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


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


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


We demonstrated theoretically that the renormalization of the electron energy spectrum near the Dirac point of graphene by a strong high-frequency electromagnetic field (dressing field) drastically depends on polarization of the field. Namely, linear polarization results in an anisotropic gapless energy spectrum, whereas circular polarization leads to an isotropic gapped one. As a consequence, the stationary (dc) electronic transport in graphene strongly depends on parameters of the dressing field: A circularly polarized field monotonically decreases the isotropic conductivity of graphene, whereas a linearly polarized one results in both giant anisotropy of conductivity (which can reach thousands of percents) and the oscillating behavior of the conductivity as a function of the field intensity. Since the predicted phenomena can be observed in a graphene layer irradiated by a monochromatic electromagnetic wave, the elaborated theory opens a substantially new way to control electronic properties of graphene with light. Since the discovery of graphene24The methodology of quantum optics lies at the basis of various exciting fields of modern physics, including quantum information81016418192021222425262728293031323435363738394044454647z axis [see 23For definiteness, we will restrict our consideration to the case of electron states near the Dirac point of a single graphene sheet subjected to an electromagnetic wave propagating perpendicularly to the graphene plane. Let the graphene sheet lie in the plane axis see . Then elv is the electron velocity in graphene near the Dirac point, e is the electron charge, and ω, lies far from the resonant frequencies of graphene, where we can obtain both the energy spectrum of electrons dressed by the electromagnetic field, For the case of the circularly polarized electromagnetic field with the vector potentialwe arrive at the energy spectrum of the dressed electrons,where signs “+” and “−” correspond to the conduction band and valence band of graphene, respectively,ω is assumed to satisfy the condition of is the field-induced band gap in graphene, π-orbitals of the two crystal sublattices of graphene)S is the graphene area, and θ is the azimuth angle of electron in the space of wave vector, where In the case of linearly polarized electromagnetic field with the vector potentialx axis, the energy spectrum of the dressed electrons reads asdirected along the and the corresponding wave functions of dressed electrons arewhereThe energy spectra of dressed electrons, (2) and 5), are pictured schematically in , are picIn order to calculate transport properties of dressed electrons, we have to solve the scattering problem for nonstationary electron states (4) and (6). Following the scattering theory for dressed conduction electronswhere wherefor the case of circularly polarized dressing field, andfor the case of linearly polarized dressing field.ω, meets the conditionIn what follows, we will assume that the wave frequency, where J, is given by the expressionLet us focus our attention on the dc conductivity of the dressed electrons. Generally, the density of the conduction electrons can be tuned by applying a bias voltage which fixes the Fermi energy, where Substituting the scattering probability of dressed electron (8) into Eq. s-wave approximationTo simplify calculations, let us consider the electron scattering within the x axis, the conductivity is plotted in where ude  see . For theIt is seen in We have shown that the transport properties of electrons in graphene are strongly affected by a dressing field. Namely, a circularly polarized dressing field monotonically decreases the isotropic conductivity of graphene, whereas a linearly polarized dressing field results in the oscillating behavior of the conductivity and its giant anisotropy. As a result, the dc transport properties of graphene can be effectively controlled by a strong high-frequency electromagnetic field. From the viewpoint of possible applications, the discussed effect can make graphene more tunable. Particularly, the switching times for conductivity of graphene controlled by a high-frequency field are expected to be shorter then for the case of conventional electrostatic control of conductivity by gate electrodes. This can create physical prerequisites for novel graphene-based optoelectronic devices.How to cite this article: Kristinsson, K. et al. Control of electronic transport in graphene by electromagnetic dressing. Sci. Rep. 6, 20082; doi: 10.1038/srep20082 (2016).


C9ORF72 gene is the most common genetic change seen in familial Frontotemporal Lobar Degeneration (FTLD) and familial Motor Neurone Disease (MND). Pathologically, expansion bearers show characteristic p62 positive, TDP-43 negative inclusion bodies within cerebellar and hippocampal neurons which also contain dipeptide repeat proteins (DPR) formed from sense and antisense RAN (repeat associated non ATG-initiated) translation of the expanded repeat region itself. ‘Inappropriate’ formation, and aggregation, of DPR might therefore confer neurotoxicity and influence clinical phenotype. Consequently, we compared the topographic brain distribution of DPR in 8 patients with Frontotemporal dementia (FTD), 6 with FTD + MND and 7 with MND alone  using a polyclonal antibody to poly-GA, and related this to the extent of TDP-43 pathology in key regions of cerebral cortex and hippocampus. There were no significant differences in either the pattern or severity of brain distribution of DPR between FTD, FTD + MND and MND groups, nor was there any relationship between the distribution of DPR and TDP-43 pathologies in expansion bearers. Likewise, there were no significant differences in the