There were 8,385 cases with a diagnosis of sarcoidosis representing 0.054%  of all hospitalizations in Switzerland. These cases were compared with age- and sex-matched controls without the diagnosis of sarcoidosis. Hospitalization and mortality rates in Switzerland remained stable over the observed time period. Comorbidity analysis revealed that sarcoidosis patients had significantly higher medication-related comorbidities compared to matched controls, probably due to systemic corticosteroids and immunosuppressive therapy. Sarcoidosis patients were also more frequently re-hospitalized , had a longer hospital stay , had more comorbidities , and had a significantly higher in-hospital mortality (2.6% [95% CI 2.3%-2.9%] vs. 1.8% [95% CI 1.5%-2.1%] (p < 0.001). A worse outcome was observed among sarcoidosis patients having co-occurrence of associated respiratory diseases. Moreover, age was an important risk factor for re-hospitalization.Sarcoidosis is a multisystem disease with an unpredictable and sometimes fatal course while the underlying pathomechanism is still unclear. Reasons of the increasing hospitalization rate and mortality in the United States remain in dispute but incriminated are a number of distinct comorbidities and risk factors as well as the application of more aggressive therapeutic agents. Studies reflecting the recent development in central Europe are lacking. Our aim was to investigate the recent mortality and hospitalization rates as well as the underlying comorbidities of hospitalized sarcoidosis patients in Switzerland. In this longitudinal, nested case-control study, a nation-wide database provided by the Swiss Federal Office for Statistics enclosing every hospital entry covering the years 2002–2012 ( Sarcoidosis is a systemic disease of unknown cause resolving in complete remission in many cases but sometimes leading to substantial and even fatal organic dysfunctions . NowadayThe aim of this study was to investigate the hospitalization rate and in-hospital mortality among sarcoidosis patients over the last years in Switzerland, as well as the associated comorbidities and risk factors of this disease.http://www.who.int/classifications/icd/en/). The coding version ICD-10 was uniformly used all along the study time period.All our observations were derived from a dataset provided by the Swiss Federal Office for Statistics, which offers a nation-wide coverage of all hospitalized patients since 1998. In this database, the patient information is fully anonymized. No written informed consent was given to the patients who were unidentifiable due to the anonymization. The data belong to the Swiss Federal Office for Statistics  who provides regulated access to the data for research purpose. In analogy to the study of Deubelbeiss and colleagues  or Baty For the current study, sarcoidosis was defined by the ICD-10 code D86*. The cases were defined as any hospitalizations with the diagnosis of sarcoidosis either as main or concomitant diagnosis. Geriatric, obstetric, psychiatric hospitalization and cases of rehabilitation were excluded from the study. The controls consisted of hospitalizations not including sarcoidosis in the main or concomitant diagnoses. Controls were extracted from the hospitalization database using a random procedure and matching the cases 1:1 for age, gender, and month of hospitalization.Sarcoidosis-comorbidities were defined as any additional diagnosis coded by the ICD-10 nomenclature. The Charlson’s comorbidity index  was usedp-values from likelihood ratio tests. Time to first re-hospitalization was defined as the time elapsed between the first and second hospitalization record of a given sarcoidosis patient in the database. Data from patients without re-hospitalization, i.e. recorded only once in the database, were censored (lost to follow-up censoring). Time—to—event data were analyzed using Kaplan-Meier estimates, log-rank tests and Cox-proportional hazards regression. Results were reported as hazard ratio (HR) with 95% confidence intervals (CI) together with the Wald test statistic. Hazard ratios provide the percentage of increase in the hazard rates for an increase of one unit in the explanatory variable. Generalized estimating equations (using working independence correlation structure) was used to test associations with one or several covariates while accounting for within-patient variability.Descriptive statistics were reported as median and inter-quartile-range (IQR). The enrichment of sarcoidosis-associated comorbidities was tested using conditional logistic regression in order to account for the way the controls were sampled in our nested case-control design. Results were reported as odds-ratio (OR) with 95% confidence intervals (CI) together with Principal component analysis (PCA) was used to explore correlations among sarcoidosis-associated comorbidities. PCA depicts how comorbidities are related to each other and how sarcoidosis cases are grouped according to comorbidities. A data-matrix summarizing the presence/absence (1/0) of comorbidities in sarcoidosis cases was created. Scaled PCA was applied to the comorbidity matrix. The role played by external explanatory variables, including age, length-of-hospital stay, gender, in-hospital death, hospitalization rate, and number of comorbidities, was further explored using vector fitting procedure to identify the directions of maximal correlation with the external variables in the PCA space . PCA ressurvival, geepack, Epi, multipleNCC, ade4 and vegan.All analyses were done at the hospitalization level, except for the re-hospitalization analysis which was done at the patient level. All statistical computations were done using the R statistical software (version 3.1.2) . The folThe number of hospitalized cases with a diagnosis of sarcoidosis either as main or secondary diagnosis between 2002 and 2012 was 8,385 corresponding to 0.054%  of all hospitalizations in Switzerland, and representing 4,664 unique patients . OverallThe proportion of hospitalizations due to sarcoidosis was age-dependent. A higher percentage of hospitalization was found among patients aged 40–50 years (1%) and elderly patients defined as more than 70 year old (6.3%). Re-hospitalizations occurred 1.80 times per patient on average .In 30% of cases, sarcoidosis was the main reason for hospitalization (primary diagnosis).p < 0.001)  .p < 0.001) and (70–96] (p < 0.001). The in-hospital mortality rate (lower panel) did not increase significantly in any age category over the observed time period (2002–2012), although there was a trend towards an increase of cases in the older category (age >70) (p = 0.085) (= 0.085) .p < 0.001), had a longer hospital stay , had more comorbidities , and had a significantly higher in-hospital mortality (2.6% [95% CI 2.3%-2.9%] vs. 1.8% [95% CI 1.5%-2.1%] (p < 0.001).The characteristics of sarcoidosis cases and matched controls are summarized in p < 0.05). Of these, 57 with the lowest p-values (p < 1 × 10−10) were kept in the analysis  and a lower rate of cancer disease . Under-represented comorbidities among sarcoidosis cases consisted of orthopedical, gestational, geriatric, and mental disorders.Sarcoidosis cases had a higher rate of pulmonary embolism . Cases with the combined diagnosis were older  and tended to be more frequent among females (sex ratio: 0.42). They had a worse outcome with more comorbidities , a longer in-hospital stay  and a higher chance of in-hospital mortality  in comparison to sarcoidosis cases without pulmonary embolism.Overall, there were 119 hospitalized cases with sarcoidosis and pulmonary embolism (ICD-10 code: I26*), corresponding to 1.4% of all sarcoidosis hospitalizations. Compared to the control group, sarcoidosis cases had a higher rate of pulmonary embolism . The risk of re-hospitalization was also significantly higher in patients having a larger amount of comorbidities with an increase of 5.4% in the hazard rates for each additional comorbidity . No statistically significant difference was found between females and males . In this longitudinal, nested case-control study, we investigated the recent mortality and hospitalization rates as well as the underlying comorbidities of hospitalized sarcoidosis patients in Switzerland from 2002 to 2012. Furthermore, we analyzed possible risk factors and predictors of outcome compared to matched controls.Overall, our analysis showed stable mortality and hospitalization rates among sarcoidosis patients in Switzerland over the observed time period. This is in contrast to recent findings of a significant mortality increase, at least for Caucasians, in the United States –6. ReasoBy clearly displaying the burden of the disease, our data reveal that sarcoidosis patients showed a significantly worse outcome compared to matched controls regarding re-hospitalization, in-hospital mortality and length of hospital stay. Similar results were recently found when comparing sarcoidosis patients with pedestrian traffic accident patients . In the In our study, we identified possible predictors and risk factors associated with a worse outcome. We found that age was a significant prognostic factor of the disease, which is supported by other previous studies . There wSpecific comorbidities possibly contributing to a worse outcome were also detected. For instance, associated respiratory diseases were significantly correlated to a longer hospital stay and a higher number of comorbidities. This defines a phenotype of sarcoidosis patients with a higher morbidity. Among these associated respiratory diseases, there was a higher risk of pulmonary embolism. A link between sarcoidosis and pulmonary embolism was postulated in previous studies  while thMedication-related events seem to substantially affect sarcoidosis patients leading to a significantly higher hospitalization rate as well as to a shorter time to re-hospitalization. Long-term benefits of the corticosteroidal and immunosuppressive therapy are often discussed controversially . Our finOne of the strength of our study is the comprehensive number of patients registered by the Swiss coding system, providing good data quality. In addition, this study is one of a few European studies investigating hospitalizations and mortality rates among sarcoidosis patients and the only one displaying risk factors for re-hospitalization. As a limitation, our analysis is based on a database which is essentially hospitalization-oriented. Our cohort only included hospitalized sarcoidosis cases while a high fraction of patients is typically treated in an out-patient setting. Further information such as medication or laboratory data were not available nor was it possible to contact patients to complete or confirm data.Overall, the current study is showing stable mortality and hospitalization rates among sarcoidosis patients over the observed time period. However, the burden of the disease becomes apparent, as patients with sarcoidosis show a significantly worse outcome compared to matched controls. This could be due to the disease itself as well as to a cluster of specific comorbidities and complications namely associated respiratory diseases. On the other hand, there is also evidence that side effects of more aggressive treatment approaches could explain this poorer outcome, probably especially affecting patients at older age.A better knowledge of these prognostic factors could lead to a better understanding of the disease and guide to a more specific monitoring as well as more individualized therapeutic approaches. Further research should be done to evaluate this and maybe contribute to a better outcome.


