The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFβ was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease. Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by excessive matrix deposition that disrupts the normal architecture of the lung parenchyma. The key pathological features of IPF include fibroblastic foci that are highly synthetic, areas of epithelial cysts associated with the honeycombing appearance of the lung, and mild lymphoplasmacytic interstitial inflammation that is associated with areas of type II cell hyperplasia in vivo system for investigating candidate therapies. Its failure as a prognostic tool may be ascribed to the fact that the model has not been well characterized in terms of identifying the clinically relevant molecular changes and when they occur. Bleomycin is well understood to induce lung injury that results in an acute inflammatory response that is unlikely to reflect the processes driving the disease in patients. The inflammatory phase is, however, followed by fibrotic changes that replicate certain pathological features consistent with those associated with IPF. Therefore, some have argued that it may be more appropriate to evaluate compounds after or during the onset of the fibrosis phase of the response (i.e. using therapeutic dosing regimens), which may be a more disease-relevant paradigm The lack of effective therapies is arguably due to an incomplete understanding of the molecular mechanisms driving the disease and the failure of preclinical experimental models to correctly predict the clinical efficacy of several molecules Using classic histopathology and physiology methods, we report that the repetitive model offered no significant improvement over the single challenge model. Integrative bioinformatic and pharmacological approaches revealed corresponding molecular changes in the lungs of bleomycin-treated mice and IPF patients, especially in genes associated with mitosis and extracellular matrix signaling. Interestingly, these same pathways appeared to be altered in fibroblasts isolated from IPF patients with rapidly progressing, but not slowly progressing disease. It did not appear that these changes in expression were directly associated with TGFβ signaling and furthermore, an inhibitor to the TGFβR1  could not completely attenuate bleomycin-induced fibrosis in mice. These data support the premise that the bleomycin model can recapitulate many of the complex profibrotic responses that are also elevated in the lungs of IPF patients, particularly in patients with active disease.To establish a dose of bleomycin that induced fibrosis but did not result in mortality, a preliminary bleomycin dose-response study was performed. Bleomycin induced a dose-dependent increase in lung fibrosis . SignifiIn response to repetitive bleomycin administration, we observed increased inflammatory cell infiltrate in the BALF of mice . InteresH (lung tissue elastance) and work of inflation (WoI) correlated significantly to collagen levels in the lung , although the changes were relatively modest and significant at 35 days post-administration , an active fibrosis phase (days 7–14) and a late fibrosis phase (days 21–35), with saline-treated samples clustering separately . Peak geGenes altered during the inflammation phase generally consisted of immune response and wound healing genes including various cytokines and chemokines. In addition to genes associated with immune and wound healing categories, genes altered during the active fibrosis phase were also associated with cell cycle-related processes. These changes in gene expression were validated by the increased expression of inflammatory cytokines early in the response to bleomycin , followeClustering analysis of genes involved in fibrosis-related pathways revealed preferential up-regulation of genes during the active fibrosis phase. Matrix metalloprotease (MMP) genes including MMP12, MMP19, as well as lysyl oxidase-like (LOXL) genes implicated in the pathogenesis of fibrotic diseases were induced from 7 days onwards following bleomycin treatment . PositivThe translational relevance of the bleomycin response signatures were assessed in clinical IPF datasets using gene set enrichment analysis (GSEA). Gene expression data from two publicly available clinical IPF datasets were compiled: GSE2052 in vivo datasets, GSEA also revealed that gene sets defined by changes in the clinical IPF datasets were enriched within the expression profiles of lung samples generated from the bleomycin model (data not shown). These results further highlight commonalities in genes altered in response to bleomycin treatment with those altered in clinical IPF samples.Closer examination of the leading edge genes  shows up-regulation of a common subset of genes across different clinical datasets . Genes iNext, we prospectively compared the gene expression profiles of fibroblasts obtained from patients with rapidly progressing and slowly progressing IPF to fibroblasts obtained from non-IPF control subjects, as these cells are centrally involved in the disease pathogenesis Given that several pathways associated with fibrotic processes were elevated in response to bleomycin-induced lung injury, we wanted to assess whether TGFβ was the dominant driver of fibrosis in this model. To this end, we assessed an ALK-5 inhibitor, SB525334, under both prophylactic and therapeutic conditions. SB525334 significantly inhibited bleomycin-induced fibrosis changes under both conditions . The maxThe data presented in this manuscript provide clear evidence that the bleomycin model of lung fibrosis induces molecular changes directly relevant to IPF and in particular may be more relevant for modeling “active” rather than slowly progressing disease. Additionally, these data indicate that there are multiple pathways involved in eliciting the pathophysiological changes in the model that are consistent with those observed in the lungs of patients with IPF. As such, it would appear that therapies aimed at affecting several of these key signaling networks either by targeting “nodes”, using combination therapy approaches, or broad spectrum inhibitors of key enzymes may be the only means of making a significant impact on disease progression in all patients with IPF.The response to bleomycin occurred in three distinct phases, which we have termed the (1) inflammation phase, (2) active fibrosis phase, and (3) late fibrosis phase. The inflammation phase was largely characterized by the presence of neutrophils in the airways and the increased expression of immune processes in the lung tissue. While some have proposed that neutrophils may be playing a role in IPF H) and our new measure of WoI Another commonly stated limitation of the bleomycin model is that the fibrosis is not progressive and in fact, resolves with time. Interestingly, we observed that there was still measureable fibrosis and changes in pulmonary mechanics in the late fibrosis phase (out to 5 weeks post-bleomycin). Furthermore, we took samples 56 days after bleomycin instillation in a separate study (data not shown) and measureable fibrosis was still present contrary to previous reports and dogma As an attempt to improve the single challenge system we performed repetitive bleomycin instillations, but found that repetitive exposures provided no additional value in terms of producing a more substantial or progressive fibrotic response in the lung. The one difference to the single challenge model was that epithelial remodeling was more evident at most of the time points assessed in the repetitive challenge system. Epithelial remodeling in IPF is associated with the deterioration of the lung parenchyma and the honeycombing pattern of the lung characteristic of IPF The most interesting observations from these studies occurred during the active fibrosis phase of the response to bleomycin. This phase was characterized by increased levels of (protein and lipid) pro-fibrotic mediators associated with increased matrix deposition in the lung that correlated with alterations in pulmonary mechanics. These changes were also temporally correlated with the increased expression of a variety of fibrosis-related gene sets including TGFβ signaling, matrix proteases, and structural proteins – observations that confirm previous findings reported by others Here, we have shown for the first time a direct correlation between the molecular changes observed in the lungs of bleomycin-treated mice and patients with IPF using novel translational and quantitative bioinformatics approaches. GSEA allows one to define gene signatures from one experimental system and subsequently quantify whether that signature is enriched in another biological system. The gene sets defined by changes during the active fibrosis phase (days 7–14) of the mouse bleomycin model generated the highest enrichment scores amongst the samples from IPF patients obtained from two separate publicly available data-sets. In fact, the changes occurring in the late fibrosis phase were also enriched in the clinical samples, an observation that is consistent with the lack of fibrosis resolution at these later times. To understand the biology behind the correlative gene changes, we overlapped a union of the leading edge gene lists with canonical signaling pathways. Interestingly, the leading edge genes were largely involved in mitotic processes likely due to the proliferation of mesenchymal cells. Additionally, there were changes in signaling pathways  associated with fibroblast proliferation and signaling in response to changes to the extracellular matrix. These data highlight the multifactorial molecular mechanisms involved in the pathogenesis of IPF Budd et al, under review). Additionally, the cells from rapidly progressing patients are able to induce substantial lung fibrosis when injected into immunocompromised mice (versus a mild response when using slowly progressing IPF patient fibroblasts) Although these are interesting observations, additional clinical information such as lung function data, high resolution computed tomography scores, and rate of disease progression associated with publicly available clinical datasets would improve the interpretation of these datasets. Such information would provide greater insights about whether these molecular changes that correlate with the model are