extent of TDP-43 pathology between FTLD patients bearing an expansion in C9ORF72 and non-bearers of the expansion. There were no association between the extent of DPR pathology and TMEM106B or APOE genotypes. However, there was a negative correlation between the extent of DPR pathology and age at onset. Present findings therefore suggest that although the presence and topographic distribution of DPR may be of diagnostic relevance in patients bearing expansion in C9ORF72 this has no bearing on the determination of clinical phenotype. Because TDP-43 pathologies are similar in bearers and non-bearers of the expansion, the expansion may act as a major genetic risk factor for FTLD and MND by rendering the brain highly vulnerable to those very same factors which generate FTLD and MND in sporadic disease.A hexanucleotide (GGGGCC) expansion in The online version of this article (doi:10.1186/2051-5960-2-70) contains supplementary material, which is available to authorized users. MAPT) [GRN) [CHMP2B[C9ORF72[C9ORF72 gene, occurring in patients with either FTLD or MND, or a combination of both [Frontotemporal Lobar Degeneration (FTLD) is a clinical, pathological and genetically heterogeneous condition. The major clinical syndromes principally involve personality and behavioural change  or language alterations of a fluent (semantic dementia) or non-fluent (progressive non-fluent aphasia) nature . All thrMAPT) , progranT) [GRN) , 4, CHMP [CHMP2B, and mos[C9ORF72–8. This  of both –8, and cPathologically, most FTLD cases with the expansion , 10–13, TMEM106B genotype has been claimed to be a genetic modifier of FTLD [C9ORF72 carriers from FTD [APOE) genotype since there have many studies claiming an increased frequency of APOE ϵ4 allele in FTLD   , and 7 with MND  bore expansions in C9ORF72 had a pure/predominant bvFTD phenotype, whereas the other 6 showed a mixed bvFTD and MND phenotype. Five of the FTLD patients without known mutation showed bvFTD phenotype, 3 had a progressive non-fluent aphasia (PNFA) phenotype and 6 showed a mixed bvFTD and MND phenotype. Pathologically, among the C9ORF72 expansion bearers with FTLD, all 8 patients with bvFTD had FTLD-TDP type A histology, whereas all 6 patients with FTD + MND had FTLD-TDP type B histology . These were usually skein-like in appearance, but occasionally more rounded, solid-appearing inclusions were also present.From clinical and neuropsychological assessments, 8 of the 14 FTLD cases with an expansion in al. 2011 ). Of theParaffin sections were cut (at a thickness of 6 μm) from formalin fixed blocks of representative regions of brain to include (where available) frontal cortex (BA8/9), temporal cortex (BA21/22), cingulate gyrus, insular cortex, motor cortex, inferior parietal and occipital (BA17/18) cortex. Blocks were also cut from the amygdala and posterior hippocampus, basal ganglia , substantia nigra (to include oculomotor nucleus), pons , medulla , cerebellum (with dentate nucleus) and cervical and lumbar spinal cord (where available).8 peptide with cysteine at N-terminus, conjugated to m-maleimidobenzoyl-N-hydrosuccinimide ester-activated thyroglobulin. The thyroglobulin-peptide complex (200 μg) emulsified in Freund’s complete adjuvant was injected subcutaneously into a New Zealand White rabbit, followed by 4 weekly injections of peptide complex emulsified in Freund’s incomplete adjuvant, starting after 2 weeks after the first immunization. Immunoreactivity of the antisera was characterized by ELISA as follows. The peptide immunogens were coated onto microtiter plates. The plates were blocked with 10% fetal bovine serum (FBS) in PBS, incubated with the rabbit antisera diluted in 10% FBS/ PBS at room temperature for 1.5 h, followed by incubation with HRP-goat anti-rabbit IgG (Bio-Rad) at 1:3000 dilution, and reacted with the substrate, 0.4 mg/mL o-phenylenediamine, in citrate phosphate buffer . The absorbance at 490 nm was measured using Plate Chameleon (HIDEX). In addition, sections of temporal cortex with hippocampus from 11 (non-expansion bearing) cases with other histological and genetic forms of FTLD, other neurodegenerative disorders and healthy controls (see Table Sections from each brain region were immunostained with a poly-GA antibody (courtesy of M Hasegawa), as described previously . The antFurther sections of frontal cortex and temporal cortex with hippocampus were immunostained for both phosphorylated (at Ser 409/410), and non-phosphorylated, TDP-43 .0 = no DPR immunostained NCI present in any field.0.5 = rare/single DPR immunostained NCI present in entire section.1 = a few DPR immunostained NCI present, in some but not all fields.2 = a moderate number of DPR immunostained NCI present in each field.3 = many DPR immunostained NCI present affecting many cells in each field.4 = very many DPR immunostained NCI present, affecting nearly all cells in every field.The presence of DPR immunostained NCI within nerve cells was assessed at ×20 magnification in those brain regions where all cases could be represented, according to:C9ORF72 expansions. Brain regions were grouped on an anatomical or a ‘functional’ basis. Hence, scores from frontal, temporal, cingulate, insular, parietal and occipital cortical regions were summated to generate a total ‘cortical’ score for each case. Scores from hippocampus and adjacent regions of subiculum, entorhinal cortex and fusiform gyrus were summated to give a total medial temporal lobe score for each case. Scores for caudate nucleus, putamen, globus pallidus, thalamus, substantia nigra, locus caeruleus and dorsal raphe were summated to give a total ‘subcortical’ score. Scores in motor cortex and in X and XII cranial nerve nuclei were summated to give a total ‘motor’ score. Scores in cerebellar granule and Purkinje cells, and in cells of the dentate nucleus, inferior olives and pontine nuclei were summated