As a result, thin magnesium hydroxide layers spontaneously grew, giving rise to characteristic volume expansion effects, which depended on the size of the particles. Rounding of the originally sharp cube edges leads to a significant loss of the morphological definition specific to the MgO cubes. Comparison of different regions within one sample before and after exposure to liquid water reveals different transformation processes, such as the formation of Mg(OH)2 shells that act as diffusion barriers for MgO dissolution or the evolution of brucite nanosheets organized in characteristic flower-like microstructures. The findings underline the significant metastability of nanomaterials under both ambient and high-vacuum conditions and show the dramatic effect of ubiquitous water films during storage and characterization of oxide nanomaterials.Physisorbed water originating from exposure to the ambient can have a strong impact on the structure and chemistry of oxide nanomaterials. The effect can be particularly pronounced when these oxides are in physical contact with a solid substrate such as the ones used for immobilization to perform electron or ion microscopy imaging. We used helium ion microscopy (HIM) and investigated morphological changes of vapor-phase-grown MgO cubes after vacuum annealing and pressing into foils of soft and high purity indium. The indium foils were either used as obtained or, for reference, subjected to vacuum drying. After four days of storage in the vacuum chamber of the microscope and at a base pressure of  Knowledge about the stability of engineered nanomaterials in aqueous systems is critical for predicting their functionality under environmental conditions. For example, for transient electronics it has become a major challenge to understand and control the factors of materials transformation in aqueous systems –2. UnderAmong the many microscopic techniques available –6 scanniAdditionally, metal oxides can also be damaged by an electron beam . Indeed,2 (from ρmagnesia = 3.5 g·cm−3 to ρbrucite = 2.3–2.4 g·cm−3) generates a significant volume expansion. For refractory materials, related phenomena can have desired [The density mismatch that follows the conversion from MgO to Mg(OH) desired  as well  desired . In the There is growing awareness in the scientific community  that a vT = 1173 K for a minimum duration of one hour in order to eliminate surface adsorbates including residual hydroxyls of high thermal stability [−7 mbar) at room temperature for four days. The latter procedure was applied in order to remove physisorbed species such as weakly bound water that forms during exposure of the foil to the ambient. The shape change of the cubes was quantified by image analysis, using the edge length increase factor, defined as L2/L1, where L1 and L2 are the edge lengths before and after high-vacuum treatment in the HIM chamber, respectively. Since it is not possible to simultaneously measure the three edge lengths of the cubes, we calculated the corresponding volume increase factor, defined as (L2/L1)3. More than 45 edge lengths were measured before and after high-vacuum treatment, for both types of samples . The cubes selected for the measurements had an estimated maximum tilt angle of 30° relative to the incident ion beam. This does not change the edge length increase factor (L2/L1), but induces a maximum error of less than 15% in edge length (L1). To verify that the tilt did not, indeed, affect our measurements, we measured cubes that were lying exactly flat on the surface (tilt = 0°) and found essentially the same dependence of L2/L1 on L1 . The microscope is equipped with an Everhardt–Thornley (ET) detector to record secondary electron images. The acceleration voltage of the ions and the beam current were 30 kV and approx. 0.25 pA, respectively. The MgO cube powders were vacuum annealed at −7 mbar, HIM imaging reveals that important morphological changes of the MgO cubes have occurred 2 layer coats the MgO cubes, leading to a partial fusion of the particles.After four days under high-vacuum conditions at a base pressure lower than 3·10occurred . As showL2/L1 of 1.14 ± 0.10 (standard deviation of the measurement) when the indium foil was used as received, where L1 and L2 are the edge lengths before and after high vacuum treatment, respectively. This corresponds to a volume increase factor (L2/L1)3 of 1.52 ± 0.44 . In the case of a complete transformation of MgO cubes into Mg(OH)2 cubes, a similar volume increase factor, i.e., 1.52, would be expected. The high value of the volume expansion measured is attributed to the Kirkendall effect, which has been observed many times in the case of solid-to-solid transformations [2+, and H2O molecules/hydroxide ions) are thus expected to lead to the growth of the Mg(OH)2 layer outwards the cubes, explaining the large increase in volume measured here. The proposed transformation process, which is schematically shown in Figure S2 of As shown in rmations –28. SuchFurthermore, a clear size dependence is observed as shown in Interestingly, contrast changes between the different imaging sessions are quite evident . In this15 cm−2. Because the defects mainly occur close to the stopping range of the ions, which is ca. 160 nm according to the SRIM software [The build-up of carbon on the sample areas during imaging may appear similar. As illustrated in , the secsoftware , it is c2+ and OH− ions and lamellar Mg(OH)2 structures crystallize and give rise to flower-like morphologies  [We performed exploratory experiments to address the impact of liquid water on thermally activated MgO cubes. A comparison was performed on sample spots having cubes of different sizes put in contact with a water droplet  , as a mi (BaSO4) –38. Inde (BaSO4) ,39. The 2 core–shell structures as a result of the Kirkendall effect.This work underlines the metastability of metal oxide nanostructures and reports drastic material changes during storage under high vacuum conditions. We observed significant and size-dependent volume expansion effects of the MgO cubes that were grown and processed in dry vacuum environments prior to imaging with helium ion microscopy. The volume expansions observed are attributed to surface reactions between the MgO cubes and water molecules originating as physisorbed species from an indium foil that was employed as a conductive substrate for imaging. Depending on their size the MgO-based cubes  become sThese results are significant and should be relevant for everyone working with nanomaterials under ambient and high-vacuum conditions. Conductive substrates require appropriate drying and surface cleaning prior to sample immobilization and measurement. Apart from the transformation behavior of cubic oxides such as MgO, NiO and CdO and its relevance for the synthesis of hydroxides with controlled morphologies –41, the File 1Additional experimental data.


The exposure to graphene oxide did not induce significant changes in the body weights, organ weights, and food consumption during the 14 days of recovery time. The microalbumin and lactate dehydrogenase levels in the bronchoalveolar lavage (BAL) fluid were not significantly changed due to the exposure. Similarly, total cell count, macrophages, polymorphonuclear leukocytes, and lymphocytes were not significantly altered in the BAL fluid. Plus, the histopathological examination of the rat lungs only showed an uptake of graphene oxide in the alveolar macrophages of the high-concentration group. Therefore, these results demonstrate that the single inhalation exposure to graphene oxide induce minimal toxic responses in rat lungs at the concentrations and time points used in the present study.Graphene is receiving increased attention due to its potential widespread applications in future. However, the health effects of graphene have not yet been well studied. Therefore, this study examined the pulmonary effects of graphene oxide using male Sprague-Dawley rats and a single 6-hour nose-only inhalation technique. Following the exposure, the rats were allowed to recover for 1 day, 7 days, or 14 days. A total of three groups were compared: control (fresh air), low concentration (0.46 ± 0.06 mg/m Graphene is a two-dimensional graphitic carbon nanomaterial with unique physical and chemical properties that enable a wide variety of applications . Plus, gα (TNF-α), tumor growth factor-β (TGF-β), and interleukin-1β (IL-1β) and IL-6, were significantly elevated in the bronchoalveolar lavage (BAL) fluid during the recovery period of 28 days. In another study, graphene exposure increased the polymorphonuclear neutrophil (PMN) levels in the BAL fluid from male Wistar rats following exposure to graphene at target concentrations of 2.5 or 10.0 mg/m3 for 6 hours per day for 5 consecutive days with 7 or 28 days of recovery [β were also increased at day 7 in a dose-dependent manner, while graphene-loaded alveolar macrophages were observed in the lungs of all the treated animals in a concentration-dependent manner. Meanwhile, when using a pharyngeal aspiration technique with female C57BL/6 mice, graphene nanoplatelets (50 μg) were found to increase the pulmonary PMN number and proinflammatory cytokines, such as monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and IL-1β, in the BAL fluid [As a result, several toxicological studies have recently been performed to determine the toxicity of graphene nanomaterials. For example, graphene nanoplatelets were found to accumulate in mouse lungs for up to 28 days after a single intratracheal instillation at a dose of 2.5 or 5.0 mg/kg . Plus, aAL fluid , 6. in vivo and in vitro studies, the inhalation of graphene nanomaterials would be expected to exert toxicity in humans, as in experimental animals. However, since only a small amount of data with limited experimental designs has so far been reported, more data obtained from a variety of experimental settings needs to be accumulated. We also hypothesized that relatively lower concentration of graphene exposure would represent more valuable outcome in order to translate into a human exposure scenario. Also, the selected concentrations were based on the previous experimental setting employed in inhalation toxicity studies for graphene nanomaterials [3 or 3 mg/m3) for 6 hours using a nose-only inhalation chamber and then allowed to recover for 1 day, 7 days, or 14 days. The results showed that the graphene oxide nanomaterials produced minimal toxic effects in the male rats for the experimental settings used in this study.Thus, according to these findings obtained fromaterials . In addiThe graphene oxide nanomaterials were kindly provided by Dr. Heon Sang Lee of Dong-A University . A transmission electron microscope equipped with an energy-dispersive X-ray analyzer (TEM-EDX) was used to measure the graphene oxide nanomaterials based on National Institute for Occupational Safety and Health (NIOSH) analytical