generic amongst all IPF patients or whether the correlation is strongest within certain IPF patient sub-populations. Interestingly, many of the overlapping changes between the model and clinical samples were likely to be due to changes in fibroblast activation and proliferation. These same processes were enriched in fibroblasts isolated from the lungs of IPF patients with rapidly progressing disease. Surprisingly, these processes did not appear to be altered when comparing fibroblasts from patients with slowly progressing IPF to non-IPF controls. Together, these observations would suggest that the bleomycin model may be more effective at mimicking active disease processes that may be more associated with patients with a rapidly progressing disease course. That said, there are caveats to these data that need to be taken into account. First, the patients’ cells are not freshly isolated or laser capture microdissected, but rather they have been in culture for several passages, which may confound interpretation. Second, the controls are not healthy individuals, but cancer patients undergoing lung resection. Finally, these data were generated from 4–6 patients within each group and additional biological replicates would improve confidence in these observations. That said, it is still very interesting to note that the cells from the rapidly progressing IPF patients do have enhanced responses to PDGF  and TGFβ , whereas these responses are not as profoundly altered in fibroblasts from slowly progressing IPF patients relative to the non-IPF controls  was only enriched in the IPF patients who had experienced an acute exacerbation compared to non-IPF control subjects, possibly indicating its importance in active disease. Similarly, the down-regulated TGFβ gene set (comprised mainly of negative regulators of the TGFβ pathway) was only down-regulated in one clinical data-set. Although no one would discount TGFβ’s contribution to the lung remodeling associated with IPF, it does highlight the fact that it is unlikely to be the only pathway driving disease pathogenesis. These observations are also consistent with the data demonstrating that the ALK-5 inhibitor, SB525334, was not capable of fully blocking the fibrotic response elicited by bleomycin. This molecule consistently attenuates the response in our laboratory as well as in others The data described in this paper contradict current dogma and suggest that bleomycin is able to induce clinically meaningful molecular responses in the lungs of mice. Using innovative bioinformatic approaches we directly linked the molecular changes occurring in the bleomycin model to those occurring in the lungs of IPF patients in a quantitative manner. Genes involved in mitotic processes were expressed at higher levels in lung tissues from bleomycin-treated mice and IPF patients as well as fibroblasts isolated from IPF patients with rapidly progressing disease. These data along with pharmacological data indicate that bleomycin triggers complex, multifactorial (TGFβ dependent and independent) responses that result in pathophysiological changes in mice that are consistent with IPF. Furthermore, our analysis suggests that the changes in this model best represent changes occurring in patients with active (i.e. rapidly progressing) disease.ad libitum. All animal care and experimental procedures were approved by the Roche Animal Care and Use Committee, which is a facility accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC).Male C57BL6/J mice (7–9 weeks old) were purchased from the Jackson Laboratory , housed under pathogen-free conditions with food and water Bleomycin sulphate  was dissolved in 0.9% saline and loaded into a IA-1C liquid MicroSprayer  as previously described 2O positive end-expiratory pressure).Invasive lung function measurements were taken at specified time points following saline or bleomycin administration as previously described Exercise capacity was measured based on a method previously published by Koch and colleagues Immediately following lung function measurements, mice were exsanguinated, bronchoalveolar lavage fluid (BALF) was collected, and total and differential cell counts were performed, as previously described For the analysis of 4-hydroxyproline (HP), BALF samples were extracted with acetonitrile at volume of 1:6, respectively. After centrifugation, acetic acid (0.1% in water) was then added to the supernatants at volume of 1.5:1, respectively. Samples were mixed and centrifuged before injection for LC/MS/MS analysis. LC/MS/MS analysis was conducted by gradient HPLC with selective reaction monitoring (SRM). The calibration range was 40- 2,000 ng/ml. Since the control BALF matrix contained a basal level of HP (approximately 200 ng/mL), the calibration curve is corrected with the basal concentration determined by the standard addition. The LC/MS/MS system used for the analysis consisted of an AB-Sciex API4000 with an electrospray source connected to an Agilent 1200 pump, an Waters 2777 autosampler, and an Agilent 1100 series column oven. Chromatographic analysis was conducted by HILIC HPLC with an Ascentis Express HILIC  column. The SRM transition is m/z 132 to m/z 41.2O pressure with 10% neutral buffered formalin through the tracheal cannula and immersed in formalin for at least 24 h. After being processed into paraffin blocks, the lungs were sectioned (5 µm) and stained with either hematoxylin and eosin (H&E) or immunolabeled with an anti-collagen I antibody , to assess fibrotic changes in the lungs as previously described After performing lung function measurements, whole lungs  were inflated under 25 cm HAdditionally, staining for alpha-smooth muscle actin , and heat-shock protein-47 (HSP-47)  was done using a 3-step biotin-streptavidin-HRP detection method, and DAB  as the detection chromogen. Tissue sections were counterstained with hematoxylin, dehydrated, and mounted with Permount mounting media.Total RNA was isolated from the lung tissue of bleomycin and saline treated mice across 7 time points . The mice used for genomic profiling were not used for measuring lung function, performing bronchoalveolar lavage, and they were not used for histopathology analysis. The entire left and right lung tissues were homogenized in QIAzol reagent. Purified total RNA was amplified and labeled using NuGen Ovation kits  and samples were hybridized to Affymetrix Mouse 430 2.0 arrays. Array washing, staining and scanning was performed according to standard Affymetrix protocols . Probe level data was curated by first mapping individual probe sequences to their most current mouse genome sequences. Probes which were non-uniquely mapped to specific genes or contained outdated mappings were discarded, and the remaining probes were summarized into probesets and normalized using RMA. Potential outlier samples were assessed using principal component analysis (PCA) on all normalized probesets across all samples, resulting in 1 sample  being removed from subsequent analysis. Probes level data was subsequently summarized to unique genes based on a variance filter, yielding one expression value per unique gene across 111 samples. Mouse genes were then mapped to their human orthologs for subsequent pathway analysis. This resulted in 14643 unique mouse genes which mapped to human orthologs and were subsequently used for analysis. All data is MIAME compliant and all raw.CEL files from the arrays are deposited in NCBI’s GEO database (GSE40151).Differentially expressed genes were determined using an ANOVA, with pairwise comparisons between bleomycin and saline treatment at each time point. P-values for differentially expressed genes in pairwise comparisons were adjusted using a Benjamini-Hochberg correction to account for multiple hypothesis testing http://pid.nci.nih.gov/index.shtml). This repository includes pathway data imported from BioCarta and Reactome. Enrichment of functional ontologies and pathways for all DEGs was determined by a hypergeometric test, with p-values and FDR calculated to determine significance of enrichment.Gene ontology (GO) functional analysis and pathway enrichment analysis were performed on DEGs between saline and bleomycin treated mice at each time point using internally curated GO biological process annotations. Similarly, pathway enrichment for all DEGs was determined using internally curated data from the NCI Pathway Interaction Database , collagens (COLs), TGFβ signaling , alternative macrophage activation, Wnt signaling, and an Gene set enrichment analysis (GSEA) Clustering analysis was subsequently performed on leading edge genes from GSEA, in order to more closely examine genes within bleomycin signatures most contributing to enrichment of the signatures in clinical datasets. A union of leading edge genes from GSEA of IPF versus control comparisons from two datasets  was compiled and expression of these genes was assessed in the two clinical datasets with IPF-only subjects versus controls.In addition, GSEA was used to determine enrichment of genes differentially expressed in clinical IPF datasets in bleomycin versus saline treated mice. DEGs were determined in each IPF dataset between IPF and non-IPF groups . DEGs were determined based on being 1.5-fold altered between groups with an FDR <0.05. A union of DEGs from comparisons in each dataset was compiled resulting in two gene sets consisting of 30 genes . Enrichment of these gene sets was determined against genes from each time point in the mouse bleomycin data, ranked based on a composite of fold-change and FDR differences between bleomycin and saline treated samples per time point, with FDRs of gene set enrichment determined as described above.GSEA was also used to determine enrichment of internally curated canonical pathways from the NCI Pathway Interaction Database in both the mouse bleomycin and cultured human fibroblast datasets. Canonical pathways enriched in the bleomycin dataset were defined by ranking genes based on a composite score of fold-difference and FDR between bleomycin and saline-treatments for the 14 day time point (active fibrosis phase). Canonical pathways were then used as gene sets for GSEA, with FDR values being determined for gene set enrichment by permuting genes. Canonical pathway enrichment was similarly determined in the fibroblast dataset by generating ranked gene as described above in rapid progressing IPF vs. non-IPF samples. Significantly enriched canonical pathways  were then compared between the bleomycin-treated 14 day time point and fibroblasts from rapid progressing IPF patients.Primary cultures of LFs were isolated from the distal parenchyma of patients from unaffected donor controls (lung cancer patients) and patients with IPF as previously described Data are expressed as mean ± SEM unless specified otherwise. Unless stated otherwise elsewhere in the methods, statistical analyses of time course studies were determined using a Student’s t-test using GraphPad Prism (version 5.03).Figure S1Bleomycin induces dose-dependent increases in lung fibrosis. Fibrosis scores evaluated in H&E stained lung sections 21 days after a single instillation of bleomycin. Data expressed as mean ± SEM of n = 8 except for the 5 U/kg group where n = 5. Significance relative to the saline treated was determined using a one-way ANOVA and Dunnett’s post-hoc test and is denoted as, *p<0.05; or ***, p<0.001.