to give a total ‘cerebellar’ score. Due to the unavailability of tissue in all cases, it was not possible to include scores for amygdala (absent from 5/21 cases) or spinal cord (absent from 10/21 cases) within the medial temporal lobe or motor region analyses, respectively.Scores per assessed area were summated across those regions where these were available for all 21 individuals with 0 = no TDP-43 immunostained NCI and/or neurites.0.5 = rare/single TDP-43 immunostained NCI and/or neurite present in entire section.1 = very few TDP-43 immunostained NCI and/or neurites.2 = a moderate number of TDP-43 immunostained NCI and/or neurites.3 = many TDP-43 immunostained NCI and/or neurites, affecting many cells in every field.4 = very many immunostained NCI and/or neurites present, affecting nearly all cells in every field.The frequency of TDP-43 pathological changes  in each of frontal and temporal cortex  and hippocampus (dentate gyrus granule cells), was scored semi-quantitatively according to:TDP-43 pathology cores per assessed area were summated across those regions where these were available for all individuals. Hence, scores from dentate gyrus of hippocampus, and from frontal and temporal cortex, were summated  to give a total TDP-43 pathology score for each case.TMEM106B assay was genotyped by allelic discrimination using the Applied Biosystems pre-developed assay cat number C_7604953_10. Genotyping was carried out using the Applied Biosystems 7900, and genotypes were assigned automatically using the SDS 2.3 software. APOE was genotyped according to Wenham [DNA was extracted from blood or frozen brain tissue by routine phenol-chloroform extraction. The o Wenham .Rating data was entered into an excel spreadsheet and analyzed using Statistical Package for Social Sciences (SPSS) software (version 17.0). Kruskal-Wallis or Mann–Whitney test was used to compare inclusion scores between several groups or pairs of groups, respectively. All correlations were performed using Spearman rank correlation test. In all instances, a p-value of less than 0.05 was considered statistically significant.C9ORF72 associated FTD, FTD + MND and MND groups, FTD associated with GRN mutation, non-mutational FTD, FTD + MND and MND groups are shown in Table Mean ages at onset, death and duration of illness for 6,51 = 0.27, p = 0.946) or death , though duration of illness differed between the groups . As would be expected, post-hoc analysis showed that duration of illness was significantly less in C9ORF72 cases with MND than C9ORF72 associated FTD (p = 0.014), but not less than C9ORF72 associated FTD + MND (p = 0.281), nor was there any difference in disease duration between C9ORF72 associated FTD and C9ORF72 associated FTD + MND (p = 0.815). Again, as expected, non-mutational MND showed a shorter duration of illness than non-mutational FTD (p = 0.022) and non-mutational FTD + MND (p = 0.013). However, there were no significant differences in duration of illness between C9ORF72 associated FTD, non-mutational FTD or GRN associated FTD , or between C9ORF72 associated FTD + MND and non-mutational FTD + MND (p = 0.900), or between C9ORF72 associated MND and non-mutational MND (p = 0.605).ANOVA comparison of age at onset, death and duration of illness between all 7 diagnostic groups revealed no significant differences in age at onset  or duration of illness , though duration of illness tended to differ between the 3 groups .ANOVA comparison of age at onset, death and duration of illness between All cases had been previously classified on the basis of the type and distribution of TDP-43 immunoreactive changes according to Mackenzie et al. 2011 [C9ORF72, but none were seen in any of the cases bearing GRN mutations, nor in any of the other FTLD cases or MND cases not known to be associated with any FTLD or MND linked mutation.In cases of FTD and FTD + MND, DPR were observed to be most frequent within the cerebral neocortex, hippocampus and cerebellum. They were infrequent or absent in basal ganglia regions, and were rare or usually absent in mid brain, brainstem, medulla and spinal cord regions. DPR were present in neuronal cytoplasmic inclusions (NCI) throughout all cortical layers in all regions of the cerebral cortex examined. In outer cortical layers they were mostly present in small non-pyramidal neurons appearing as dots or clusters of granules , hippocampal , motor , subcortical  or cerebellar  regions, or for total score summated across all 5 subregions  , hippocampal , motor  and for total score summated across all 5 subregions , but not for subcortical  or cerebellar  regions. However, there was no significant correlation between the severity of DPR pathology and duration of disease, except weakly so in cerebellum .When all cases were grouped, there were significant correlations between the severity of DPR pathology and age at onset of disease for cortical . No significant differences in DPR severity scores were found between ϵ4 allele bearers and non-bearers, either overall (p = 0.240) or according to subregion , hippocampal (p = 0.189), motor (p = 0.887), subcortical (p = 0.506) or cerebellar (p = 0.640).Comparisons of DPR severity scores according to possession of at least one copy of TMEM106B genotypes were only available for 7 of the 14 FTLD cases, and 1 of the 7 MND cases, bearing C9ORF72 expansions, 9 of the 12 GRN mutation carriers, and 10 of the 14 FTLD cases and 1 of the 20 MND cases without known mutation. Because TMEM106B SNPs rs1020004, rs1990622 and rs6966915 were in complete linkage disequilibrium, pathological analyses were confined to rs 1990622. Unfortunately, only one of the FTLD, and none of the MND cases, irrespective of type or presence of mutation, was homozygous for the minor allele at any SNP. Of the C9ORF72 expansion bearers, 4 of the FTLD cases were homozygotes for the major allele and 3 