method 7402 [3 and 3 mg/m3 for the low- and high-concentration chamber, respectively.Male Sprague-Dawley (SD) rats were exposed to the graphene oxide nanomaterials using a nose-only exposure system . The nose-only exposure system provided reliable exposure condition with accuracy and consistency. Thus, this exposure system was extensively used for inhalation of carbon-based nanomaterials including graphene in our laboratory and by others –10. The  ad libitum. During the acclimation period, the animals were trained to adapt to the nose-only inhalation chamber. The rats were divided into 3 groups: control , low-concentration group (n = 12), and high-concentration group (n = 12). The low- and high-concentration groups were exposed to the graphene oxide for a single period of 6 hours, while the control group received filtered fresh air. The animals were examined daily for any evidence of exposure-related toxic responses. The body weights were measured at the time of purchase, at the time of grouping, after the 6 h inhalation exposure, and before necropsy. The food consumption (g/rat/day) was measured once a week. After the single 6 h inhalation exposure to graphene oxide, the rats were allowed to recover for 1 day, 7 days, or 14 days (n = 4 per treatment group for each time period) to investigate the toxic responses. At sacrifice, gross observations of the organs were recorded, and the testes, kidneys, spleen, liver, lungs, and brain were all carefully removed and weighed. All the animal protocols were approved by Hanyang University Institutional Animal Care and Use Committee.The six-week-old male specific-pathogen free SD rats were purchased from OrientBio  and acclimated for 2 weeks before initiating the inhalation exposure. During the acclimation and inhalation exposure, the rats were housed in polycarbonate cages (maximum of 3 rats per cage) installed in individually ventilated cage racks. The rats were kept under a controlled temperature (21.8 ± 0.17°C) and humidity (49.16 ± 2.37%) and a 12 h light/dark cycle. The rats were fed a rodent diet  and filtered waterThe concentrations of graphene oxide in the chambers were measured using polycarbon filters connected to a MAS Escort ELF sampling pump  at a flow rate of 1.0 L/min. The weight difference of the polycarbon filter before and after sampling was calculated. The size distribution of the graphene oxide was then measured directly using a scanning nanoparticle spectrometer  connected to a condensation particle counter  and dust monitor . The volume of sheath air and polydispersed aerosol air used in the SNPS and CPC was 15 and 1.5 L/min, respectively.α (TNF-α), interleukin- (IL-) 1β, IL-18, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF) in the BAL fluid was measured using a Bio-Plex Rat 23-Plex assay  according to the manufacturer's protocol. The level of tumor growth factor β1 (TGF-β1), matrix metallopeptidase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) was measured using Quantikine ELISA kit  according to the manufacturer's instructions.At sacrifice, the right lungs were 4 times with 3 mL aliguots of cold 0.9% NaCl solution . The BAL fluid samples were then centrifuged for 7 min at 500 ×g, and the BAL cells were collected and resuspended in 1 mL of 0.9% NaCl solution for evaluation. The total cells number was determined using a hemocytometer. The cells were first smeared and then stained with Wright Giemsa Sure Stain for counting the total number of cells, macrophages, polymorphonuclear leukocytes (PMNs), and lymphocytes. The BAL levels of microalbumin and lactate dehydrogenase (LDH) were also measured using reagents obtained from Randox Laboratories Ltd.  and Waco Pure Chemical Industries, Ltd. , respectively. The level of inflammatory cytokines such as tumor necrosis factor-At sacrifice, the left lungs were removed and fixed in a 10% formalin solution containing neutral PBS under 25 cm water pressure followed by embedding in paraffin and staining with hematoxylin and eosin. The stained lung tissue sections were examined under a light microscope for histopathological evaluation.P < 0.05 and P < 0.01.The statistical analysis was performed using SPSS (Version 19) and the statistical evaluation performed using an analysis of variance (ANOVA) following multiple comparison tests using Duncan's method. The level of statistical significance was set at θ = 26.4°, corresponding to the interlayer spacing (d = 0.34 nm). The peak in the XRD pattern for the dried graphene oxide particles showed broadening, as well as a shift to a lower angle (2θ = 9.5°), indicating that the interlayers were 0.93 nm apart due to intercalation by the hydroxyl, carbonyl, and epoxide groups and moisture. The DLS measurement showed the average flake size of the graphene oxide in a solution. Plus, the equivalent hydrodynamic diameter of the graphene oxide was estimated using Stokes-Einstein equation to be 150–250 nm (The field emission TEM (FE-TEM) analysis characterized the graphene oxide as a folded and stacked platelet structure Figures . The TEM0–250 nm .3) measured based on the weight of the polycarbon filter before and after sampling were 0.46 ± 0.06 and 3.76 ± 0.24 for the low- and high-concentration groups, respectively (3) in the chambers was measured directly using SNPS and was 3.33 × 106 ± 2.95 × 105 and 6.17 × 106 ± 4.13 × 105 for the low- and high-concentration groups of graphene oxide, respectively (2/cm3) of the graphene oxide were 50.6 ± 1.82 nm and 6.45 × 1010 ± 7.04 × 109 nm2/cm3, respectively, for the low-concentration chamber and 72.9 ± 2.02 nm and 2.72 × 1011 ± 3.70 × 1010 nm2/cm3, respectively, for the high-concentration chamber . No significant body weight changes were observed for the low- and high-concentration groups during the exposure and recovery periods (Supplement 2). The examination of the rat organs, including the testes, kidneys, spleen, liver, lungs, and brain, also revealed no significant clinical signs or organ weight changes during the observation period (Supplements 3–11).No significant gross effects were observed during the exposure and recovery periods. Also, there were no significant differences in food intake between the control and graphene oxide-treated groups  in order to observe the causality and dose-dependent toxic responses in animals. Concentrations of graphene oxide were chosen based on preliminary data which presented dose-dependent toxic responses after 5-day repeated inhalation of graphene nanomaterial. Moreover, our concentration is more relevant to worker exposure levels compared to other previous study which used up to 10 mg/m3 [μg/m3 in the work area sampling [Since graphene nanomaterials are experiencing rapid development with predicted widespread application, concerns about human exposure have also been recently increasing. However, safety information, particularly the inhalation toxicity of graphene-based nanomaterials, remains limited . Therefosampling .3, 3.05 mg/m3, and 10.1 mg/m3) and a longer period of inhalation (6 hours per day for 5 consecutive days) and similar concentration (0.68 and 3.86 3.05 mg/m3) and exposure period (6 hours per day for 5 consecutive days and 28 days after exposure) [Overall, no significant clinical changes due to graphene oxide exposure were observed during the 6-hour inhalation and 14-day recovery period, unlike other carbon nanomaterial toxicity studies such as MWCNTs and SWCNTs , 15, 16.α, IL-1β, IL-18, G-CSF, M-CSF, VEGF, TGF-β1, MMP-9, and TIMP-1) demonstrated that only MMP-9 (day 1) and IL-18 and TGF-β1 (day 7) were significantly elevated due to exposure of graphene oxide at high concentrations. These elevated parameters returned to baseline level after 14-day recovery period. This temporary elevation of MMP-9, IL-18, and TGF-β1 may be due to phagocytosis of graphene oxide by alveolar macrophage and other types of lung cells [μg of unoxidized graphene platelets using a pharyngeal aspiration technique and the results demonstrated significant increases in LDH and the total cells and PMNs numbers at 24 h after exposure [μg graphene (approximately 2.5 mg/kg mouse) used in the previous study was significantly higher than the doses used in the present study, where the approximate deposited doses were 0.026 mg (0.087 mg/kg rat) for the high-concentration group and 0.003 mg (0.01 mg/kg) for the low-concentration group assuming a deposition rate of 10% and a minute ventilation of 0.19L/min in a 300 g rat [3 × (0.19 L/min) × 6 h × 0.1 = 0.003 mg deposited a day (0.46/1000 × 0.19 × 360 min × 0.1 = 0.003); high-concentration group, 3.76 mg/m3 × (0.19 L/min) × 6 h × 0.1 = 0.026 mg deposited a day (3.76/1000 × 0.19 × 360 min × 0.1 = 0.026). Another previous study also showed markedly elevated damage parameters in the BAL fluid [3 for 6 h/d, for 5 days with 7 or 28 days of recovery) showed an elevated number of total cells and lymphocytes at 10.01 mg/m3 and PMNs at 3.05 mg/m3. Yet, the particle concentrations were significantly higher than the concentrations used in the present study. Interestingly, the lowest concentration  in the previous study did not increase the lung toxicity parameters, and this concentration is similar to the high concentration  used in the present study in terms of the approximate total deposited dose in the rat lungs. The previous study also found a dose-dependent increase in the LDH levels in the BAL fluid after exposure to graphene. Thus, when taken collectively, while the experimental designs differed significantly between the two studies, the dose would seem to be the major factor determining the toxicity of graphene.The present study also observed several toxicological indicators to determine the toxicity of graphene oxide in the exposed rats. However, the results showed limited level of toxicity in the lungs of the exposed rats. BAL fluid evaluation of nine inflammatory parameters . Therefore, this data demonstrated that the exposure concentration was an important factor determining the amount of graphene oxide accumulated in the alveolar region of the rat lungs, and the graphene oxide used in this study persisted in the lungs up to 14 days after exposure. However, no other histopathological changes were found in the lungs of the exposed rats. Similarly, Ma-Hock et al.  also fouIn conclusion, the results of this study suggest that the single inhalation of graphene oxide in male Sprague-Dawley rats exerted minimal toxic responses, indicating that the selected graphene oxide was relatively inert at the concentrations used. However, further studies with varying experimental settings are still needed to determine the toxicity of graphene oxide nanomaterials.Supplement 1: shows food intake of rats.Supplement 2: shows body weight of rats before and after the exposure.Supplement 3-5: shows gross findings of rats during the recovery period.Supplement 6-11: shows absolute and relative organ weight of rats during the recovery period.