(TIF)Click here for additional data file.Figure S2Bleomycin induces inflammatory cell infiltrate in the airways of mice. The numbers inflammatory cells increase in the BALF fluid after a single (A – C) or repetitive (D – F) bleomycin (black bars) or saline (white bars) administration. Differential cell counts revealed that neutrophils (A and D), lymphocytes (B and E), and macrophages (C and F) were all elevated in the BALF. Data are expressed as mean ± SEM of n = 7–8 mice. Significance (relative to the time-matched control at each time point) was determined using a Student’s t-test and is denoted as follows: *p<0.05; **p<0.01; and ***p<0.001.(TIF)Click here for additional data file.Figure S3Bleomycin induces collagen deposition in the lung. Measureable changes in matrix remodeling were observable after a single (A and B) or repetitive (C and D) bleomycin (black bars) or saline (white bars) administration. Lung collagen (A and C), and BALF hydroxyproline levels (B and D) were all elevated in response to bleomycin. Data are expressed as mean ± SEM of n = 7–8 mice. Significance (relative to the time-matched control at each time point) was determined using a Student’s t-test and is denoted as follows: *p<0.05; **p<0.01; and ***p<0.001.(TIF)Click here for additional data file.Figure S4Changes in lung mechanics correlate to changes in lung fibrosis. Changes in H (A) and volume normalized work of inflation (WoI) (B) after a single bleomycin instillation significantly correlated to the amount of lung tissue stained positive for collagen I as determined by Pearson correlation coefficients (R). The correlations for H (C) and WoI (D) with collagen I were similar after repetitive bleomycin instillations.(TIF)Click here for additional data file.Figure S5Bleomycin increased collagen deposition and myofibroblast activation. Serial lung tissue sections from one representative animal illustrate that areas positive for collagen I (D) were also positive for α-smooth muscle actin (E), as well as HSP47 (F). As a reference, sections from saline treated animals stained for collagen I (A), α-smooth muscle actin (B), and HSP47 (C) are also shown. Images were captured at 200X magnification.(TIF)Click here for additional data file.Figure S6Exercise capacity is reduced in bleomycin treated mice. The exercise capacity of mice treated with saline (filled circles) or 2 U/kg bleomycin (filled squares) was measured on a motorized treadmill. Data expressed as mean ± SEM of n = 19–20. Significance relative to the saline treated was determined using a one-way ANOVA and Dunnett’s post-hoc test and is denoted as, *p<0.05; or ***, p<0.001.(TIF)Click here for additional data file.Figure S7Bleomycin induces cytokine, chemokine, and pro-fibrotic mediator secretion in the BALF. BALF levels of KC (A), IL-16 (B), CCL17 (TARC) (C), total IL-12 (D), TGFβ (E), and LPA (18:1 isoform) (F)were measured. Saline treated controls represented by white bars and bleomycin treated animals by black bars. The following cytokines had no measurable change: IFN-g, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-10, GM-CSF, VEGF, IL-13, IL-17 (data not shown). Data are expressed as mean ± SEM of n = 8 mice. Significance (relative to the time-matched control at each time point) was determined using a Student’s t-test and is denoted as, *p<0.05.(TIF)Click here for additional data file.Figure S8Supervised hierarchical clustering of custom panels of genes across all mouse samples. Clustering of genes was performed for a panel of (A) genes involved in Wnt signaling, (B) genes altered downstream of PI3 kinase, and (C) genes involved in alternative macrophage activation. Samples are ordered based on treatment and time point.(TIF)Click here for additional data file.Figure S9Heatmap of gene set enrichment of custom signatures in clinical IPF datasets. GSEA was performed for gene sets of custom gene panels from fibrosis-related mechanisms including MMP, LOXL, collagen, and TGFβ signaling. Enrichment of each gene set (denoted in rows) was determined against ranked lists of genes from clinical datasets comparing IPF vs. non-IPF conditions from two datasets . Enrichment scores were plotted in a heatmap where gene sets enriched in IPF samples  were denoted in red while gene sets enriched in non-IPF samples were denoted in blue. Intensity of each cell was based on enrichment score .(TIF)Click here for additional data file.Figure S10Plasma exposure levels for SB525334 in bleomycin treated mice after initiating a diet using Purina Rodent Chow #5001 mixed with SB525334.(TIF)Click here for additional data file.Data File S1A union of leading edge genes from GSEA of bleomycin-induced gene sets in IPF vs. non-IPF subject comparisons from two clinical cohorts . This alphabetized list of genes corresponds to a union of the genes highlighted in (PDF)Click here for additional data file.Data File S2Canonical pathways enriched among the leading edge genes from GSEA of bleomycin-induced gene sets in IPF vs. non-IPF subject comparisons from two clinical cohorts . The canonical pathways, p value of the enrichment of leading edge genes in these pathways, and the leading edge genes contained within the pathways are highlighted in this table.(PDF)Click here for additional data file.


The role of extracellular signal-regulated protein kinase (ERK) in intestinal ischemia/reperfusion (I/R) injury has not been well investigated. The aim of the current study was to examine the effect of inhibition of the ERK pathway in an in vitro and in vivo model of intestinal I/R injury.ERK1/2 activity was inhibited using the specific inhibitor, U0126, in intestinal epithelial cells under hypoxia/reoxygenation conditions and in mice subjected to 1 hour of intestinal ischemia followed by 6 hours reperfusion. In vitro, cell proliferation was assessed by MTT -2,5-diphenyl tetrazolium bromide) assay, apoptosis by DNA fragmentation, and migration using an in vitro model of intestinal wound healing. Cells were also transfected with a p70S6K plasmid and the effects of overexpression similarly analyzed. In vivo, the effects of U0126 on intestinal cell proliferation and apoptosis, intestinal permeability, lung and intestinal neutrophil infiltration and injury, and plasma cytokine levels were measured. Survival was also assessed after U0126. Activity of p70S6 kinase (p70S6K) was measured by Western blot.In vitro, inhibition of ERK1/2 by U0126 significantly decreased cell proliferation and migration but enhanced cell apoptosis. Overexpression of p70S6K promoted cell proliferation and decreased cell apoptosis. In vivo, U0126 significantly increased cell apoptosis and decreased cell proliferation in the intestine, increased intestinal permeability, intestinal and lung neutrophil infiltration, and injury, as well as systemic pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β. Mortality was also significantly increased by U0126. Inhibition of ERK1/2 by U0126 also abolished activity of p70S6K both in vitro and in vivo models.Pharmacologic inhibition of ERK1/2 by U0126 worsens intestinal IR injury. The detrimental effects are mediated, at least in part, by inhibition of p70S6K, the major effector of mammalian target of rapamycin pathway. Intestinal ischemia/reperfusion (I/R) occurs in a wide range of clinical scenarios, including small bowel transplantation, midgut volvulus, major trauma, thrombosis of the superior mesenteric artery and vein, hemorrhage and cardiopulmonary bypass. Intestinal I/R leads to the release of cytotoxic substances from the ischemic tissues and the accumulation of inflammatory mediators in the intestine. This results in mucosal injury, hyperpermeability, and inflammation. The release of proinflammatory factors and bacteria-derived endotoxins from the injured intestine may also instigate a systemic inflammatory response and lead to multiple organ dysfunction syndrome -3. ThereThe extracellular signal-regulated protein kinases (ERK) 1 and 2 (ERK1/2) signaling pathway is a cascade consisting of at least three families of protein kinases, including Raf (MAPKKK or MEKK), MAPKKs (MEK 1 and MEK 2), and MAPK (ERK 1 and ERK 2 or p42/p44 MAPKs). Each molecule acquires protein kinase activity after phosphorylation, and is subsequently able to phosphorylate and then activate the next member of the signaling cascade. The ERK pathway not only regulates a wide range of cell behaviors, such as, growth, proliferation, migration, differentiation, apoptosis and autophagy, but also mediates inflammatory responses ,6. The E2-adrenoceptor agonist, in transient cerebral I/R [ERK1/2 has been implicated in tissue injury induced by I/R, but results have been inconsistent. Zhu et al. demonstrated that activation of ERK1/2 mediated neuroprotection of dexmedetomidine, a potent and highly selective αbral I/R . Similarbral I/R . Howeverbral I/R . Similarbral I/R . There ibral I/R . Therefol-glutamine, 1% penicillin and 1% streptomycin in the incubator with a humidified atmosphere of 5% CO2/95% air at 37°C. Cells were used between passages 17 and 25.The intestinal epithelial IEC-6 cell line which was derived from adult rat intestinal crypts was purchased from the American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 2 mM Cells were transfected with empty vector pRK7 plasmid, p70S6K plasmid (wild-type p70S6K