were heterozygotes, with the MND case being homozygous for the major allele. Of the GRN mutation bearers, 5 of the FTLD cases were homozygotes for the major allele and 4 were heterozygotes. Of the non-mutational FTLD cases, 6 were homozygotes for the major allele, 2 were heterozygotes and 1 was homozygous for the minor allele.Due to availability of DNA, Nonetheless, comparisons of DPR severity scores were performed between FTLD cases heterozygous (TC) and homozygous (TT) for the major allele. No significant differences in DPR severity scores were found between bearers of TC or TT genotypes, either overall (p = 0.881) or according to subregion , hippocampal (p = 0.549), motor (p = 0.495), subcortical (p = 0.546) or cerebellar (p = 0.878).C9ORF72, FTLD cases without expansions, and FTLD cases with GRN mutation for either frontal cortex , temporal cortex  or dentate gyrus of hippocampus , or as total TDP-43 severity score . Moreover, there were no significant differences in the severity of TDP-43 pathology scores between FTLD cases with type A histology bearing an expansion in C9ORF72, FTLD cases with type A histology but without expansion in C9ORF72 mutation, and FTLD cases with GRN mutation and type A histology for either frontal cortex , temporal cortex  or dentate gyrus of hippocampus , or as total TDP-43 severity score . Similarly, there were no significant differences in the severity of TDP-43 pathology scores between FTLD cases with type B histology bearing an expansion in C9ORF72 and FTLD cases with type B histology but without expansion in C9ORF72 mutation for either frontal cortex (p = 0.570), temporal cortex (p = 0.147) or dentate gyrus of hippocampus (p = 0.344), or as total TDP-43 severity score (p = 0.254).Composite rating scores for TDP-43 pathology are shown for each case in Table C9ORF72 expansion carriers, there were no significant correlations between the severity of TDP-43 pathology and either age at onset of disease, or duration of illness, for either cortical, hippocampal or total scores. Neither was there any correlation between TDP-43 scores and clinical phenotype (ie FTD versus FTD + MND).Within the APOE ϵ4 allele were performed on 36/39 FTLD cases (for whom DNA was available for genotyping), irrespective of the presence or absence of mutation. No significant differences in the severity of TDP-43 pathology scores were seen between FTLD cases with APOE ϵ4 allele and FTLD cases without APOE ϵ4 allele for either frontal cortex (p = 0.187), temporal cortex (p = 0.293) or dentate gyrus of hippocampus (p = 0.171), or as total TDP-43 severity score (p = 0.261). Similarly, comparisons of TDP-43 severity scores according to heterozygosity (TC) or homozygosity (TT) for major allele in TMEM106B were performed on 24/39 FTLD cases (where these data were available), irrespective of the presence or absence of mutation. No significant differences in the severity of TDP-43 pathology scores between FTLD cases with TC and TT genotypes were seen for either frontal cortex (p = 0.301), temporal cortex (p = 0.592) or dentate gyrus of hippocampus (p = 0.768), or as total TDP-43 severity score (p = 0.519).Comparisons of TDP-43 severity scores according to possession of at least one copy of C9ORF72, neither the topographical distribution, nor the relative severity, of DPR differs between cases of FTD alone, FTD + MND or MND alone. Secondly, neither the morphological appearance, nor relative severity, of TDP-43 immunoreactive changes differ in cases of FTLD bearing an expansion in C9ORF72 from that in non-expansion bearing cases of FTLD (ie sporadic forms of FTLD), or from cases of FTLD bearing mutations in GRN. These findings support the observations of Mackenzie and colleagues [per se in causing FTLD or MND.There are two major outcomes from this study. Firstly, that in cases of FTLD or MND bearing expansions in lleagues  who likeC9ORF72 have been postulated to invoke disease by 3, mutually non-exclusive, mechanisms: firstly involving a loss of C9orf72 protein through haploinsufficiency [Expansions in ficiency , secondlficiency , 21, andficiency , 22, 23.Firstly, in both the present, and in other studies , the disSecondly, it is hard to conceive how a seemingly identical topographical pattern of DPR pathology could determine, or even predispose towards, such diverse clinical phenotypes as FTD or MND. To explain such an apparent paradox, Mackenzie et al. 2013  invoked per se. Although, there is no a priori reason to suspect that patients with an expansion in C9ORF72 should ‘naturally’ carry a higher burden of TDP-43 pathology than non-expansion carriers, it is nonetheless important to observe that such differences do indeed not exist. Moreover, if the expansion were directly driving a TDP-43 proteinopathy, it is hard to reconcile this with observations of different TDP-43 histologies. FTLD-TDP Type B  is the most common TDP-43 histological subtype associated with the expansion [The second observation from this study indicates that as far as the TDP-43 proteinopathy is concerned, there are again no distinctions in pathology between expansion bearers and non-bearers of the expansion, either overall or more specifically in respect of FTLD-TDP type A or type B histologies. This also supports observations by Mackenzie et al.  and furtxpansion , 24, thoxpansion , and rarxpansion . How sucC9ORF72 could be that the expansion acts as a risk factor for FTLD and MND, though does not drive the (TDP-43) pathological process directly, acting more as a ‘gatekeeper’ to disease, rendering the brain susceptible to the ‘development’ of all sporadic forms of FTLD-TDP, and sporadic forms of MND associated with TDP-43. Such a scenario could accommodate the present observations of the different histological types of TDP-43 proteinopathy being associated with disease, and the relative proportions with which they