Two-dimensional (2D) materials usually have a layer-dependent work function, which require fast and accurate detection for the evaluation of their device performance. A detection technique with high throughput and high spatial resolution has not yet been explored. Using a scanning electron microscope, we have developed and implemented a quantitative analytical technique which allows effective extraction of the work function of graphene. This technique uses the secondary electron contrast and has nanometre-resolved layer information. The measurement of few-layer graphene flakes shows the variation of work function between graphene layers with a precision of less than 10 meV. It is expected that this technique will prove extremely useful for researchers in a broad range of fields due to its revolutionary throughput and accuracy. Work function plays an important role in high-performance electronic devices such as transistors678510116Secondary electron (SE) contrast is promising in surmounting these barriers to work function characterisation. SEs are excited by a charged beam of energetic particles, e.g. electrons or ions at the position where the beam enters the specimen, which might be used for structure modification15171819212324Here, we present a method to extract quantitative information from the SE contrast of graphene. We have established imaging parameters to acquire reproducible SE contrast from graphene samples. Atomic level identification of graphene thickness up to 10 layers is demonstrated. A model based on illumination by both the primary beam and backscattered particles was established to explain the observed layer-dependent SE contrast. Furthermore, by subtracting the contribution from SE attenuation, this model allows the quantitative determination of work function variations. The validity of the model is further supported by the obtained graphene SE energy spectra. Our work shows a high throughput, high resolution way to effectively determine the work function of graphene, and extends the application fields of SE contrast to quantitative analysis.+ ions) or the substrates.We first evaluate the visibility of graphene in the SE imaging and compare it with optical imaging. The images shown in 26g) is defined as the intensity difference between graphene (Ig) and its substrate (Is): Cg = (Ig/Ig−1)×100%. The contrast varies with imaging conditions. For example, 2 (SiO2/Si). They were acquired with electron beam energies ranging from 0.5 keV to 20 keV. The graphene appears “brighter” than the substrate at a low energy of 0.5 keV with a contrast ratio of 9%. At 1.5 keV the monolayer graphene exhibits a similar SE intensity to the substrate, i.e. the SE contrast ratio is 1%. The graphene is still discernible since its edge exhibits a visible contrast (7%). At higher energies (>2 keV), the graphene appears “darker” than the substrate (~2/Si and Au) is sketched as a function of electron beam energy. For the SiO2/Si substrate, a contrast maximum (~20%) appears around a beam energy of 5 keV and a minimal contrast (<5%) at c.a. 1.5 keV. On the contrary, the SE contrast of graphene on several substrates  of SEs in graphene. Therefore, the SE contrast for the N-layer graphene is given by:Based on the consistent SE contrast of graphene, we now demonstrate quantitative extraction of the work function of graphene from SE imaging. The SE intensity N ≥ 4 and nm, which is quite close to the value estimated by the Seah and Dench modelN > 10), and the substrate attenuation effect can be ignored Equation Most importantly, the devation from linearity for graphene thinner than four layers see  indicaten = 4.6 for graphene (see the discussion below). Therefore δg is determined by the work function, and we can extract the work function directly from the observed contrast  filmGraphene on a highly p-doped SiOSecondary electron images of few-layer graphene were obtained from both scanning electron microscopes (Carl Zeiss Supra and FEI Strata DB235) and a helium ion microscope (Carl Zeiss Orion Plus).13 to ~1.2 × 1014 e−cm−2. The emitted SEs were collected by a through-the-lens (in-lens) detector to minimize the influence of back-scattered electrons and chamber generated SEs. The collection parameters were kept constant during the whole experiment  was used to reduce the beam irradiation damage+ beam energy was kept constant at 30 keV. The working distance was fixed at 10 mm and the field of view was fixed at 80 μm. An Everhart-Thornley detector collected SEs from graphene with negligible back-scattering ions. The beam current was kept at 1 pA which resulted in an irradiation dose of approximately 5 × 1011 ions/cm2 for each scan. Only small amounts of defects would be generated at this irradiation dose2/Si substrate, the charge neutralization system (electron flood gun) was always turned on to compensate positive charges introduced by He+ irradiation . The base chamber vacuum was approximately 5 × 10−7 mBar.In the HIM imaging process, SE images were obtained from a Carl Zeiss Orion Plus scanning helium ion microscope. The HeThe measurement of graphene SE energy spectra was carried out using a home-built energy filter in both SEM (Zeiss Supra) and HIM (Zeiss Orion Plus). We placed a TEM grid with periodic holes of 400 μm in diameter on top of the graphene sample. The spacing between graphene and the top of grid was maintained as 0.7 mm. The graphene sample was connected to ground, while a voltage source meter was used to apply a negative voltage from 0 V to How to cite this article: Zhou, Y. et al. Quantitative secondary electron imaging for work function extraction at atomic level and layer identification of graphene. Sci. Rep.6, 21045; doi: 10.1038/srep21045 (2016).


Metamaterials and plasmonics are powerful tools for unconventional manipulation and harnessing of light. Metamaterials can be engineered to possess intriguing properties lacking in natural materials, such as negative refractive index. Plasmonics offers capabilities of confining light in subwavelength dimensions and enhancing light–matter interactions. Recently, the technological potential of graphene-based plasmonics has been recognized as the latter features large tunability, higher field-confinement and lower loss compared with metal-based plasmonics. Here, we introduce hybrid structures comprising graphene plasmonic resonators coupled to conventional split-ring resonators, thus demonstrating a type of highly tunable metamaterial, where the interaction between the two resonances reaches the strong-coupling regime. Such hybrid metamaterials are employed as high-speed THz modulators, exhibiting ∼60% transmission modulation and operating speed in excess of 40 MHz. This device concept also provides a platform for exploring cavity-enhanced light–matter interactions and optical processes in graphene plasmonic structures for applications including sensing, photo-detection and nonlinear frequency generation. Realizing tunable metamaterials across a broad spectral range is of great interest. Here, Liu et al. introduce hybrid structures comprising graphene plasmonic resonators strongly coupled to conventional split-ring resonators and reach 60% transmission modulation with an operating speed above 40 MHz. Since the turn of the century, research on metamaterials has progressed rapidly with substantial expansion of both the scope of novel functionalities and the operating frequency range enabled by different types of artificial structures1234567891012131516192122232426272829303132Here, we demonstrate as proof-of-concept a type of electrostatically tunable hybrid metamaterial employing graphene plasmonic resonators strongly coupled to conventional metal-based metamaterials. In addition to their strong electromagnetic response and high tunability, such hybrid metamaterials also provide an interesting platform for exploring cavity-enhanced optical processes and light–matter interactions in graphene plasmonic structures for applications including sensing35x direction26x direction. Therefore, embedding the GR in the middle of the C-SRR capacitor gap is an effective way to achieve strong near-field coupling of the two structures, and the resulted hybrid structure forms a coupled oscillator system. When the localized SP resonance of the GR is tuned to approach and subsequently surpass the C-SRR LC-resonance by electrostatically varying the carrier density , the spectral response of the hybrid metamaterial is modulated in the frequency range containing the two resonances. Such a mechanism of transmission modulation is fundamentally different from those exploiting free-carrier absorption12134142414235The proposed hybrid metamaterial concept can be applied to different types of structures, but in this work the specific realization is based on GRs and electric-field-coupled complementary split-ring resonators (C-SRRs)38392/Si substrate, which is also utilized as the back-gate for electrostatic control of the graphene carrier density. Electron-beam lithography is employed for patterning the structures (see Methods). Following the above design principle, we have developed and experimentally investigated multiple different hybrid metamaterial structures targeting two different operating frequency ranges, that is, ∼10 and ∼4.5 THz. To achieve this, GR widths of ∼400 nm and ∼1.8 μm, respectively, are chosen to ensure that the SP resonance can be electrostatically tuned across the C-SRR LC resonance2/Si substrate is ∼45% in this frequency range, and its reflection is estimated to be ∼30%). The bare C-SRR array exhibits two resonances in the measured frequency range due to the presence of an optical phonon mode of the underlying SiO2 layer13 cm−2, in which two resonances originating from the hybridization of the intrinsic graphene SP resonance and the SiO2 optical phonon mode are present2 optical phonon. Both graphene SP resonances interact with the corresponding C-SRR resonances with similar frequency; however, the interaction between the lower frequency ones (∼10 THz) are more effective, thanks to the relatively stronger resonances and better frequency matching at high graphene carrier density. Focusing on the spectral range near the lower frequency LC-resonance, it is evident that at the CNP, the transmission spectrum of the device is close to that of the corresponding reference C-SRR array, whereas large transmission modulation in both amplitude and line shape is realized by varying the carrier density, with more than 40% relative modulation of the peak transmission achieved with HM1. This is in sharp contrast to previous investigations where a continuous layer of graphene is brought into contact with SRRs41To investigate the carrier density-dependent spectral response of the fabricated C-SRR-GR hybrid metamaterial devices, their transmission spectra are characterized employing Fourier transform infrared spectroscopy (FTIR) with the normally incident radiation polarized perpendicular to the GRs (see Methods). Moreover, the transmission spectra at high carrier densities display a double-peak feature. This is a characteristic feature of the strong coupling46β is a parameter determining the coupling strength, and may also be interpreted with an equivalent circuit model as the relative strength of a capacitive coupling between two LC-resonators (see ω+– ω−) when ωSP=ωC−SRR is approximately βωC−SRR, and the coupling strength  is found to be ∼1.2 THz in both devices, more than 10% of the individual resonance frequency and is therefore also within the ultrastrong coupling regimedescribing the anti-crossing behaviour of two strongly coupled resonancestors see  and 5. TEF|=0.3 eV  increases proportional to the device area, the total resistance (R) associated with the entire C-SRR-GR array scales inversely proportional to the device area, facilitating high-speed operation of large-area devices. The demonstrated C-SRR-GR hybrid metamaterials can be utilized as efficient modulators and switches. Since fast modulation is highly desired for many applications such as real-time compressive imaging11The demonstrated concept of coupling graphene-based plasmonic structures with conventional metal-based metamaterials to achieve highly tunable hybrid metamaterials can be straightforwardly extended to other frequency ranges  and different graphene plasmonic and/or metameterial structures , to further broaden the scope of novel functionalities. The presented device structures also provide a platform for further exploring intriguing cavity-enhanced light–matter interactions  and a lift-off process defines the C-SRR arrays, which are in direct contact with the GRs to enable electrostatic control of the carrier density using the Si substrate as the back-gate, as well as direct characterization of the electrical properties of the GRs. The devices operating around 10 THz have a surface dimension of 2 mm × 2 mm or 0.6 mm × 0.6 mm, and the devices operating around 4.5 THz have various surface dimensions: 2 mm × 2 mm; 1 mm × 1 mm or 0.5 mm × 0.5 mm.The C-SRR-GR hybrid metamaterials are fabricated from pre-transferred large-area CVD grown monolayer graphene on 300-nm thick SiO−1 for the ∼10 THz devices and 2 cm−1 for the ∼4.5 THz devices. The low-frequency measurement range is limited to ∼1 THz by the DTGS detector, while the high-frequency measurement range is significantly above the operating frequencies of the investigated devices.Transmission spectra of the C-SRR-GR hybrid metamaterial devices are characterized with FTIR spectroscopy. The measurements are performed at room temperature in the vacuum chamber of a Bruker Vertex 80v FTIR, with the normally incident radiation linearly polarized perpendicular to the GRs using a wire–grid polarizer. The resolution is 8 cmThe modulation speed measurement is conducted with a 4.7 THz quantum cascade laser as the light source, a function generator as the voltage source for modulating the hybrid metamaterial devices (up to 80 MHz), and a superconducting hot electron bolometer as the fast detector (with response up to 200 MHz), feeding the output signal to a lock-in amplifier with demodulation frequency up to 50 MHz (limiting the frequency range of the measurement). The output voltage from the function generator is set to be a sinusoidal signal with 10 V amplitude (limited by the equipment). The modulation depth at any specific frequency in EF is significantly higher than the corresponding energy of the frequency range investigated. In most of the simulations, the carrier relaxation time is assumed to be 50 fs which is realistic for the graphene material available for our experimental demonstration2 layer (300 nm thick) is computed taking into account the surface optical phonon mode at ∼14.5 THz .Supplementary Figures 1-8, Supplementary Table 1, Supplementary Notes 1-7 and Supplementary References.