expressing vector pRK7-HA-S6K1-WT)  using th2/5% CO2/94.5% N2 for indicated periods of time, cells were reoxygenated by placing cells into in an incubator under 37°C/ 5% CO2 conditions with 10% serum (normoxic conditions) for indicated periods of time. To inhibit ERK1/2 phosphorylation/activity, cells were treated with 10 µM of U0126 (Sigma) [A well-established hypoxia/reoxygenation (H/R) model was employed using IEC-6 cells to mimic I/R in vivo . After i (Sigma) ,16, a sp3 cells were added into each well in a 96-well plate. After 24 hours, cells were cultured in medium without FBS under hypoxic conditions for 12 hours in a hypoxic chamber, and then 10 µM of U0126 or vehicle was added into cells. After 1 hour, 10% FBS was supplemented to medium. Cells were transferred to an incubator under normoxic conditions for 3 days. Cells were then incubated with MTT (Sigma)  for 3 hours. After removal of medium, dimethyl sulfoxide (200 µl/well) was added to wells. A microplate reader was used to measure the absorbance of dissolution sample at 570 nm and subtracted background at 670 nm.MTT -2,5-diphenyl tetrazolium bromide) assay was employed for cell growth assessment . Briefly4 cells/well). After 24 hours, fresh FBS-free medium containing U0126 (10 µM) or vehicle was added. Cells were cultured under hypoxic conditions for 24 hours followed by incubation in normoxic conditions for 6 hours. To measure the DNA fragmentation, the medium was discarded, and the attached cells were washed twice with PBS and lysed with lysis buffer (200 µl /well). Cell lysates were collected into 1.5ml-tubes and centrifuge at 200 Χ g for 10 minutes. A 100-µl supernatant of the cell lysate per well was taken and added into the ELISA plate and measured the immunocomplex at 405/490 nm in a microplate reader. The value was normalized by cell numbers in corresponding wells in parallel experiments. Cells grown in basal conditions served as untreated control.A Cell Death Detection ELISA (enzyme-linked immunosorbent assay) kit (Roche) was used to measure the DNA fragmentation in cells . Cells w5 cells in a 12-well plate for 24 hours. Cells were then cultured with FBS-free medium under hypoxic conditions for additional 12 hours. The confluent cell monolayer was scraped with a pipette tip across the diameter of the well. After removal of cell debris by washing with PBS two times, cells were then treated with U0126 (10 µM) or vehicle for 1 hour followed by adding 10% FBS. Cells were cultured for another 24 hours under normoxic conditions. The wound area at the beginning of wounding and at the end of treatment was photographed respectively under an inverted phase-contrast microscope with an attached camera. The wound area was measured using OPTIMAS 6.1 image analysis software (Optimas Corp.). The recovered surface area was calculated by subtracting the wound area at the end of treatment from the corresponding original wound area.A H/R model of in vitro intestinal wound healing was employed to mimic I/R in vivo . Each weAll animal protocols were approved by the University of Texas, Houston Medical School Animal Welfare Committee.2 asphyxiation immediately and recorded as dead.Male, 8-10 weeks old C57BL/6J mice were randomly assigned into 4 groups (n=5/group): sham, sham-U0126, IR, IR-U0126. Mice were fasted with free access to water overnight before laparotomy. U0126   was giveIntestinal and lung tissues were fixed in buffered formaldehyde solution (10% in PBS) for 24 hours and embedded in paraffin. The tissue was then sectioned, deparaffinized, stained with hematoxylin and eosin (HE). To evaluate histopathologic changes in the intestine, Park/Chiu scoring system was used in a blinded manner using a light microscope on a 0 to 5 scale, where 5 is the most severely injured ,18. HistA commercial Myeloperoxidase (MPO) Colorimetric Activity Assay Kit (BioVision Research Products) was employed to quantitatively measure the MPO activity in the intestinal and lung tissue. The MPO activity reflects the extent of neutrophil infiltration into the tissue. The MPO value was normalized to milligrams of protein determined by Bio-Rad Protein Assay (Bio-Rad Laboratories).The relative level of cytokines TNF-α, IL-6 and IL-1β in plasma was determined by enzyme-linked immunosorbent assay with a Mouse Cytokine ELISA Strip I for Profiling 8 Cytokines kit (Signosis) according to manufacturers’ instruction.The ex-vivo isolated everted ileum sac method  was usedIntestinal tissues and IEC-6 cells were quickly lysed with radioimmunoprecipitation assay buffer in the presence of protease inhibitors. Western blot analysis was performed as previously described . AntibodCell apoptosis in the intestinal tissue was evaluated using an In Situ Cell Death Detection Kit (Roche) as described previously .Ki-67 is a nuclear protein that is expressed in proliferating cells. It has been used as a marker for cell proliferation. Immunohistochemistry for Ki-67  (Merck Millipore) was performed using the Dako-Cytomation EnVision + System-HRP (AEC) kit (Dako) in paraffin-embedded intestinal sections as described previously . Ki-67 pData is presented as mean ± SEM and were compared by one way analysis of variance with the nonparametric Mann-Whitney test used to determine differences between groups. The Mantel–Haenzel log rank test was used to compare survival rates. A p value less than 0.05 was considered statistically significant.ERK1/2 is activated by phosphorylation. To test the efficacy of U0126 inhibition of ERK1/2 in intestinal cells under H/R conditions and after I/R in the intestines, we detected protein levels of phosphorylated ERK1/2 by Western blot. U0126 virtually abolished the phosphorylation of ERK1/2 in IEC-6 cells under H/R conditions . In vivoUnder H/R conditions, there was no difference between control and vehicle treated groups in cell proliferation (control vs. vehicle: 2.40±0.10 vs. 2.33±0.15. P>0.05), migration (control vs. vehicle: 101.67±13.31 vs. 95.67±10.69. P>0.05) or apoptosis (control vs. vehicle: 6.07±0.73 vs. 6.53±1.27. P>0.05) . U0126 sIn sham groups, treatment of U0126 did not affect intestinal cell apoptosis  or proliferation (Ki-67 proliferation index) . However, after intestinal I/R, U0126 significantly enhanced intestinal cell apoptosis  and reduced cell proliferation  . These dMPO activity, an index of neutrophil infiltration, was similar in the sham and sham-U0126 groups (sham vs. sham-U0126: 0.25±0.06 vs. 0.24±0.16. P>0.05). However, MPO activity was markedly increased in the intestine after I/R (sham vs. IR: 0.25±0.06 vs. 0.96±0.07. p<0.01) and was further increased by U0126 treatment (IR vs. IR-U0126: 0.96±0.07 vs. 2.24±0.26. p<0.01) .Permeability was similar in the intestine between sham and sham-U0126 groups (sham vs. sham-U0126: 17.64±1.99 vs. 22.86±5.90P>0.05), but was increased after I/R  and U0126 treatment exacerbated the intestinal permeability (IR vs. IR-U0126: 81.30±18.16 vs. 157.08±23.30. p<0.01) .Similarly, there was no difference between sham and sham-U0126 in mucosal injury (sham vs. sham-U0126: 0.10±0.22 vs. 0.20±0.27. P>0.05), but I/R induced severe mucosal injury in the intestine (sham vs. IR: 0.10±0.22 vs. 2.50±0.50. p<0.01), and treatment with U0126 further worsened intestinal injury (IR vs. IR-U0126: 2.50±0.50 vs. 4.50±0.50. p<0.01) .Consistent with previous reports ,21, systIntestinal I/R caused significant lung inflammation (sham vs. IR: 0.56±0.07 vs. 5.57±1.59. p<0.01) and injury (sham vs. IR: 0.20±0.45 vs. 3.30±0.48. p<0.01). U0126 further exacerbated both inflammation (IR vs. IR-U0126: 5.57±1.59 vs. 11.11±2.53. p<0.01) and injury (IR vs. IR-U0126: 3.30±0.48 vs. 4.38±0.41. p<0.05). There were no differences in inflammation (sham vs. sham-U0126: 0.56±0.07 vs. 0.59±0.20. p>0.05) or injury (sham vs. sham-U0126: 0.20±0.45 vs. 0.20±0.45. p>0.05) between sham groups .Since inhibition of ERK1/2 activity by U0126 led to more severe local and remote organ (lung) damage, we assessed the impact of U0126 on mortality and demonstrated a marked increase in mortality in U0126-treated animals (p<0.01) . MortaliSimilar to ERK pathway, the mTOR/p70SK pathway is also a major regulator of cell growth, proliferation and motility -24 as weU0126 is a signal transduction inhibitor that specifically targets the ERK pathway in mammalian cells . U0126 hERK1/2 activation controls cell proliferation and apoptosis . StudiesInhibition of ERK1/2 may also increase inflammation. Our results showed that inhibition of ERK1/2 facilitated neutrophil infiltration into the intestinal tissue and promoted the production of proinflammatory factors systemically during intestinal I/R. Furthermore, ERK1/2 inhibition exacerbated inflammation-related remote organ damage. Consistent with our results, Noh et al. reported that upregulation of ERK1/2 kinase activity promoted the anti-inflammatory cytokine IL-10 expression in lipopolysaccharide-stimulated conditions .The lung is frequently injured after intestinal I/R. Once activated by proinflammatory mediators, neutrophils are able to penetrate the endothelial layer and migrate into the alveolar space ,36. RecrThe precise molecular mechanism of ERK1/2 in regulation of intestinal injury and inflammation after I/R remains to be elucidated. PPARγ is an anti-inflammatory mediator in the intestine and contains a mitogen-activated protein kinase site. Phosphorylation of this site by ERK1/2 resulted in inhibition of PPARγ . HoweverIt is well-known that ERK1/2 regulates mTOR /p70S6K pathway ,27. We hIn conclusion, we have demonstrated that ERK1/2 activation is crucial to protection against intestinal I/R injury. Inhibition of ERK1/2 decreased cell proliferation, suppressed cell migration, and increased cell apoptosis, as well as exacerbating the breakdown of the intestinal barrier. These effects were due, at least in part, to increased local and systemic inflammation. Additionally, p70S6K mediated the effects of ERK1/2 inhibition on intestinal I/R injury. This finding enhances the understanding of the mechanisms responsible for intestinal I/R injury and may potentially be translated into new therapeutic approaches for prevention and recovery of intestinal I/R damage. The results of this study may also be applicable to other organs damaged by I/R since ERK1/2 is universally expressed by most tissues/organs, such as lung, kidney, liver and heart.