occur, and with findings that the extent of TDP-43 pathology is the same in expansion and non-expansion bearers both in terms of either type A or type B histologies.An alternative explanation of the role of C9ORF72. Indeed, there has been one Belgian case with C9ORF72 expansion and clinical FTLD which lacks detectable TDP-43 pathology, but shows FTLD-UPS pathology [GRN with C9ORF72[MAPT with C9ORF72[GRN or MAPT) mutation prevails.There is other evidence in support of this latter line of argument. Firstly, although rare, pathologies other than FTLD or MND, such as corticobasal degeneration , 17, Alz C9ORF72, 55 or M C9ORF72–57  has been claimed to be a genetic modifier of FTLD in both GRN and C9ORF72 mutation carriers [C9ORF72 carriers from FTD [TMEM106B allele with FTLD-TDP type A histology appeared to have a lower TDP-43 burden than homozygous carriers of the major allele. Therefore, we investigated whether variations at this locus might influence both the distribution and severity of both DPR and TDP-43 pathologies in FTLD cases associated with either C9ORF72 expansions or GRN mutations. Unfortunately, no homozygous bearers of TMEM106B minor allele were available in the present study for either mutation, but we did not find any differences in DPR and TDP-43 pathology between heterozygotes and those homozygous for the major allele suggesting that heterozygosity, at least, does not confer a ‘partial’ protection in either C9ORF72 or GRN mutation carriers. Similarly, although possession of at least one copy of APOE ϵ4 allele has been (variably) claimed to increase risk of FTLD [carriers , 28, 29,from FTD , 28, 29. of FTLD –33, we fin vivo, and in vitro, as well as the role RNA foci might play in causing the disease. However, there is evidence of C9orf72 haploinsufficiency in expansion carriers [RAB38 as a risk factor for bvFTD [GRN[TMEM106B[How the expansion might act in a ‘gatekeeper’ role is not clear. Much attention has been levied towards a determination of the nature and effect of DPR carriers , though carriers , 23, 58,carriers . In thisor bvFTD . RAB38 hor bvFTD , 62 and or bvFTD , and impor bvFTD . Indeed,FTD [GRN and TMEMTMEM106B. ConsideC9ORF72 are not without ‘pathological expression’, what DPR might translate into in clinical terms is not clear, and whether these structures confer anything beyond diagnostic utility still remains to be demonstrated.Nonetheless, if such a ‘gatekeeper role’ is true, then the almost complete penetrance of the mutation needs to be explained. In this respect the expansion might be viewed as providing an ‘open door’ to disease, but without actually causing disease directly, and in this regard the DPR pathology may be a ‘red herring’ without pathogenetic consequence. Clearly, until we know more about the normal function of C9orf72 protein, what cells it is present in, and where in the cell it is located, all is mere speculation. The concept of the expansion being a risk factor for disease, rather than a cause, is tempting since it would neatly accommodate the observed diversity of clinical and histological subtypes associated with the expansion. However, were the expansion to act as a risk factor, it appears to act  selectively for FTLD and MND since expansions are only infrequently, and then maybe only coincidentally, seen in more common disorders such as Alzheimer’s and Parkinson’s disease –53. TherSelected clinical, pathological and genetic details for 40 cases of Frontotemporal Lobar degeneration and 27 cases of Motor Neurone disease.(XLSX 18 KB)Additional file 1:


PD-L1-negative tumors assessed by immunohistochemistry often still respond to PD-1 blockade. PD-L1 is inducible by interferon, therefore, absolute negative tumors are the ones unable to up-regulate PD-L1 in response to interferons. Genetic mutations in the interferon receptor signaling pathway leading to loss of PD-L1 up-regulation were hypothesized to exhibit innate resistance to PD-1 blockade.After optimization, 50 primary human melanoma cell lines were exposed to interferons  and PD-L1 expression was measured. Interferon signaling was assessed by single cell phospho-proteomics, pSTAT1 (Y701), pSTAT3, pSTAT5, pSTAT6 expression level by flow cytometry (FACS LSRII). Western blot assessed JAK1/JAK2/IRF1 as well as STAT/pSTAT expression and Whole exome sequencing was performed by next generation sequencing for three selected melanoma cell lines and on biopsies from 25 patients with advanced melanoma treated with anti-PD-1 therapy.Three out of 50 melanoma cell lines were unable to up-regulate PD-L1 in response to interferon gamma; two of them had disruptive mutations in JAK1 or JAK2, and a third one had a defect in expression of IRF1 in response to interferons. Western blot analysis confirmed loss of function for the JAK1/JAK2 mutations and loss of downstream IRF1/STAT1/3/5 phosphorylation events. Whole exome sequencing of biopsies from 15 patients with metastatic melanoma who had objective response to PD-1 blockade (pembrolizumab) showed no homozygous inactivating mutations in interferon signaling pathway genes. Interestingly, one patient with the highest mutational load out of 10 patients without clinical response to PD-1 blockade had an amplified allele of JAK1 with a P429S mutation in the src-homology (SH2) domain. A cell line derived from this patient showed lack of sustained up-regulating of PD-L1 expression in response to interferon gamma by, and Western blot confirmed loss of JAK1 expression. Immunohistochemistry of tumor biopsy for this patient showed few CD8+ T cells.This study has defined genetic mechanisms of innate resistance to PD-1 blockade which lead to inhibition of adaptive PD-L1 expression in patients with advanced melanoma. This work suggests lack of interferon-gamma induced PD-L1 upregulation has the potential to be a negative selective marker for PD-1 blockade therapy.