Optoelectronic devices utilizing graphene have demonstrated unique capabilities and performances beyond state-of-the-art technologies. However, requirements in terms of device quality and uniformity are demanding. A major roadblock towards high-performance devices are nanoscale variations of the graphene device properties, impacting their macroscopic behaviour. Here we present and apply non-invasive optoelectronic nanoscopy to measure the optical and electronic properties of graphene devices locally. This is achieved by combining scanning near-field infrared nanoscopy with electrical read-out, allowing infrared photocurrent mapping at length scales of tens of nanometres. Using this technique, we study the impact of edges and grain boundaries on the spatial carrier density profiles and local thermoelectric properties. Moreover, we show that the technique can readily be applied to encapsulated graphene devices. We observe charge build-up near the edges and demonstrate a solution to this issue. Graphene grain boundaries and charge inhomogeneities limit its electronic properties. Here the authors combine scanning near-field optical microscopy with electrical read-out to image these defects at the nanoscale under an encapsulation layer, and show that charges build up along the edges of the flake. As large scale integration and wafer scale device processing capabilities of graphene have become available1234567910121415161718A major challenge is that many of the available characterization techniques are invasive1321141521232414152123Here we demonstrate fully non-invasive room-temperature scanning near-field photocurrent nanoscopy2829303132333435363739in situ28293031323334353637The measurement principle is sketched in 26IPC and referred to as near-field photocurrent and is obtained together with near-field optical and topography information. A typical map of IPC, obtained by scanning the tip over a CVD graphene device, is shown in The near and far fields impinging on the device induce charge flows in the device (by mechanisms discussed below), and drive currents into an external current amplifier via contacts on the device. We isolate the part of the current that is induced by near fields by demodulating the current at the second harmonic of the tip tapping frequencyIPC falls to zero for tip locations away from the graphene on a similar length scale as the topography, demonstrating the successful isolation of near-field contributions. In IPC as the tip is moved over the edge of graphene. The full-width at half maximum of the photocurrent peak at this location is ∼100 nm, matching the rise distance in the topographic signal. This resolution is far below any limits relating to the 10.6 μm free space light wavelength.We can assess the spatial resolution of the photocurrent maps by comparing a region near the edge  with a tS to drive charge currents within the device and into the contacts (see Methods section). Therefore, we interpret the variations of IPC in terms of microscopic variations in S. The Seebeck coefficient, which depends on material properties such as carrier density and mobility, is a measure of the electromotive force driven by a temperature difference in a material. A complete description of IPC needs to take into account the carrier cooling length45κ the sheet thermal conductivity in plane and g the interfacial thermal conductivity out of plane to the heat sinking substrate, describes how far heat propagates through the charge carriers, before dissipating to the environment ∝SG(VBG)−SGB(VBG).To gain more insight in the Seebeck coefficient at the grain boundary, we tune the carrier density by a global gate . We obseSGB is the ratio of the first- and zero-order Onsager coefficients , using the polymer-free van der Waals assembly technique3956While monitoring the photocurrent of such encapsulated devices, we observe indications of strong carrier density variations near the edges over micrometre scales. These variations are influenced by lighting conditions , gate voVBG=VD. We observe photocurrent at the junction in We exploit the observed edge doping to create a natural p–n junction along the edge of the device. For this we apply a backgate voltage at which the edge of the graphene is p-type and the bulk n-type. This is similar to the situation in 2 due to the surface of the h-BN being not completely conformal with the underlying SiO2 substrate. This water molecule penetration then leads to trapped charges is responsible for the observed edge doping. We also present here a technique to completely eliminate the edge doping. We place encapsulated graphene on top of a local conductive gate, such as a 15 nm AuPd alloy sketched in 2  we consider that the heat spreads in two dimensions with heat sinking to lattice and substrate, producing a T(x) profile described by a modified Bessel function of the second kind, with a finite tip size correction /Pd (35 nm). The graphene was transferred onto deposited contacts.The CVD graphene, grown on copper, was transferred onto a self-assembled monolayer2(300 nm) wafer, used as backgate. The Cr(0.8 nm)/Au(80 nm) contacts were defined using electron beam lithography.The exfoliated graphene device was fabricated on a Si/SiO2(300 nm)/h-BN(46 nm)/Graphene/h-BN(7 nm) and the Si/SiO2(300 nm)/AuPd(15 nm)/h-BN(42 nm)/Graphene/h-BN(13 nm) stacks, were fabricated using the polymer-free van der Waals assembling techniqueThe Si/SiOHow to cite this article: Woessner, A. et al. Near-field photocurrent nanoscopy on bare and encapsulated graphene. Nat. Commun. 7:10783 doi: 10.1038/ncomms10783 (2016).Supplementary Figures 1-10, Supplementary Notes 1-2 and Supplementary References


Erythema nodosum is a panniculitis which may have various causes, such as drugs, infections, sarcoidosis, inflammatory bowel disease, tuberculosis or can be idiopathic. We here report a case of a womandiagnosed with pulmonary tuberculosis whose first symptom was erythema nodosum. A thorough clinical and laboratory investigation of the patient presenting with erythema nodosum is always required, in order to assess a possible systemic, underlying condition. Erythema nodosum is a panniculitis, characterized by red, tender nodules usually located on the extensor surfaces of the legs. It may have various causes, such as drugs, infections, sarcoidosis, inflammatory bowel disease, tuberculosis or can be idiopathic . In up tWe here report a case of a 78 year old woman, with free medical history, who presented to the Emergency Department of our Hospital with erythema nodosum on her both shins . She alsThe commonest causes of erythema nodosum reported in the literature, are streptococcal infection in children and streptococcal infection and sarcoidosis in adults . Its couIn conclusion, a thorough clinical and laboratory investigation of the patient presenting with erythema nodosum is always required, in order to assess a possible systemic, underlying condition.