TLR2 might be important in a small group of Caucasian sarcoidosis patients. The present study aimed to establish whether these findings are relevant to the Japanese population.Sarcoidosis is a systemic inflammatory disease characterized by the formation of non-caseating granulomas, with varied clinical manifestations. The common etiology of sarcoidosis is uncertain, but it is thought to be triggered by an exogenous antigenic stimulus, such as some bacterial proteins. Toll-like receptors (TLRs) recognize microbial components and elicit innate as well as adaptive immune responses. It has been reported that polymorphisms in TLR2 and assessed the allelic diversity between 257 Japanese sarcoidosis patients and 193 Japanese healthy controls.We genotyped 5 single-nucleotide polymorphisms (SNPs) in TLR2 alleles and haplotypes in the sarcoidosis cases were found in comparison with the controls. However, marginal associations were observed for TLR2 at rs3804099 and rs3804100 in sarcoidosis patients with cutaneous manifestations.No significant differences in the frequency of TLR2 polymorphisms are not significantly related to the pathogenesis of sarcoidosis in the Japanese population.Our results suggest that  Sarcoidosis is a systemic inflammatory disorder resulting in non-caseating granulomas in multiple organs, such as: lung, skin, eye, lymph nodes, central and peripheral nervous system, and heart -3. JapanThe exact cause of sarcoidosis is unknown, but the fact that the frequency and course of the disease varies widely among racial groups suggests that genetic factors may be the basis of disease susceptibility. African Americans are more commonly and severely affected by sarcoidosis than Caucasian Americans. The annual sarcoidosis incidence for African Americans is threefold higher, compared with Caucasian Americans; at 35.5 versus 10.9 cases per 100,000, respectively . In the Mycobacterium tuberculosis and Propionibacterium acnes has been detected in some sarcoid lesions by using polymerase-chain-reaction (PCR) methods [Environmental factors are also thought to contribute to the disease progression. The DNA of  methods -13. Rece methods . These sTLR polymorphisms are closely associated with many autoimmune diseases [TLR2 are associated with impaired responses to bacterial infection in human [TLR2 promoter lesion (rs4696480) was associated with sarcoidosis in a Dutch Caucasian population, but could not confirm this in their validation cohort. They inferred from these findings that a TLR2 variant could play a role in a small percentage of patients. The association between TLR2 polymorphisms and sarcoidosis needs to be confirmed by further replication studies, particularly in other ethnic groups. In the present study, we therefore evaluated the association of multiple SNPs in TLR2 in Japanese patients.Toll-like receptors (TLRs) recognize microbial components and elicit innate as well as adaptive immune responses. Stimulation with TLR ligands induces the production of proinflammatory cytokines and type I interferons in cells of the innate immune system through intracellular signaling cascades -17. Accudiseases -20. Amondiseases ,22. Polyin human -27. Recein human  reportedTwo hundred fifty-seven unrelated patients with a diagnosis of sarcoidosis and 193 healthy controls were recruited from Yokohama City University, Hokkaido University, Fujita Health University, Tokyo University, Keio University, and Kumamoto City hospital. All patients and control participants were of Japanese ethnicity. Sarcoidosis patients were diagnosed according to the diagnostic criteria developed by the Japanese Society of Sarcoidosis and Other Granulomatous Disorders (JSSOG) previously described . Uveitisrs1898830, rs11938228, rs3804099, re3804100, and rs7656411 . Probe fluorescence signals were detected by TaqMan Assay for real-time PCR  following the manufacturer’s instructions.Peripheral blood lymphocytes were collected, and genomic DNA was extracted from peripheral blood cells using the QIAamp DNA Blood Maxi Kit . We evaluated five single-nucleotide polymorphisms (SNPs): s7656411 . These S2 test. The Haploview 3.32  program was used to compute pair-wise linkage disequilibrium (LD) statistics [Hardy–Weinberg equilibrium was tested for each SNP among the controls. Differences in allele and genotype frequencies between cases and controls were assessed by the χatistics . Standaratistics . HaplotyTLR2 gene: rs1898830, rs11938228, rs3804099, re3804100, and rs7656411 . The allele frequencies of the five SNPs in both the cases and controls are listed in rs3804099 and rs3804100 were higher when compared with the frequency in 193 controls . However, these statistical differences disappeared after the Bonferroni correction was applied (p>0.05).Linkage disequilibrium (LD) blocks of five SNPs in  defined . The TLRTLR2 polymorphisms affect the development of sarcoidosis in the Japanese population. Our results showed that all the TLR2 polymorphisms so far examined were not significantly associated with any clinical subtype of sarcoidosis including ocular involvement in the Japanese population. However, a marginally significant p-value was observed for the SNPs rs3804099 and rs3804100 in patients with cutaneous manifestations, in comparison with the healthy control group. Healthy normal human skin contains two distinct major subsets of resident dendritic cells: Langerhans cells (LCs) and dermal DCs (DDCs). These DCs are located in the outer skin layers of the epidermis and play a critical role as the first line of defense against pathogens invading the skin. These DCs in the skin, especially DDCs, express TLR2 as well as TLR1 and TLR6. DDCs recognize bacteria and trigger the innate immune response [TLR2 polymorphisms and skin manifestations of sarcoidosis. The variants of the TLR2 gene in DDCs may play a causative role in the development of cutaneous sarcoidosis in a site-specific manner.The current study was designed to determine whether response . Our resSeveral reports have suggested that genetic variants of innate immune receptors might be associated with the risk of developing sarcoidosis . Innate TLR4 polymorphisms in the Caucasian population [TLR4 polymorphisms and sarcoidosis in the Japanese population [TLR4 is a major receptor for lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls. TLR4 and TLR2 signal transduction results in the activation of inflammatory pathways involving nuclear factor-kappa B (NF-kB). Conflicting reports about the association between TLR4 and sarcoidosis have recently been published. It was shown that there is a significant association between patients with chronic sarcoidosis and pulation . Whereaspulation ,37. Our pulation .NOD2 with early-onset sarcoidosis and Blau syndrome. However, no significant associations between the genetic polymorphisms in the NOD2 gene and the risk of adult sarcoidosis were detected [Nucleotide-binding oligomerization domain 2 (NOD2), a member of the NLR (Nod-like receptor) family, is an intracellular microbial sensor. This protein detects muramyl dipeptide (MDP); a component of bacterial peptidoglycans, and induces innate immune responses. Several reports have suggested there may be an association of polymorphisms in detected ,38-41.In the present study, genetic variations in TLR2 did not affect ocular sarcoidosis risk. However, some types of genetic predispositions underlying the pathogenesis of sarcoidosis can lead to ocular inflammation. Microbial pathogens have long been suspected as the cause of sarcoidosis. Therefore, further studies are needed to analyze other genes involved in the innate immune response against bacterial antigens.TLR2 do not appear to be significantly relevant to sarcoidosis in the Japanese population. However, in cutaneous sarcoidosis, rs3804099 and rs3804100 SNPs in TLR2 are slightly associated with clinical disease. Further studies, especially in other ethnic populations, are required to elucidate what association there may be between sarcoidosis and TLR2.In summary, the minor allele frequencies of


Scar sarcoidosis is a rare and uncommon but specific cutaneous manifestation of sarcoidosis. In general it arises in pre-existing scars deriving from mechanical traumas. As most surgeons dealing with scars might not be aware of cutaneous sarcoidosis and its different types of appearance the appropriate staging and treatment might be missed or at least delayed. To our knowledge this is the first case in literature of scar sarcoidosis on a finger.We present a case of a 33-year-old carpenter who developed scar sarcoidosis on his right index finger 4 years after the tendon of the long digital flexor got accidentally cut by an angle grinder. He was referred due to a swelling of the finger suspected to be a malignant soft tissue tumour. The circumference of the affected finger had almost doubled, adding up to 94 mm. Incision biopsy revealed typical noncaseating granulomas. Further investigation showed a systemic extent of the disease with involvement of the lung. A systemic treatment with oral steroids led to an almost full regression of the swelling with restoration of function and resolution of lung infiltrates.In case of a suspicious and/or progressive swelling a definite diagnosis should be achieved by biopsy within a short time to enable a proper treatment. If scar sarcoidosis is proven further investigation is necessary to exclude a systemical involvement. A surgical treatment of the swelling is not indicated. Sarcoidosis is a systemic inflammatory disorder with formation of noncaseating granulomas. The incidence of this multisystem disease is 10–15 cases per 100.000 per year. Broad American studies show that sarcoidosis is 10–17 times more common in African American than in Caucasians-6. Scar We report on a 33-year-old carpenter with a solid swelling in the area of the palmar base and middle phalanx of his right index finger Figure A and B. The patient told us that this scar derived from an injury 4 years ago when the tendon of the deep digital flexor was cut by an angle grinder. After suturing the tendon appearance and function of the finger had been fully restored.Apart from enlarged soft tissue X-rays of the finger were unsuspicious. MRI-scans showed a considerable palmar soft tissue swelling with an enhancement of contrast medium with contact