Graphene can be synthesized from polyacrylonitrile (PAN) polymer through pyrolysis. A metal catalyst such as nickel (Ni) is required for the conversion of the polymer to graphene. The metal catalysts can be placed either atop or underneath the polymer precursor. We observed that spatially non-uniform and disconnected graphene was fabricated when PAN film coated with a Ni layer was pyrolyzed, resulting in flake-like graphene. Formation of the flake-like graphene is attributed to the dewetting of the Ni layer coated on the PAN film. Dewetting phenomenon can be reduced by decreasing the pyrolysis temperature, and hence, more uniform graphene could be prepared. The effects of Ni coating thickness and the pyrolysis temperature on the fabricated graphene have been experimentally analyzed. Ever since the discovery of graphene , researc2 is required for the application to electronic device [The solid-phase method employs transition metals such as Ni and Cu as a catalyst to form graphene from solid-state carbon sources such as polymer, SiC, small molecule, and self-assembled monolayer. Particularly, in the method using polymer as a precursor of graphene, various polymers like polyacrylonitrile (PAN), polystyrene, and polymethylmethacrylate were used, and the polymer placed either atop or underneath metal catalysts were pyrolyzed in a reductive gas to form graphene -23. Whenc device ,24. Thisc device ,23.2/Si substrate from PAN coated with a Ni film through pyrolysis. The Ni coating layer tends to be aggregated to form particulates due to dewetting [Here, we present systematic experimental results to synthesize graphene on a SiOewetting  at a higMw =150,000) (0.5 wt.%) dissolved in N,N-Dimethylformamide  was spin-coated on 1 × 1 cm2 SiO2 (300 nm thickness)/Si wafers. Subsequently, a Ni layer was coated on the spin-coated PAN/SiO2/Si substrates with a magnetron sputtering system. The sputtering rate was approximately 10 nm/min, and the thickness of the Ni layer was changed by the sputtering time. The Ni-coated PAN/SiO2/Si samples were pyrolyzed in a high-vacuum furnace; the vacuum level in the furnace was roughly 10-5 Torr.Polyacrylonitrile  is easily agglomerated in the vacuum atmosphere. So the temperature of 1,050°C was selected as the maximum temperature. In the case of the minimum temperature range, the temperature where graphene is formed and the agglomeration of the nickel layer is suppressed was selected as the minimum temperature. Therefore, the temperature ranged from 1,050°C to 700°C was selected for the pyrolysis).After the pyrolysis, the coated Ni layer was removed with a Ni etchant  at approximately 35°C for 2 to 4 h.The synthesized graphene on the substrate was characterized by Raman spectroscopy  and Raman mapping . The surface morphologies of the products were observed using a field-emission scanning electron microscope   and approximately 1,590 cm-1 (G peak) and no second-order Raman peak at 2,600 to 2,800 cm-1 corresponding to 2D peak of graphene was observed. This indicates that the PAN film was converted to amorphous carbon on the surface of the SiO2/Si substrate [-1 appeared  continuously decreased  of the Raman spectra shown in Figure -1, and the intensity ratio of G to 2D peaks (IG/I2D) was approximately 2  if tThe authors declare that they have no competing interests.HK carried out the experiments, contributed to the interpretation of the data and drafted the manuscript. JMH contribute to measure Raman spectrum. SHY and GA assisted in the experiments. SOC supervised the whole study. All authors read and approved the final manuscript.