A method is reported for collecting room-temperature data from protein crystals under vacuum by protecting them with a thin graphene layer. in vacuo holds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is side-stepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment.The measurement of diffraction data from macromolecular crystal samples held  This is owing to developments in beamline instrumentation, such as pixel-array detector (PAD) technology  to establish a proof of principle for using graphene in this way. Our results demonstrate that graphene acts to maintain crystal hydration during exposure to these harsh conditions, allowing the measurement of high-quality diffraction data at room temperature.2.2.1.2.1.1.−1. Sitting drops were made by mixing 1 µl lysozyme solution and 1 µl reservoir solution  and were equilibrated against 300 µl reservoir solution at 20°C. Crystals (100 × 100 × 50 µm) grew within 3 h and were found to belong to space group P43212, with unit-cell parameters a = b ≃ 78, c ≃ 38 Å. The crystals did not require cryoprotection before being flash-cooled in liquid nitrogen.Commercial lyophilized lysozyme from hen egg white (Sigma–Aldrich) was resuspended in Milli-Q water to a concentration of 100 mg ml2.1.2.Streptomyces murinus (Sigma–Aldrich) was resuspended in solution  to a concentration of 16.3 mg ml−1. Sitting drops were made by mixing 2 µl protein solution and 2 µl reservoir solution  and were equilibrated against 300 µl reservoir solution at 20°C. Crystals (120 × 80 × 80 µm) grew within 7 d and were found to belong to space group I222, with unit-cell parameters a ≃ 93, b ≃ 98, c ≃ 103 Å. The cystals did not require cryoprotection before being flash-cooled in liquid nitrogen.Commercial glucose isomerase (GI) from 2.1.3.Thaumatococcus danielli (Sigma–Aldrich) was resuspended in Milli-Q water to a concentration of 40 mg ml−1. Sitting drops were made by mixing 4 µl protein solution and 2 µl reservoir solution  and were equilibrated against 500 µl reservoir solution at 20°C. Crystals (200 × 200 × 100 µm) grew within 7 d and were found to belong to space group P41212, with unit-cell parameters a = b ≃ 59, c ≃ 151 Å. The crystals did not require cryoprotection before being flash-cooled in liquid nitrogen.Commercial thaumatin from 2.2.http://acsmaterial.com). The thickness of the PMMA was measured by micro-interferometry on a Bruker Contour GT-X and was found to be 100 ± 10 nm . It is possible to entirely remove the PMMA by pipetting a few drops of acetone onto the completely dry graphene/PMMA, ensuring that the surface of the graphene/PMMA is completely covered. The removal of the PMMA layer was confirmed by infrared spectroscopy and micro-interferometry . However, without support the remaining graphene layers are too fragile and are destroyed if further manipulated  (PMMA), with a quoted PMMA thickness of ∼500 nm (2.3.via a vacuum gauge to measure the pressure within the chamber (diaphragm vacuum pump from KNF). In general, pumping took a few seconds to reach around 2000 Pa.In order to investigate whether graphene/PMMA can protect crystals at room temperature in vacuum, a simple chamber was designed in-house and then printed in three dimensions . As a control, one crystal of thaumatin was also placed under vacuum without a graphene/PMMA wrap.2.4.et al., 2009A high-precision crystal humidifier/dehumidifier (HC1b) from Arinax was used to expose the crystals to different relative humidities (Sanchez-Weatherby I222 to P21212) occurs in dehydrated crystals of GI when exposed to a relative humidity of between 70 and 90%  or 20 × 20 µm (0.7 × 1011 photons s−1) (defined using apertures). Diffraction images were measured with a PILATUS 6M-F area detector. For samples within the vacuum chamber, data collections were limited to a total oscillation of approximately 60°, whereas crystals outside the chamber were exposed for a total oscillation of 180°. The oscillation range for all samples was 0.10° per image, with an exposure time of 40 ms per image. Crystals within the chamber were collected at approximately 295 K with a vacuum pressure of ∼2000 Pa. Samples outside the chamber were collected at 100 K cooled with an Oxford Cryosystems Cryostream.Diffraction data were collected on beamline I04 at Diamond Light Source using an X-ray energy of 12.658 keV and a beam size of 90 × 45 µm  > 2 was applied to the data.Several data sets were measured for wrapped thaumatin crystals under vacuum. For brevity, the results for only one representative data set are displayed in Table 1et al., 20021kwn) was stripped of all nonprotein atoms and was used for rigid-body refinement with REFMAC5  > 2 was applied.For the HC1b experiments, at least three data sets were measured for each condition and the results in Table 3I/σ(I) > 2.For the crystals wrapped in graphene/PMMA and then exposed to acetone, the data were processed as above using a resolution cutoff of 3.a) and 3b) and Figs. 3c) and 3d) show the diffraction of thaumatin wrapped in graphene/PMMA in the chamber under air or vacuum, respectively. Under air reflections are visible to approximately 1.8 Å resolution and under vacuum reflections are visible to approximately 1.92 Å resolution. From these diffraction images there is a slight degradation in the diffracting power of the crystals wrapped in graphene/PMMA placed under vacuum, but no degradation in the quality of the reflections. This is also confirmed by the results in columns 2 and 3 of Table 1e) and 3f) show diffraction from unwrapped thaumatin placed under vacuum. Although diffraction is still observed, the quality is visibly worse compared with those wrapped in graphene. The diffraction quality also drops off much more quickly in these crystals, with limited diffraction being visible in the last image of the data collection when compared with the same image for those crystals protected by graphene/PMMA. The results for this scenario are shown in column 4 of Table 1Rmeas are higher. There is a marked reduction in the unit-cell parameters, most notably in the a and b dimensions, which decrease from approximately 59 Å for wrapped crystals to 54 Å for unwrapped crystals exposed to vacuum.Representative diffraction patterns of thaumatin within the vacuum chamber are shown in Fig. 3B factors from the refinement can be compared with a similar room-temperature data collection without graphene/PMMA and vacuum. For the wrapped thaumatin data collected here, the average atomic B factor is reported to be 38.57 Å2, while for an equivalent thaumatin room-temperature data set the average atomic B factor is 24.21 Å2 (unpublished work). This difference in B values might be attributable to the additional manual handling required during the wrapping process and may point to a potential problem with the current procedure.Table 2I222 to P21212. This is also accompanied by a decrease in the crystal quality, as observed by an increase in the value of Rmeas and an increase in the B factor. As a comparison, GI crystals exposed to 65% relative humidity which are wrapped in graphene/PMMA show no phase transition, and the data quality remains consistent with the data sets where the change in relative humidity has not been applied. The changes in unit-cell parameters can be observed in Fig. 5HC1b experiments were carried out on crystals of GI and the results of these data collections can be seen in Table 3Rmeas and B-factor values are much higher. In fact, in two out of the seven data sets treated with these conditions a reduction in the unit cell is also observed (Supplementary Table S8).The results from the data collections carried out using the HC1b with lysozyme can be found in Table 4B factor and a drop off in resolution when compared with a control data set in Table 4The data-processing statistics for the lysozyme crystal wrapped in graphene/PMMA and soaked in acetone can be seen in Table 54.The results outlined in this manuscript have demonstrated how effective multilayer graphene/PMMA can be at protecting protein crystals from dehydration once removed from their mother liquor. This was confirmed by several different methods. Although the PMMA was retained along with the graphene to aid in its manipulation, the permeability of PMMA to water has been well documented  and 3f) is degraded compared with that in Figs. 3(a), 3(b), 3(c) and 3(d), where the crystals are protected by graphene/PMMA.The first experiments outlined how graphene/PMMA can be used to protect crystals when exposed to vacuum without having to cryocool the crystals. The data sets where the crystals were wrapped in graphene/PMMA and then either exposed to vacuum or left in air were comparable in quality. When the crystals were left unwrapped and exposed to vacuum, the data quality was evidently worse. The unit-cell parameters in this case also showed a reduction, which is consistent with crystal dehydration. Surprisingly, however, these crystals still diffracted even under vacuum. This is probably explained by the retention of a small amount of mother liquor around the crystal during the mounting procedure that could act as a temporary barrier against dehydration. This was, however, not as effective as graphene/PMMA (see Table 1The vacuum chamber provided a convenient method for testing samples under a rough vacuum; however, the chamber was not optimized for routine crystallographic data collection. The entrance and exit windows of the chamber limited the angular rotation to approximately 60°, but still allowed the collection of >95% complete data. The scatterguard and beamstop were also not optimally set up, with air gaps between these and the entrance and exit windows of the chamber. For this reason, analysis of the potential advantages of vacuum for reducing the background was not performed.B factors were higher compared with similar structures determined from data collected using more standard means. This suggests that either the additional manual handling step is harming the crystals or that if the crystals were not completely sealed by the graphene/PMMA then slow dehydration might be taking place.The results from graphene/PMMA-wrapped crystals in vacuum show relatively good data quality. However, the current process of mounting crystals in graphene/PMMA includes several manual handling steps that might be detrimental to the crystal and hence the data quality. We noticed that the quality of some data sets was reduced and refined atomic I222 to P21212 is observed, which in this case is a known side effect of dehydration.Tests carried out using the HC1b effectively demonstrate how graphene/PMMA can protect crystals from dehydration. For both the lysozyme and GI examples, there are significant changes in the lattice properties and data quality for unwrapped crystals exposed to lower relative humidity. The equivalent data sets for graphene/PMMA-wrapped crystals produced no such lattice changes or significant degradation in data quality. For lysozyme there is approximately a 3.5 Å reduction in the unit-cell axes, strongly suggesting dehydration of the crystal. For GI a phase transition from et al., 2013The results that we have presented support the claim that this method (Wierman In all of these experiments the material used was multi-layer graphene coated with a 100 nm layer of PMMA. The PMMA acts as a support for the graphene, without which it is almost impossible to manipulate it for the purpose described here. We have demonstrated that multi-layer graphene/PMMA can be used as a covering to protect protein crystals. The samples studied here were on the scale of several tens of micrometres, making the 100 nm