to the flexor tendon Figure A and B. For diagnosis and exclusion of the existence of a malignant and/or local aggressive soft tissue tumour we performed a small incision biopsy. The result of the histopathological examination was scar sarcoidosis with detection of typical noncaseating granulomas Figure . The patOral corticosteroid therapy with an initial daily dose of 40 mg prednisone was started and led to an efficient diminution of the swelling of the right index finger. Hilar lymphadenopathy and lung infiltrates resolved. After 12 months therapy with a stepwise decrease of the corticosteroid dosage the swelling of the finger had almost vanished and function and range of motion was totally restored Figure A and B. Besides lymphadenopathy an involvement of lung or other organs is a feature of sarcoidosis. Even destruction and deformation of bones have so far been described. In a reScar sarcoidosis is a form of cutaneous sarcoidosis. It occurs after mechanical harms of the skin like cuts, but even a venous puncture or a tattoo seem to be sufficient to cause it,3,7,10. Every suspicious or progressive swelling needs a complete work-up with consideration of differential diagnoses and radiological examinations . In case of cutaneous or subcutaneous swelling and a discoloration in the area of an old scar beside keloid a scar sarcoidosis is a possible differential diagnosis. For histological proof and exclusion of a malignant process a biopsy is to be performed. The biopsy offers the right diagnosis and thus leads to an appropriate, reasonable and successful treatment of sarcoidosis. In most cases satisfactory clinical results can be achieved by systemic or topic application of corticosteroids. Sometimes a long term therapy is needed, even if the local manifestation seems to be gone. Further local surgical procedures are not indicated.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.MH and JH are responsible for conception and writing of the manuscript, AS and GG have been involved in drafting and revising the manuscript. CS and CD were responsible for acquisition and analysis of data and findings. All authors read and approved the final manuscript.


Furthermore, the quantum capacitance increases very rapidly with the gate voltage for SLG, but this increase is much slowed down when n becomes greater. On the other hand, the magnitude of the EFM phase shift with respect to the SiO2 substrate increases with n at the sample bias of +2 V but decreases with n at -2 V. The difference in both quantum capacitance and EFM phase shift is significant between SLG and BLG but becomes much weaker between MLGs with a different n. The layer-dependent quantum capacitance behaviors of graphene could be attributed to their layer-dependent electronic structure as well as the layer-varied dependence on gate voltage, while the layer-dependent EFM phase shift is caused by not only the layer-dependent surface potential but also the layer-dependent capacitance derivation.The nanoscale electrical properties of single-layer graphene (SLG), bilayer graphene (BLG) and multilayer graphene (MLG) are studied by scanning capacitance microscopy (SCM) and electrostatic force microscopy (EFM). The quantum capacitance of graphene deduced from SCM results is found to increase with the layer number ( Graphene is drawing an increasing interest nowadays since its debut in reality  as it is2 layer. Although many novel methods have been used to fabricate graphene [C between the tip and the underlying semiconductor in response to a change in the applied ac bias ΔV could be obtained. The detailed operational modes of EFM and SCM have been reported elsewhere [The graphene samples were prepared by the mechanical exfoliation method  and depographene ,22, mechgraphene  is stillgraphene , tappinglsewhere . All the2 substrate. The profile of the marked line is shown in Figure 2 substrates are obtained in the same way. As the height of a graphene layer on top of graphene is close to the interlayer distance of graphite [h) as a function of the assigned layer number (n) by a straight line: h = nt + t0, as shown in Figure t = 0.37 nm which is in close agreement with the interlayer distance of graphite (approximately 0.335 nm) and the offset t0 = 0.15 nm which may be caused by the different interaction between tip-graphene and tip-SiO2 [0.37 + 0.15 = 0.52 nm, which is in agreement with the results of SLG reported in the literatures [h-n linear fitting results, area A is termed as SLG, area B as BLG, and area C (four-layer) and D (eight-layer) as MLG.Figure graphite ,25 we fieratures ,15. FromC/dV amplitude at sample DC biases of 0 V and +3 V are shown in Figure C/dV amplitude does vary with the number of graphene layers, and the differences between SLG, BLG, and MLG can be obviously observed from both images. As the ac voltage variation (ΔV) is kept constant in all measurements, the capacitance variation (ΔC) obtained by multiplying dC/dV amplitude with ΔV was adopted afterwards instead of the dC/dV amplitude. The line profiles of ΔC obtained on SLG and BLG are shown in Figure C values of SLG are slightly smaller than those of BLG, but at the DC bias of +3 V, the ΔC values of SLG are larger than those of BLG. Figure C with respect to the SiO2 substrate for SLG, BLG, and MLG obtained at 0 V and +3 V, respectively. The results show that at the DC bias of 0 V, the ΔC measured on graphene increases with n. The increase is fast when n increases from 1 to 4, and it slows down when n increases from 4 to 8. On the other hand, ΔC decreases with n for the case of +3 V DC bias. Moreover, the ΔC values measured on graphene layers are always smaller than those measured on the SiO2 substrate for both biases.SCM measurements were carried out on graphene with different layer numbers, and the images of dCtot) could be written as:As the capacitance measured on graphene is composed of two series capacitance: the quantum capacitance of graphene and the capacitance of the underlying oxide layer, according to the previous studies -16, the rs is the radius of the disk on which the nonstationary electron/hole charge is distributed). C'MOS and C'q are the unit area capacitance for tip/SiO2/Si structure and graphene, respectively. By considering the contact area, the capacitance measured on SiO2 substrate is Cq can be derived as:where Ctot and CMOS are the capacitances measured on the top of graphene layers and on the SiO2 substrate, respectively, but the ratio Atip/Aeff could not be obtained from the experiments. For FLG, the ratio was found to vary with the gate voltage, as well as the SiO2 thickness [2 existed; this ratio for FLG was approximately equal to 1 at the gate voltage of 0 V and changed slightly with the gate voltage, but its relation with n is not clear. As a rough approximation, we took Atip/Aeff = 1 for all grephene layers, thus the values of Cq can be calculated from Equation 2. The calculated values for different graphene layers at both DC voltages of 0 and 3 V are shown in Table In Equation 2, hickness -16. As rhickness , in the n. With +3 V bias applied, all quantum capacitance variations are much larger than their corresponding values at 0 V. The increase is mostly significant for SLG, which increased about 280 times. The increase magnitude, as shown in Table n. Therefore, the change of graphene quantum capacitance with the DC biases is dependent on n, resulting in the different layer-dependent quantum capacitances of graphene at 0 V and +3 V. Since SCM has been performed in the contact mode where the tip contacts with the surface, the DC bias applied between the tip and sample backside acts as the gate voltage. So our results indicate that the capacitance variations increase with the gate voltage for different graphene layers, and the increase magnitude decreases as n increases. In previous studies, both the SCM measurements on FLG [Atip/Aeff = 1 is used for different graphene layers, it may cause errors for the obtained Cq values, especially at a DC bias of +3 V. Nevertheless, the different quantum capacitance behaviors for graphene with different n are definite. As the quantum capacitance represents the density of states (DOS) at Fermi level [n [n, as shown in Table et al. that the work function could be tuned by the gate voltage, where they found that SLG showed larger work function changes with gate voltage than BLG did [EF) in graphene, which was different for SLG and BLG. Our results can be interpreted in the similar viewpoint. Different changes of EF with gate voltage for different graphene layers could result in different carrier density changes with gate voltage, so are the changes of the quantum capacitance with gate voltage.From Table s on FLG -16 and ts on FLG  showed tmi level ,27 and t BLG did . They exn at the sample biases of +2 V and -2 V at a lift height of 20 nm are shown in Figure 2 substrate is smaller than that between BLG and SiO2, while for the bias of -2 V, SLG has a larger phase shift with respect to SiO2 than BLG. Detailed correlations of the phase shift with n obtained at +2 V and -2 V are shown in Figure 2 substrate increases with n at +2 V but decreases with n at -2 V. In a previous report [et al. measured the EFM phase shifts on FLG ranged from 2 to 18 layers, and also observed the similar phase shift reverse for two-layer and three-layer graphene at tip biases of -2 V and +3 V. They suggested that the phase shift difference was related with the layer-varied surface potential. This suggestion is doubtful, since the phase shift of electrostatic force is composed of two factors, which could be written as [Meanwhile, the EFM results measured on graphene with different s report , Datta eitten as :k is the stiffness of the cantilever, Q is the quality factor, z is the tip-sample distance and C is the tip-sample capacitance. VDC is the applied bias, and Vsurf is the surface potential correlated with the difference between the tip and sample work functions (Vsurf = (Wtip-Wsample)/e). Hence, both surface potential and capacitance derivation (∂2C/∂z2) will contribute to the phase shift of electrostatic force. First let's estimate the surface potential contribution (Vdc - Vsurf)2 to the different phase shift between SLG and BLG. The work function different between SLG and BLG was reported by Yu et al. [2 substrate is about 5.0 eV and the same tip is applied , SLG should have a larger phase shift difference with respect to the SiO2 substrate than that of BLG for both biases. In other words, the difference in phase shift behavior between SLG and BLG could not only be attributed to their different surface potentials. Thus, the capacitance derivation should be another contribution to the phase shift. Our SCM results aforementioned do indicate that the quantum capacitance of graphene varies with n, and it is significantly dependent on the sample biases, which could be expected to induce different EFM phase shifts for different graphene layers at different samples biases.where u et al. , which iIn conclusion, the nanoscale electrical properties of graphene with different number of layers have been studied by SCM and EFM, and the layer dependences of capacitance variation and EFM phase shift are obtained. SLG, BLG, and MLG exhibit obvious differences in electrostatic force and capacitance behaviors. The different electrical properties obtained on different number of graphene layers could be mainly attributed to their different electronic properties.The authors declare that they have no competing interests.SHZ carried out the experiments. YL participated in the SCM and EFM studies. SHZ and XJY interpreted the results and wrote the manuscript. All authors read and approved the final manuscript.