In particular, the Dirac peaks shift very rapidly when the pressure decreases from ~2 × 10−3 Torr to ~5 × 10−5 Torr within 5 minutes. These Dirac peak shifts are known as adsorption and desorption of environmental gases, but the shift amounts are considerably different depending on the fabrication process. The high gas sensitivity of the device fabricated by shadow mask is attributed to adsorption on the clean graphene surface.To pattern electrical metal contacts, electron beam lithography or photolithography are commonly utilized, and these processes require polymer resists with solvents. During the patterning process the graphene surface is exposed to chemicals, and the residue on the graphene surface was unable to be completely removed by any method, causing the graphene layer to be contaminated. A lithography free method can overcome these residue problems. In this study, we use a micro-grid as a shadow mask to fabricate a graphene based field-effect-transistor (FET). Electrical measurements of the graphene based FET samples are carried out in air and vacuum. It is found that the Dirac peaks of the graphene devices on SiO Also, an inhomogeneity of electron-rich and hole-rich puddles arises due to the charged impurity and substrate-induced disorder. This inhomogeneity32 surface, and trapped charges19142 and hBN were investigated under various conditions.Graphene123567910111213141516Employing e-beam or photolithography while patterning electrical contacts unnecessarily exposes the graphene surface to chemicals . These chemical residues have been reported to be nearly impossible to completely remove, thereby degrading graphene device quality22232 substrates, where p+ doped Si (ρ ∼ 3 × 10−3 Ωcm) was used as the bottom gate electrode and SiO2  as the gate dielectric. First, the Si/SiO2 substrates were sonicated in acetone, then in isopropanol, and later rinsed by DI water. Highly oriented pyrolytic graphite  and natural Kish graphite were used to produce graphene flakes by mechanical exfoliation via adhesive tape . The graphene based FETs were also fabricated on the hBN substrate. For graphene on hBN fabrication, hBN flakes were first deposited on the Si/SiO2 wafer using the mechanical exfoliation method. Then, a microscope was utilized to select one appropriate hBN flake, which was the substrate for fabricating graphene FET. A graphene layer was transferred to the hBN substrate using the dry transferring method14The graphene channel of FETs was supported on Si/SiOElectron-beam evaporation was used to make electrodes with Ti (1 nm) followed by Au (80 nm) for electrical contacts at both ends of the graphene FET channel (‘source’ and ‘drain’). The electrodes were evaporated onto the graphene through square holes in a commercial grid for transmission electron microscopy (TEM), which was used as a shadow mask. Two kinds of TEM grids  were employed, and the specifications are listed in Table 1 of the 2/Si substrate is shown in 2. To pattern the electrodes, we used a polydimethylsiloxane (PDMS) slab as a supporting layer carrying the TEM grids. The PDMS slab has a dimension of 5 × 5 × 1 mm3 with a punched hole (1–2 mm dia.), where the center of the TEM grid was located. After that, the PDMS slab was affixed to a glass slide. The slide with the PDMS slab was inverted and attached to a micromanipulator. The micro manipulator was then utilized to position the TEM grid covering the chosen part of the graphene flake on the hBN substrate. Using a micromanipulator while monitoring the area through an optical microscope, the TEM grid was carefully positioned on the graphene flake of interest  so that one of the TEM bars covered the graphene, leaving its opposite side ends uncovered between the bar of the mask. After the first evaporation, a square array pattern of the fine TEM grid was created as shown in A schematic drawing of our back-gated graphene FET supported on a SiORtot versus Vg curves were found at a significantly positive Vg  for most of the fabricated samples. This positive Dirac peak shift of the graphene devices on the SiO2 substrate has been observed in other studies128As our device fabrication process involves shadow-masking to make electrical contacts, this method has the crucial advantage of being a resist-free process. After finishing the fabricating process, no other treatments , the doping polarity was completely reversed at a pressure of 1 × 10−5 Torr without heating. The Dirac point was shifted down to Vg < −40 V at high vacuum conditions. This n-doping source in vacuum should be different from p-type dopant at ambient. The p-type dopants acting as electron acceptors are gases (or water vapor) adsorbed on graphene surface and at the interface between the graphene substrate under ambient conditions. The adsorbates were desorbed in a high vacuum without annealing, and the p-doping effect disappeared.The graphene channel (10 μm long and 3 μm wide) supported on the SiO2 surface act as gas adsorption sites to adsorb gas and water molecules2O, O2, and NO2 lead to the p-type doping of graphene, while ammonia leads to the n-type1030−3 Torr in The Dirac peak at a positive gate voltage implies that the major carrier holes dominate the electrical transport at zero gate voltage. It is known that the defects at the SiO2 surface or directly adsorbed on the graphene surface2) and NO2 gases could serve as additional dopants existing in air, as they also adsorb weakly to graphene, acting as an electron acceptor33Usually, under ambient conditions oxygen and water molecules can be adsorbed on the SiO2. The graphene FETs supported on hBN of a few nm thickness were fabricated by the same method with TEM grids as shadow masks. The p-type doping behavior also occurred in the graphene on hBN as shown in Vg > +40 V at room temperature. When the chamber pressure decreased to 1 × 10−3 Torr, the peak shifted to lesser Vg, but the VD was still over +40 V. The results were very similar to those obtained with graphene on SiO2. The sample was then kept at 1 × 10−3 Torr at room temperature for 7 hours and measured again. The Dirac peak was still higher than +40 V, confirming that the desorption process does not depend on the duration time but the pressure mainly. This p-doping effect of the graphene on hBN can be explained by adsorbed gases and water moisture from the surrounding environment, which is similar to the graphene on SiO2. The mobility of this sample was estimated as 1.96 × 103 cm2/V/s from the Drude formula.To study the effect of the underlying substrate on doping graphene, hBN was employed as a substrate along with SiORtot/Vg curves of the sample measured under ambient conditions at 1 × 10−3 and 1 × 10−5 Torr at room temperature are depicted in 2/V/s, which is somewhat lower than that of the device fabricated by the TEM grid. This device also displayed a p- doped state in air, but the p-doping effect was weaker than that of the devices fabricated using the shadow mask. This difference may be due to the residue remaining on the graphene surface  were fabricated depositing the metal contact pads using e-beam lithography. The face