layer of PMMA negligible by comparison with respect to its impact on background scatter; however, the thickness of the PMMA could begin to play a role when working with micrometre-sized or smaller crystals.Attempts were made to remove the PMMA from the graphene using a Kapton loop as a support. Here, a crystal of lysozyme was wrapped in graphene/PMMA and mounted on the Kapton loop. The mounted crystal in graphene/PMMA was soaked in acetone to remove the PMMA; however, some degradation in crystal quality was observed. The statistics in Table 5in vacuo environments, a crystal size of <50 µm is typically used to limit absorption by the crystal itself (Liu et al., 2013The experiments carried out here used relatively large crystals. For long-wavelength beamlines requiring 5.The results presented here demonstrate the use of graphene/PMMA as a layer to protect protein crystals from dehydration when exposed to vacuum. We have shown that it is possible to collect room-temperature data from protein crystals under vacuum, which has applicability in the development of new MX beamlines with in-vacuum sample environments. We have also confirmed these findings by collecting data from different samples at varying relative humidities and shown that no significant change in data quality is observed when the samples are wrapped in graphene/PMMA. Finally, we have shown that initial investigations into removal of the PMMA layer indicate that it may be feasible to use acetone post crystal-wrapping. Graphene has the potential to form a chemically resistant barrier for crystals, opening up possibilities for new types of experiments. It is interesting to consider that graphene/PMMA can create a sealed environment around the crystals that could be exploited as an anaerobic environment, for example.in vacuo using graphene/PMMA wraps that minimize the solvent surrounding the crystal.With the recent renewed interest in the use of evacuated sample environments, it is timely to demonstrate a method for mounting and measuring data at room temperature from protein crystals in vacuum. The expectation is that significant gains in the signal-to-noise ratio from such data are possible now that the integrity of crystals can be maintained thaumatin wrapped in graphene, 4zxrPDB reference: 10.1107/S1399004715014194/tz5077sup1.pdfSupporting Information.. DOI: Click here for additional data file.10.1107/S1399004715014194/tz5077sup2.mp4Video showing the mounting of crystals in graphene/PMMA.. DOI:


We determine the spin-lifetime anisotropy of spin-polarized carriers in graphene. In contrast to prior approaches, our method does not require large out-of-plane magnetic fields and thus it is reliable for both low- and high-carrier densities. We first determine the in-plane spin lifetime by conventional spin precession measurements with magnetic fields perpendicular to the graphene plane. Then, to evaluate the out-of-plane spin lifetime, we implement spin precession measurements under oblique magnetic fields that generate an out-of-plane spin population. We find that the spin-lifetime anisotropy of graphene on silicon oxide is independent of carrier density and temperature down to 150 K, and much weaker than previously reported. Indeed, within the experimental uncertainty, the spin relaxation is isotropic. Altogether with the gate dependence of the spin lifetime, this indicates that the spin relaxation is driven by magnetic impurities or random spin-orbit or gauge fields. Studying the spin lifetime anisotropy in graphene, which provides information on spin-orbit interaction and is relevant to spintronic applications, poses important experimental challenges. Here, the authors study the spin lifetime anisotropy using spin precession under oblique magnetic fields. The spin-lifetime anisotropy, which can be quantified by the ratio between the spin lifetimes for perpendicular and parallel spin components to the graphene plane ζ<1  and, therefore, it is expected to be useful for sufficiently large n only, owing to the large magnetoresistive effects that are present in graphene at low carrier densities , circumventing spurious magnetoresistive phenomena. In our approach, based on non-local spin devices, B⊥, which causes the spins to precess exclusively in-plane as they diffuse from the injector (F1) towards the detector (F2). Because the magnetic field required to generate the spin precession is relatively weak , the magnetizations of the injector and detector are assumed to remain in-plane over the whole-field range, due to magnetic shape anisotropy.Our measurement scheme  allows uβ  substrate from highly oriented pyrolytic graphite. Two inner ferromagnetic Co electrodes and two outer normal Pd electrodes contacting the graphene flake are fabricated using a single-electron-beam lithography step and shadow evaporation3030In our devices , we use μ=1.7 × 104 cm2 V−1 s−1 and a residual carrier density n0=1.5 × 1011 cm−2, which sets the region where electron-hole puddles result in an inhomogeneous carrier density Vg=VCNP, but remains below 3% for all gate voltages when B⊥≤0.2 T  to antiparallel (↓↑) configuration, or vice versa. Vg; the observed variation nearby the CNP is associated with a faster spin relaxation (see below) and a resistive, but not tunnelling, interface between the metal and graphene4We first characterise the graphene charge transport properties by means of standard four-terminal local measurements, from which we estimate an average electron/hole mobility Ds as a function of Vg. To this end, we fit the data to the solution of the Bloch equations4Ds are presented in A typical precession curve for magnetic fields perpendicular to the graphene plane is shown in Bd≲0.1 T. This condition is met at a large-enough injector-detector separation L. For typical graphene devices it is sufficient that λs is the graphene spin-relaxation length , β*=β−γ is the angle between the magnetization of the electrodes and the magnetic field, taking into consideration the small tilting γ. The factor cos2(β*) accounts for the projection of the injected spins along the direction of the magnetic field and the subsequent projection along the direction of the detector magnetization.Having determined the in-plane spin-transport properties and demonstrated the high quality of the graphene sample, we proceed with the oblique-precession experiments to extract ζ. For the general case of an anisotropic system, we determined ζ=1, it is straightforward to verify that β.where 2(β*). The representation of ζ=1, 2(β*) results in a straight line. According to ζ>1 and below it for ζ<1. In ζ∼1. The data is well-enclosed by the lines that correspond to ζ=0.8 and ζ=1.2, thereby defining rough lower and upper limits for ζ. Vg for T=150 K and 300 K by fitting the data to Vg or T.Rnl should saturate to a constant value from which n that we have studied. Indeed, although an apparent saturation of Rnl is observed at B∼1 T, further increase of the magnetic field demonstrates that Rnl is actually not independent of B, presenting a monotonous decrease, no matter the value of n. In the A comparison of the above results with those obtained with the methods shown in refs Ds  adatoms, such as gold, or the substrate, and the spin–orbit coupling caused by hydrogen-like adatoms (excluding magnetic moments)2320ζ=0.5 should hold strictly, in disagreement with our results.Considering the obtained anisotropy  and the  and Ds , we can The anisotropy can be weaker for other adatoms that induce resonances, when the intrinsic spin–orbit coupling becomes important at off-resonance energies. Such is the case for fluor-like adatoms, although here ζ is expected to depend on energy and significantly deviate from 0.5 only at positive energies. In addition, spin-relaxation mechanisms associated with spin–orbit coupling from hydrogen- and fluorine-like adatoms predict ζ<1 to ζ>1 and mask a ζ=0.5 relationζ∼1 and, at the same time, explain the magnitude and carrier-density dependence of ζ=1 as in n. However, measurements as a function of n and at low magnetic fields are necessary to discard possible artefacts  ref. . CombiniWe have thus demonstrated spin-lifetime anisotropy measurements in graphene and discussed them in light of current theoretical knowledge. We used a measurement technique that provides reliable information on spin dynamics at low magnetic fields, and in a broad carrier-density range that was not accessible before. We show that only a very limited number of models can explain our results, and we provide a route based on our methods to discriminate between them using graphene spintronic devices that are within reach of the current state of the art. In addition, the microscopic properties of the graphene used in devices originating from different laboratories are not necessarily equivalent, owing to the graphite source or the processing steps that have been used. Therefore, it is plausible that experimental results in one research group might not be directly reproduced in another11112131417243p-doped Si substrate covered with 440 nm of SiO2. The thickness of the SiO2 layer is chosen to have optimum optical contrast between single- and multi-layer graphene flakes, which were discriminated by optical means after contrast calibration with Raman measurements. The width of the flake in the device shown in w∼1 to 1.5 μm. An amorphous carbon (aC) interface is created between all contacts and the graphene flake by electron-beam (e-beam)-induced deposition before the fabrication of the contacts30Graphene flakes are obtained by mechanically exfoliating highly oriented pyrolytic graphite (SPI Supplies) onto a 2 V−1 s−1 and thus compare well with similar devices on h-BN−8 Torr. We first deposit the outer electrodes of Ti/Pd by angle deposition. Selecting an angle of ±45° from the normal to the substrate assures that no image of the lithographically produced lines reserved for the Co electrodes are deposited during the evaporation of Ti/Pd; indeed, Ti and Pd deposit onto the sidewalls of the lithography mask and are later on removed by lift-off29wo and deposited thicknesses dTi and dPd of the identical outer Ti/Pd electrodes are wo=1,500 nm, and dTi=5 nm and dPd=10 nm. The inner Co contacts have widths wiE2=150 nm and wiE3=140 nm and thickness dCo=30 nm. The centre to centre distance between the Co contacts is L=11 μm; their difference in width sets distinct coercive field strengths that allow us to control the relative orientation of their magnetization by an external magnetic field.We define the contact electrodes in our devices in a single e-beam lithography step using shadow evaporation to minimize processing contamination−4 Torr at 300 K and 1.5 × 10−5 Torr at 150 K. We characterise the graphene charge transport properties by means of three- and four-terminal measurements. The contact resistances are of the order of 10 kΩ or larger, as determined by three-terminal measurements. The devices are homogeneous and we have not observed any significant shifts of the CNP in the different regions between each pair of contacts. For the four-terminal local measurements a current I is driven between the outer Pd electrodes, E1 and E4, and the voltage measured between the inner Co electrodes, E2 and E3 , to the non-local voltage is derived within the spin-diffusion model including spin anisotropy, which is quantified by the anisotropy ratio ζ. In the isotropic limit (ζ=1), this model has been successfully used to describe spin precession phenomena in lateral devicesThe contribution of the residual spin component parallel to the field, B= can be described within a rotated cartesian axis