Phylogenetic profiling has been widely used for comparing bacterial communities, but has so far been impossible to apply to viruses because of the lack of a single marker gene analogous to 16S rRNA. Here we developed a reference tree approach for matching viral sequences and applied it to the largest viral datasets available. The resulting technique, Shotgun UniFrac, was used to compare host-associated and non-host-associated phage communities , and revealed a profound split similar to that found with bacterial communities. This new informatics approach complements analysis of bacterial communities and promises to provide new insights into viral community dynamics, such as top-down versus bottom-up control of bacterial communities by viruses in a range of systems. The phylogenetic composition of bacterial communities is primarily determined by whether they are found in host-associated or free-living environments The lack of a single marker gene in viral genomes complicates phylogenetic profiling of viral communities, a powerful technique for studying microbial communities, and previous studies have focused on profiling viral gene functions We applied Shotgun UniFrac to 130 phage metagenomes from diverse environments. As observed with microbial communities, the primary factor separating metagenomes was whether they were derived from a free-living or host-associated environment. Host-associated environments vary more than a variety of free-living communities (considering only matches to the subset of viruses in the reference tree), and phage communities from the same host species tended to cluster .−23, one-tailed t-test; p<0.001, Monte Carlo t-test with 1000 iterations), suggesting stability in distal gut phage community membership over time.Our analysis also included 26 human feces phage metagenomes from 12 individuals with between 1 and 4 metagenomes per individual  (n = 6). Four overlapping metagenomes (Ocean (db:MG-RAST) and Ocean (db:CAMERA)), were used as controls to ensure that the source database did not affect the clustering results which is possible, for example, if one required preprocessing that the other did not.The number of input metagenomes by type were: Reclaimed water at discharge point (n = 1); Reclaimed water at point-of-use (n = 2); Freshwater stromatolite (n = 2); Hot Spring, Yellowstone National Park (n = 2); Potable water (n = 1); Saltern  (n = 5); Ocean (db:MG-RAST) (n = 4); Saltern  (n = 3); Northern Islands Coral (n = 4); Marine stromatolite (n = 1); Ocean (db:CAMERA) (n = 4); Freshwater (n = 4); Human feces (n = 80); Saltern  (n = 3); Healthy human lung (n = 2); Mosquito-associated (n = 3); Cystic fibrosis human lung (n = 3); Table S1OTU assignment statistics by metagenome.(XLS)Click here for additional data file.Table S2Description of metagenome types and sources.(XLS)Click here for additional data file.Table S3Full QIIME metadata mapping file.(XLS)Click here for additional data file.Table S4Full viral OTU table . These data were used in jackknifed weighted Shotgun UniFrac calculations .(XLS)Click here for additional data file.Table S5Viral OTU table sub-sampled to 200 sequences per metagenome. These data were used in weighted UniFrac calculations .(XLS)Click here for additional data file.


First-principles study on the electronic structure and transport property of the boron nitride sheet (BNC) structure, in which a triangular graphene flake surrounded by a hexagonal boron nitride sheet, is implemented. As the graphene flake becomes small and is more isolated by the boron nitride region, the magnetic ordering of the flake increases. When the BNC structure is connected to the graphene electrodes, the spin-polarized charge-density distribution appears only at the triangular graphene flake region, and the electronic structure of the graphene electrode is not spin polarized. First-principles transport calculation reveals that the transport property of the BNC structure is spin dependent. One of the key factors in the field of spintronics is the spin filter effect, which plays a fundamental role as the spin-polarized current source in devices such as spin-field-effect transistors and single solid-state qubits. The carbon-related nanostructures have recently been fabricated experimentally and explored theoretically to clarify magnetic ordering mainly in the zigzag edge of graphene -3. TheseIn the previous study , we inveAll calculations are performed in the framework of the density functional theory using the real-space finite-difference approach, which makes it possible to carry out the calculation with a high degree of accuracy by combining with timesaving double-grid technique and the direct minimization of the energy functional -11. The In the calculation for electron transport properties, we employ the computational model in which the graphene/BNC/graphene structure is sandwiched between the two graphene electrodes. The scattering wave functions from the left electrode are written as follows:Φ′s are the bulk wave functions inside the electrode and i is the index of the propagating waves from the electrode. The reflection coefficients r, transmission coefficients t, and the wave function in the scattering region ϕ are evaluated by the overbridging boundary-matching formula under the nonperiodic condition in the z direction [where irection ,15,16. Tirection :e, and h are a transmission coefficient matrix, the electron charge, and Planck’s constant, respectively.where T, k point used in the two-dimensional Brillouin zone integration is 16. For all the calculations in this paper, a repeating sheet model is separated by 17.0 bohr in each layer. The lattice constant is 2.67 bohr, which is obtained by the bond length of the graphene sheet. Structural optimization is performed until the remaining forces are less than 0.08 eV/bohr. Table n↑(r)−n↓(r) of the BNC structures. The BNC sheet with the smallest graphene flake is found to be the largest magnetic moment, and the spin-polarized charge-density distribution accumulates at the graphene flake region.In order to investigate the effect of the size of graphene flakes on the magnetic orderings, we first consider the three BNC structures under periodic boundary conditions for all directions. Figure n↑(r)−n↓(r). From Table At the next step, for the purpose of investigating the effect of the distance between the graphene flakes on the magnetic moments, the other three models are investigated. Figure p↑ and 2 p↓ orbitals of the boron atom (1.60 eV) is smaller than that of the nitrogen atom (3.88 eV) in the local spin density approximation [By comparing the other BNC structures investigated in a previous study , where tximation , resultix direction is performed by the equidistant sampling of four k points. The calculated magnetic moment of the graphene/BNC/graphene structure is found to be 1.14 μB. Figure For the investigation of the electron transport properties of the BNC structure, the electrodes have to be positioned at both sides of the BNC structure. Since the graphene structure is employed as the electrode in our study, we need to take into account whether the magnetic moments of the BNC structure are retained after the BNC structures are sandwiched between the graphene electrodes. Figure It is important to evaluate the spin transmissions quantitatively toward the application of a spin-filter material. Based on the results in the previous subsection, the spin-polarized transport property of the graphene/BNC/graphene structure is investigated. Figure s is donated by σs(EF). The spin-polarization ratio of the graphene/BNC/graphene structure is found to be approximately 0.95 at the Fermi level, which is comparable to that obtained with ferromagnetic tunnel junctions using a transition metal [P(EF) in the present study is smaller than that in the previous study [to characterize the spin polarization of the electron current, where the conductance of spin on metal . Howeverus study  due to tus study . In the us study .We have investigated the magnetic ordering and transport property of the BNC structure suspended between the graphene electrodes by first-principles calculations. The magnetic moment of the BNC structure under the conventional periodic boundary conditions increases as the size of the graphene flake becomes small and the spin-polarized charge-density distribution accumulates at the graphene flake region. It is also found that the spin-polarized charge-density distribution is preserved at the graphene flake when the BNC structure is connected to the graphene electrodes. The magnetic moment is smaller than that of the BNC structures examined in the previous study  because The authors declare that they have no competing interests.TO (T Ota) carried out preliminary calculations and drifted the manuscript. TO (T Ono) developed the computational code, implemented the calculations, and completed the manuscript. Both authors read and approved the final manuscript.