See  and the Rtot versus back-gate bias Vg of the graphene on SiO2 device used in The 2 dielectric. This graphene layer was confirmed as a single layer graphene (SLG) by Raman spectroscope, so the thickness should be about 0.3 nm. However, the thickness of the graphene layer was estimated to be ~30 nm  adsorbed gases were removed from graphene. A high vacuum environment, along with heating, has been used to degas water vapor and adsorbed gasses on both the graphene surface and the interface between graphene and substrate33To confirm the degassing of the adsorbed gases and water vapor due to vacuum pumping, AFM scanning of the same sample was performed after it was placed in vacuum at 1 × 10M images . There iM images  was abouM images . This iset al. suggested that the observed n-type behavior is associated with a low work function of SiO2 substrate (W = 3.03 to 3.41 eV)g = 4.23 to 4.48 eV)2 substrate and the graphene layer depends on atomic configurations of SiO22 has an amorphous structure and transfers electrons to graphene. Additionally, the number of electrons transferred from the SiO2 substrate to graphene increases rapidly as the graphene is close to SiO2. In our work, the n-doping effect of graphene at high vacuum occurs with not only graphene on SiO2, but also graphene on hBN substrate. Few-nanometer-thick hBN was not enough to shield the low surface potential of SiO2, resulting in the n-doped graphene, because the Fermi level shift in graphene, due to hBN with several layers, was approximately 0.15 V from density functional theoryet al. reported an identification of a strong contamination source for graphene in vacuum systemsUnexpected n-doping behavior of graphene on hBN was occasionally found even on high quality samples by others, but no clear theoretical explanation has been suggested beyond attributing it to contamination14Rtot and Vg of the graphene on SiO2 measured at room temperature in water is shown in We also conducted a Dirac point measurement of graphene FETs in different liquid environments in order to confirm the effects of the adsorbed water layer. The relationship between resistance 2 with electric contact pads fabricated using e-beam lithography as shown in The same measurement was performed in ethanol as shown in et al. found that the top dielectric can induce significant changes in the back-gate capacitance due to the finite width of graphene deviceset al. confirmed that the back gate capacitance was increased by up to 2 orders of magnitude in water, by measuring the Hall-effect, which was dependent on the dielectric constant of the mediumThese electrical characteristics of the graphene based FETs in liquid dielectric environments can be explained by the change of effective back-gate capacitanceA lithography free method was demonstrated to eliminate polymer and solvent residues remaining on the graphene surface. In this study, we used a shadow mask to fabricate graphene based FET. The commercial TEM grids were utilized as a shadow mask for evaporation to deposit electrical metal contacts. Patterning with a shadow mask eliminates the use of polymers and solvents that are unavoidable in e-beam lithography or photo lithography. Our clean graphene device fabricated by shadow mask patterning indicated high sensitivity in both gas and liquid environments.2 and on hBN were investigated in various levels of vacuum, in ambient conditions, and in liquid. We found that graphene FETs were heavily p-doped as prepared in air, and this p-doping in electrical behavior of graphene was reversed into n-doped during vacuum pumping at room temperature. During the vacuum pumping process, the absorbed gases on the graphene surface under ambient conditions were able to be removed without annealing. From the AFM data showing inflated graphene and slow adsorption and desorption processes, the water is supposed to be a main source of the doping effect. The reversible changes at room temperature in the adsorption and desorption cycling imply the occurrence of physisorption rather than chemisorption. As water possesses a much lower saturation vapor pressure at room temperature, the majority of adsorbates should be water molecules. The n-type doping effect was found at 1 × 10−5 Torr in clean graphene surfaces supported on both SiO2 and hBN at room temperature. This n-doping effect from the substrates was explained by the work function of SiO2 being lower than graphene. The liquid environment measurement showed narrowing of the Dirac peak, which was attributed to the increment of the gate capacitance. Most results measured in our sample were similar to those reported previously by other groups, but our results were more sensitive to the environment as the sample was prepared in dry conditions without using polymer resist. As a result, the dominant doping source for the graphene device was not polymer residue, but water in air. The clean surface of graphene adsorbs surrounding water molecules strongly, while conventionally processed devices should be covered with polymer residue on top of graphene, preventing the adsorptions. Thus, the device fabricated by the resist-free method showed higher sensitivity in environmental gas than the device with polymer resist. By encapsulating the graphene in vacuum after the metal deposition using a shadow mask method, a high quality graphene device can be fabricated.The electrical properties of the graphene based FETs on SiONatural Kish graphite (Grade 300) was mechanically exfoliated on a Si wafer. The TEM grid was attached at the center of a hole punched in a PDMS slab. The TEM grid was carefully positioned using a micro-translator in the xy-plane, to locate the TEM grid into the target position. After alignment, z-directional contact made the PDMS slab stick to the wafer, due to its adhesive property. The adhesion was sustained in a high vacuum evaporation process, where Au contact pads were deposited through square holes of the TEM grid.The transport characteristics of the devices with three terminals were measured in air and vacuum at room temperature. During the vacuum pumping process, the absorbed gases on the graphene surface were able to be removed under ambient conditions in the laboratory. The semiconductor characterization system  was employed for accurate electrical measurements. A bias voltage was applied between the source and drain terminals of the graphene channel. The Si substrate was used as a global back-gate; this bias field controls the carrier concentration and polarity in the graphene layer. The voltage applied to gate terminal was swept continuously during the measurements. The output signal was converted to the resistance of the device and then used to plot the graph. The Dirac point of the device was determined from the graph. AFM measurements were performed using a commercial AFM . Pt/Cr (Multi75E-G) coated cantilevers with stiffness 3 N/m were utilized in noncontact mode for the AFM imaging.How to cite this article: Tien, D. H. et al. Characterization of Graphene-based FET Fabricated using a Shadow Mask. Sci. Rep.6, 25050; doi: 10.1038/srep25050 (2016).