system characterized by the unit vectors The spatio-temporal evolution of the spin density, ors  see  by,Ds, while β of the applied magnetic field B.where N due to the presence of the magnetic field, N=γcs × B, which results in a precessional evolution of the spin density. The constant pre-factor, |γc|=gμB/ħ, is the gyromagnetic ratio of the carriers, where μB is the Bohr magneton and g is the so-called g-factor. In the case of exfoliated graphene, we can consider the g-factor to be equal to the free-electron value and to be field independent and isotropic.The injected spins feel a torque In the limiting case in which the precessional motion is fully dephased, the spin density perpendicular to the field direction is suppressed, and the spatio-temporal evolution of withVnl at the detector is,The above equation is equivalent to that governing the spin diffusion when only one spin lifetime is relevant, which here is given by the effective spin lifetime L indicates the distance between injector and detector and I the magnitude of the injector current. The factor cos(β−γi) accounts for the projection of the injected spins along the direction of the magnetic field, corrected by a small tilting of the magnetization of the injector electrode, which is given by the angle γi. Similarly, the detector picks up the projection of the spin density along its own magnetization, whose orientation is corrected by an angle γd, leading to the cos(β−γd) factor . Therefore, it is typically assumed to be a fitting parameter.where see also . The facB=0 taking into account γi=γd=γ, we obtain,If we normalize the above result Rnl=Vnl/I.where α(B)/α(B=0)=1 =1, the pre-factor in Rnl simply follows cos2(β−γ). In contrast, when ζ≠1, the cos2(β−γ) dependence is no longer valid. Because Ds can be independently determined from the conventional spin precession measurements (β=90°), At low-enough magnetic fields, the magnetoresistance can be disregarded and 0)=1 see . By replHow to cite this article: Raes, B. et al. Determination of the spin-lifetime anisotropy in graphene using oblique spin precession. Nat. Commun. 7:11444 doi: 10.1038/ncomms11444 (2016).Supplementary Figures 1-7, Supplementary Notes 1-6 and Supplementary References


In a subset of cells, the ALOX5 inhibitor, zileuton, or the 5-lipoxygenase activating protein inhibitor, MK-886, was administered during hypoxia exposure. ALOX5 expression was measured by qRT-PCR and western blot and HPAEC proliferation was assessed. Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression. Seventy two hours of hypoxia significantly increases HPAEC ALOX5 expression, hydrogen peroxide (H2O2) release, and HPAEC proliferation. We also demonstrate that targeted ALOX5 gene silencing or inhibition of the ALOX5 pathway by pharmacological blockade attenuates hypoxia-induced HPAEC proliferation. Furthermore, our findings indicate that hypoxia-induced increases in cell proliferation and ALOX5 expression are dependent on H2O2 production, as administration of the antioxidant PEG-catalase blocks these effects and addition of H2O2 to HPAEC promotes proliferation. Overall, these studies indicate that hypoxia exposure induces HPAEC proliferation by activating the ALOX5 pathway via the generation of H2O2.Pulmonary Hypertension (PH) is a progressive disorder characterized by endothelial dysfunction and proliferation. Hypoxia induces PH by increasing vascular remodeling. A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5). While ALOX5 metabolites have been shown to promote pulmonary vasoconstriction and endothelial cell proliferation, the contribution of ALOX5 to hypoxia-induced proliferation remains unknown. We hypothesize that hypoxia exposure stimulates HPAEC proliferation by increasing ALOX5 expression and activity. To test this, human pulmonary artery endothelial cells (HPAEC) were cultured under normoxic (21% O Pulmonary Hypertension (PH) is a progressive disorder characterized by sustained increases in pulmonary arterial pressures and vascular remodeling. Although the mechanisms underlying PH remain unknown, hypoxia induces PH in experimental models and is believed to contribute to disease development ALOX5 activity and leukotriene production are regulated by numerous signaling pathways. Primarily, ALOX5 requires the presence of FLAP for leukotriene synthesis Trypan blue, fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), PEG-Catalase, and gelatin were obtained from Sigma-Aldrich . Zileuton was obtained from Patheon Pharmaceuticals . MK-886 was purchased from Calbiochem . Scrambled and silencing RNA (siRNA) for ALOX5 was obtained from Qiagen .3-IGF-1 (insulin-like growth factor), GA-1000 , and heparin. Unless otherwise stated, cells were maintained in a 37°C incubator at 5% CO2.Human pulmonary artery endothelial cells (HPAEC) were obtained from Lonza Clonetics . HPAEC were grown in EGM-2 medium (Lonza), which contains basic growth medium (EBM-2), fetal bovine serum (FBS), and antibiotics, ascorbic acid, vascular endothelial growth factor (VEGF), human fibroblast growth factor (hFGF-B), hydrocortisone, human epidermal growth factor (hEGF), R2 levels. For hypoxic conditions, HPAEC were placed in a hypoxia chamber maintained at 37°C, 1% oxygen, and 5% CO2 levels. Human pulmonary artery endothelial cells (HPAEC) were cultured in normoxic or hypoxic conditions for 24-, 48-, or 72 hours. To investigate the contribution of ROS levels, PEG-Catalase  was administered during the final 24 hours of the 72 hours of hypoxia exposure. The role of 5-Lipoxygenase in HPAEC proliferation was studied by exposing cells to 10 µM concentrations of Zileuton or MK-886 either throughout the entire exposure period or during the final 24 hours.HPAEC, passages 3-8, were exposed to hypoxia in a Biospherix exposure chamber  as previously described −ΔΔCt method.ALOX5 and FLAP mRNA levels were determined by quantitative real-time PCR (qRT-PCR) using the iCycler system . Total RNA was extracted from HPAEC using RNA-Bee. RNA concentrations were measured using the ND-1000 Spectrophotometer . RNA (1.5 micrograms) was combined with random nanomer primers (Ambion), dNTPs (New England Bio-Labs) and nuclease-free water for reverse transcription. cDNA templates were amplified with gene-specific primer sets. All transcripts were detected using SYBR Green I . Transcripts were normalized to the housekeeping gene, β-Globin. Values are expressed as percent of control. Expression changes were determined using the 25′- GGCAGGAAGACCTGATGTT -3′, target region 333–351) were designed using BLOCK-it RNAi Designer (Invitrogen). siRNA targeted to a specific noncoding gene was employed as a scrambled RNA control. At 40–50% confluence, HPAECs were transfected with scrambled or ALOX5 siRNA using GeneSilencer  transfection reagent according to manufacturer's instructions. After transfection for 6 hours, the transfection media was replaced with EGM containing 10% FBS. HPAECs were exposed to normoxia  or hypoxia  for 72 h. HPAEC lysates were then harvested and examined for ALOX5 levels using qRT-PCR and Western blots. In selected studies, HPAEC proliferation was determined using MTT assays.Human ALOX5 siRNA (NM_000698), siRNA duplexes  Cell Proliferation Assay. Briefly, proliferating cells reduce the tetrazolium MTT resulting in intracellular formazan. Detergent reagent was added to cell to solubilize formazan. Supernatants were then collected and quantified using a spectrophotometer at 562 nm. Cell proliferation was further confirmed by cell counting using a trypan blue dye exclusion assay. Briefly, following hypoxia exposure, cells were trysinized and resuspended in medium. Cells were mixed with PBS and trypan blue at a 1∶8∶1 ratio. The trypan blue-negative cells were counted using a hemacytometer.2O2) release was quantified using the Amplex Red Assay. Cells were incubated in a solution containing the Amplex Red reagent (Molecular Probes), horseradish peroxidase and a buffer solution for 30 minutes at 37°C. Supernatants were then collected and fluorescence measured at 560 nm. H2O2 concentrations were determined through standard curve extrapolation normalized to cellular protein concentration. Overall reactive oxygen species (ROS) and reactive nitrogen species were detected using the ROS-sensitive fluorescent probe 2′, 7′-dihydrodichlorofluorescein diacetate . Confluent HPAEC monolayers were loaded with 25 µg/mL DCF-DA for 1 hour at 37°C in Krebs–Ringer Phosphate Buffer . A laser-scanning confocal microscope  and fluorimeter were used to detect DCF fluorescence at excitation and emission wavelengths of 488 nm and 520 nm, respectively.Hydrogen Peroxide .in vitro, we assessed HPAEC proliferation following 24, 48, and 72 hours of hypoxia exposure. While 24 and 48 hours of hypoxia exposure had no effect on cellular proliferation, 72 hours of hypoxia increases cellular proliferation when measured by MTT assay  Recent studies demonstrate that ALOX5 contributes to HPAEC proliferation We next sought to determine whether alterations in ALOX5 activity contribute to hypoxia-induced endothelial proliferation. To confirm that ALOX5 promotes hypoxia-induced HPAEC proliferation, we measured cellular proliferation in response to hypoxia in the presence or absence of the ALOX5 inhibitor, zileuton. Inhibition of ALOX5 enzyme activity by zileuton administration during the final 24 hours of hypoxia exposure attenuates hypoxia-induced increases in endothelial proliferation (2O2) release also demonstrates that 72 hours of hypoxia exposure is required for increased in H2O2 . Although the physiological concentrations of H2O2 are unknown, the concentrations used In this study, we demonstrate that prolonged hypoxia exposure significantly increases ALOX5 expression when compared to normoxic controls. Although ALOX5 was originally believed to be expressed only in myeloid cells, previous studies confirm ALOX5 expression in pulmonary artery endothelial cells This study also reveals that prolonged hypoxia exposure stimulates endothelial cell proliferation. Although previous studies indicate that hypoxia failed to stimulate endothelial cell proliferation 2O2 contribute to the hypoxia-induced endothelial cell proliferation, as PEG-Catalase treatment prevents hypoxia-induced endothelial proliferation. Interestingly, our data demonstrate a prolonged exposure to hypoxia (72 hours) is needed to induce pulmonary endothelial cell proliferation, whereas 24- and 48- hours of hypoxia exposure do not significantly impact endothelial cell proliferation. It is plausible that accumulation of H2O2 is needed for the induction of cellular proliferation and our H2O2 measurements demonstrate a delay in hypoxia-induced increases in H2O2  as previous studies indicate that Nox4 expression is elevated in low oxygen environments Previous research demonstrates that chronic hypoxia significantly increases endothelial cell proliferation. Although our evidence indicates that hypoxia increases ALOX5 in a redox-sensitive manner, the mechanisms underlying these events remain unclear. Two major pathways are implicated in regulating ALOX5 expression: promoter methylation and transcription factor activation In summary, the current study demonstrates that hypoxia-induced ROS plays a critical role regulating ALOX5 and endothelial proliferation. Our data suggests that ALOX5 mediates hypoxia-induced endothelial cell proliferation and implicates increased ALOX5 expression and activity in vascular remodeling in experimental and clinical PH. Overall, these results suggest that ALOX5 inhibition merits additional investigation as a therapeutic target for the prevention or treatment of PH.