The results reveal that the obtained films exhibit improved mechanical properties with the enhancement of tensile strength and Young's modulus by as high as 82% and 136%, respectively. The electrical properties of graphene-Ag composite films were studied as well, with the sheet resistance of which reaching lower than approximately 600 Ω/□. The graphene-Ag composite films can be expected to find interesting applications in the area of nanoelectronics, sensors, transparent electrodes, supercapacitors, and nanocomposites.In this paper, we have reported that paper-like graphene-Ag composite films could be prepared by a facile and novel chemical reduction method at a large scale. Using ascorbic acid as a reducing agent, graphene oxide films dipped in Ag Many operties ,6, fieldoperties , sensorsoperties -10, tranoperties -14, and operties -18, havein situ thermal reduction [Large-scale preparation of paper-like graphene films has aroused much attention for their unique mechanical and electrical properties ,16,19-22eduction . As a reeduction  and as aeduction .in situ reduction of graphene oxide films together with Ag+ by ascorbic acid. The mechanical and electrical properties of the obtained graphene-Ag composite films are also investigated.Although many efforts and applications have been achieved for these novel carbon films, it is still a great challenge to develop a novel method to prepare the films at a large scale. Herein, we report a new method to prepare graphene-Ag composite films with excellent and improved properties, which are fabricated by the large-scale assembly of graphene oxide films, followed by The natural graphite powder (carbon content 99.999%) in the experiment was purchased from Qingdao Tianyuan Carbon Co. Ltd, Qingdao, China. Other solvents and reagents were of analytical reagent grade and used as received.3 aqueous solution , and the graphene oxide films were soaked in the mixed solution for 5 h. The schematic illustration of two chemical synthesis routes is described in Figure Graphene oxide was synthesized through the modified Hummers method  as stateλ = 1.5418 Å) and 2θ from 5° to 85°. The Raman spectra were obtained using a Senterra R200-L Raman spectrometer  with a 514-nm line of laser source. Fourier transform infrared (FTIR) spectra were recorded using a Vertex 70 vacuum FTIR spectrometer (Bruker Optik GmbH) and scanned from 4,000 to 400 cm−1 with KBr as background. Thermogravimetric analysis  was performed under a highly pure nitrogen atmosphere with a heating rate of 1°C to 10°C/min from 30°C to 700°C. The films with 5-mm width and 4- to 5-cm length were measured by dynamic mechanical analysis  at the room temperature. A four-probe detector  was used to measure the sheet resistance of the films.Atomic force microscope (AFM) image was taken with the Multimode Nanoscope V scanning probe microscopy system  using tapping mode with Picoscan v5.3.3 software. The morphology of the films were observed using a scanning electron microscope (SEM) using a Carl Zeiss ULTRA 55  with energy dispersive X-ray (EDX) spectrometry mode. The crystal structures of the films were examined by X-ray diffraction  with Cu Kα  and G band . The G band represents the plane vibrations with E2g symmetry and is mainly sensitive to the configuration of sp2 sites, while the D band is related to the breathing mode of κ-point phonon of A1g symmetry. As the graphite was oxidized into graphene oxide, the G band is broadened and the D band increases substantially, indicating the decrease in size of the in-plane sp2 sites, possibly because of the extensive oxidation and ultrasonic exfoliation [ID/IG intensity ratio of graphene is observed compared to that of the graphene oxide. Finally, when AgNO3 was used, the increase of D band also occurred. This change suggests an increase in the average size of the sp2 sites upon reduction of graphene oxide, which indicates that the reduction reaction has taken place and agrees well with the Raman spectrum of the graphene oxide reduced by hydrazine as reported by Stankovich et al. [The Raman scattering signals were measured on the graphite powder , the peaks seem to be similar with those of samples c and d since a few existing Ag particles do not block the reaction. However, when the amount of AgNO3 is excessive as 20 mg (sample h) and 300 mg (sample i), all peaks become stronger again, which means that the side effects will arise gradually as the amount of AgNO3 increases.FTIR is used to characterize the functional groups in the films shown in Figure on peaks ,16. As f3 from 2 to 300 mg under nitrogen atmosphere. In the left image of Figure 3 is appropriate (no more than 10 mg), the TGA curves of graphene-Ag composite films exhibited a mass loss at a temperature lower than 600°C, slightly lesser than that of graphene reduced only by ascorbic acid. However, when the amount of AgNO3 is 20 mg and 300 mg, the TGA curves of the composite films turned out to have the same trend as that of graphene oxide. The right image of Figure 3 is increased, and more than 15% weight is left at 690°C as the AgNO3 is excessive up to 300 mg. We can also find that the residue weight of samples i and j has a little difference with the EDX results. It may be due to the excessive Ag particles which aggregated and deposited nonuniformly on the surface of the graphene-Ag composite films.Thermogravimetric analysis has also been performed. Figure The mechanical properties of graphene oxide films and graphene films have also been studied, as shown in Figure 3 is no more than 10 mg, the sheet resistance decreases; on the other hand, when the amount of AgNO3 is 20 mg, the sheet resistance increases. When the optimal amount of AgNO3 is 10 mg, a minimum sheet resistance of approximately 600 Ω/□ for graphene-Ag composite films is obtained. It can be found that the conductivity of the resultant graphene-Ag composite films can be improved greatly via the uniform decoration of Ag particles.Finally, the sheet resistance of these films was measured using the four-probe detector as shown in Figure + aqueous solution, Ag particles can be uniformly distributed on the surface of graphene films using ascorbic acid as a reducing agent. The morphology of the composite films can be maintained during the reduction process. The obtained films have been characterized by AFM, SEM, XRD, Raman, FTIR, TGA, DMA, and a four-probe detector. The results show that the obtained films exhibit improved mechanical properties with the enhancement of tensile strength and Young's modulus by as high as 82% and 136%, respectively. The electrical properties of the graphene-Ag composite films were studied as well, with the sheet resistance of which reaching lower than approximately 600 Ω/□. The composite films hold a great potential for applications in the fields of nanoelectronics, sensors, transparent electrodes, supercapacitors, and nanocomposites.In summary, we have demonstrated that graphene-Ag composite films are fabricated in a large scale using a facile chemical reduction method. The graphene oxide sheets can be easily assembled to form free-standing graphene oxide films during the volatilization process on PTFE hydrophobic substrate. After dipping the graphene oxide films into the AgThe authors declare that they have no competing interests.RGG carried out the sample preparation, participated on its analysis, performed all the analyses except AFM and FTIR analyses, and wrote the paper. NTH also wrote the paper and analyzed the samples. JC performed the FTIR analysis. QRZ participated on the AFM analysis and proof corrections. ZY, YJS, LYZ, and YFZ participated in the study guidance and paper correction. All authors read and approved the final manuscript.