and 2015 were enrolled. The levels of sICAM-1 and other interstitial pneumonia markers were measured. In this study, 30 patients with stable IPF and 11 patients with acute exacerbation of IPF were collected. Mean sICAM-1 levels were 434 ± 139 ng/mL for the stable phase of IPF, 645 ± 247 ng/mL for early phase of acute exacerbation of IPF, 534 ± 223 ng/mL for connective tissue disease-associated interstitial pneumonia, 221 ± 42 for chronic obstructive pulmonary disease, and 150 ± 32 ng/mL in healthy volunteers. For the stable phase of IPF, sICAM-1 levels correlated with Krebs von den Lungen-6 (KL-6) . Mean sICAM-1 levels were significantly higher in patients with early phase of acute exacerbation of IPF than with stable phase of IPF (p = 0.0199). Multiple logistic analyses indicated that the predictors for early phase of acute exacerbation of IPF were only sICAM-1 and C-reactive protein . In patients with stable IPF, sICAM-1 levels correlated with KL-6; sICAM-1 might be a predictive indicator for prognosis. In the early phase of acute exacerbation of IPF, sICAM-1 might be more useful for diagnosis than other interstitial pneumonia markers. Intercellular adhesion molecule-1 (ICAM-1) is a glycoprotein with a molecular weight of 80–110 kD belonging to the immunoglobulin superfamily. Stimulation of inflammatory cytokines such as interferon and interleukin (IL)-1 leads to expression of ICAM-1 in vascular endothelial cells, tracheal epithelial cells, and fibroblasts within a few hours , SP-D (reference range 0–110 ng/mL), CRP (reference range 0–0.5 mg/dL), LDH (reference range 119–229 UI/L), and WBCs (reference range 3100–8800/μL) were measured. Similarly, sICAM-1 was measured in healthy volunteers and patients with CTD-associated IP and COPD.The diagnosis of acute exacerbation of IPF was based on the Japanese guidelines. Hence, after excluding other diseases such as obvious infections or heart failure, acute exacerbation of IPF was diagnosed based on the presence of all of the following: Increased severity of dyspnea, high-resolution CT findings showed honeycomb lung and new ground-glass appearances and infiltrates, and a decrease in partial pressure of oxygen in arterial blood of 10 mmHg or greater. On the day when acute exacerbation of IPF was diagnosed, blood sICAM-1, KL-6, SP-D, et al. were measured. Patients for which blood was collected on the first day of acute exacerbation of IPF were defined as “AEx IPF”. Almost AEx IPF cases underwent repeat measurements of sICAM-1 levels in the stable phase before or after acute exacerbation of IPF. All the blood samples in this study were centrifuged after clot formation. The serum samples were stored at −20 °C prior to analysis. Serum levels of ICAM-1 were measured by enzyme-linked immunosorbent assay . This study was approved by the institutional review board of the Saitama Red Cross Hospital. Written consent was received from all patients.Lung tissue samples were taken during surgery or autopsy performed at Saitama Red Cross Hospital in the past. Non-cancerous lung tissue samples were selected (two cases in each group) from patients of lung cancer with stable IPF, CTD-associated IP, and COPD receiving lung surgery. Autopsy lung tissue samples from AEx IPF cases (two cases) were used. AEx IPF and COPD lung tissue was taken from patients different from those in which serum sICAM-1 levels were measured. Resected tissue was embedded in paraffin after immersion fixation for 4 h in 10 % formalin. Lung tissue was cut into 3 μm-thick slices. The use of the tissue samples was approved by the ethical review board of our hospital.Lung tissue underwent immunohistochemical staining using a fully automated immunohistochemical stainer . For antigen activation, specimens underwent heat processing (95–100 °C) for 60 min in ethylenediaminetetraacetic acid buffer solution (pH 8.5) and we used rabbit polyclonal antibody against intracellular adhesion molecule-1  as the primary antibody. For the horseradish peroxidase reaction chromogenic substrate, 3,3′-diaminobenzidine and compared nuclear staining with hematoxylin were used.Lung tissue also underwent manual immunofluorescence staining. Paraffin-embedded lung tissue was deparaffinized, hydrated and autoclaved in citrate acid buffer . Bovine serum albumin (3 %) was used to block non-specific hydrophobic binding for 30 min and we used rabbit polyclonal antibody against ICAM-1 (1:400 dilution) as the primary antibody for 1 h. Secondary antibody with Alexa Flour 488-conjugated anti-rabbit IgG  and counterstain with DAPI were used.The unpaired t test was used for comparisons between two groups. To investigate the correlation between sICAM-1 and other interstitial pneumonia markers, Spearman’s rank correlation coefficient was used. Items suspected to be related to early acute exacerbation  were evaluated using univariate and multivariate logistic regression analyses. The analysis was performed with the Excel Statistics software .For the 30 patients with stable IPF, mean percentage forced expiratory vital capacity (%FVC) was 78.4 % and percentage diffusing capacity of the lungs for carbon monoxide (%DLco) was 74.9 %, indicating many patients with mild IPF Table . There wAmong the 11 patients with AEx IPF, mean sICAM-1 levels were 645 ± 247. Mean sICAM-1 levels were significantly higher in cases with AEx IPF than with stable IPF (p = 0.0199) Fig. . CompariICAM-1 in lung tissue was investigated using immunohistochemical and immunofluorescence staining. In healthy tissue, ICAM-1 was rarely expressed; however, in patients with stable IPF, ICAM-1 was observed, particularly, in the epithelial cells of cysts and bronchiectasis. ICAM-1 was also observed in some vascular endothelial cells. In cases with CTD-associated IP and AEx IPF, ICAM-1 expression was even stronger than that observed in cases with stable IPF Figs. , 5.Fig. This study demonstrated that sICAM-1 levels were increased in patients with stable IPF and that a positive correlation existed between sICAM-1 and KL-6. This study also indicated that sICAM-1 levels were more useful than KL-6 or SP-D for diagnosing the early phase of acute exacerbation of IPF.It has previously been reported that sICAM-1 levels are elevated in diseases other than IPF (Greve et al. Few studies have compared sICAM-1 levels with other interstitial pneumonia markers such as KL-6. In this study, sICAM-1 levels correlated with KL-6 in the stable phase. As KL-6 is a strong prognostic marker of IPF (Yokoyama et al. Acute exacerbation of IPF, which occurs in approximately 5–15 % of cases with stable IPF per year, has a poor prognosis with a mortality rate of 50–80 % (Song et al. The limitations of this study include the fact that prognosis could not be investigated due to the small sample size. A previous report demonstrated that sICAM-1 levels were a prognostic factor for IPF (Risse et al. In conclusion, sICAM-1 levels in patients of stable IPF strongly correlated with KL-6; sICAM-1 might be a predictive indicator for prognosis. In the early phase of acute exacerbation of IPF, sICAM-1 might be more useful for diagnosis than other interstitial pneumonia markers. In stable and acute phases of IPF, sICAM-1 might be an important biomarker.