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:


Idiopathic pulmonary fibrosis (IPF), the prototype of interstitial lung diseases, has the worst prognosis and is the only interstitial lung disease for which approved pharmacological treatments are available. Despite being considered a rare disease, IPF patients pose major challenges to both physicians and healthcare systems. It is estimated that a large number of IPF patients reside in BRIC countries  given their overall total population of approximately 3 billion inhabitants. Nevertheless, the limited availability of chest imaging in BRIC countries is considered a chief obstacle to diagnosis, since high-resolution computed tomography of the chest is the key diagnostic test for IPF. Further, obtaining reliable lung function tests and providing treatment access is difficult in the more rural areas of these countries. However, IPF might represent an opportunity for BRIC countries: the exponentially increasing demand for the enrollment of IPF patients in clinical trials of new drugs is predicted to face a shortage of patients – BRIC countries may thus play a crucial role in advancing towards a cure for IPF. Luca Richeldi Fig. Fig. 1LucIdiopathic pulmonary fibrosis (IPF) is the prototype of interstitial lung diseases (ILDs), a group of pulmonary diseases also referred to as “rare lung diseases”. A rare disease is defined by the European Union as one that affects less than 5 in 10,000 of the general population; as a consequence, a single rare disease may affect only a handful of patients, whereas another may affect as many as 250,000. Therefore, the concept of “rare” needs to be further defined in order to more accurately address diseases and their interventions. Diseases (both common and rare) do not present boundaries or geographic preferences; this is particularly true for diseases, such as IPF, for which risk factors linked to a particular racial background or a specific, defined geographic area or environment have not been identified to date. Thus, it is likely that the burden of disease will be concentrated in the most densely populated regions of the globe. In this context, BRIC countries , with an estimated 2.9 billion inhabitants overall, may comprise 1 million cases of a rare disease, thus easily representing a major medical need.Defining the exact epidemiology of a rare disease is a challenge: IPF is not an exception to this rule. Different data collection approaches have been applied in measuring the incidence and prevalence of IPF, and the findings of these studies vary widely . A recenLuca Richeldi was a consultant for and participated in advisory committees of AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Promedior, Roche Genentech, Sanofi-Aventis, and UCB. Further, he was a speaker for Boehringer Ingelheim, Cipla Pharmaceuticals, and InterMune. He received research support paid to his institution from InterMune.Adalberto Sperb Rubin Fig. Fig. 2AdaIPF is the most commonly diagnosed ILD in Brazil. Despite epidemiological studies on the Brazilian population being rare , IPF hasIn Brazil, as in many countries, the initial challenge of IPF is based on its proper diagnosis, since this process has significant therapeutic implications. Therefore, IPF, which has as a histologic substratum of usual interstitial pneumonia, must be distinguished from other chronic fibrosing lung diseases as well as from other ILDs. Indeed, in Brazil, cases of chronic hypersensitivity pneumonitis with characteristics very similar to IPF have been recorded . TherefoFor a number of years, we have followed various international directive agreements and implemented an anti-inflammatory treatment strategy as part of the management of IPF. Recent studies performed in a Brazilian population indicated the possibility that a combination of corticoids and immunosuppressant drugs had beneficial effects . The PANThe relationship between gastroesophageal reflux disease and IPF has long been under evaluation in Brazil , as in oAs with patients with other chronic lung diseases, IPF patients are included in pulmonary rehabilitation programs, and studies have demonstrated an improvement in various outcomes associated with their quality of life, with the 6-minute walk test used as one of the markers of this benefit .Lung transplantation, recommended in cases where the symptoms are progressive and where functional loss indicates reduced survival, is one of the few therapeutic strategies that has a real impact on survival with IPF . HoweverNew England Journal of Medicine [In 2014, two publications in the Medicine , 14 presMedicine ; it has As is the case in all BRIC countries, the greatest challenges lie in the availability of computed tomography (CT) and the equipment necessary to test lung function in the less developed parts of the country. Further, Brazil lacks a large enough number of pathologists and radiologists with experience in the precise diagnosis of ILDs, something which is fundamental to managing IPF. Nevertheless, there has been rising interest for IPF and its appropriate management within the national medical community, which, along with the availability of new therapies and further exchange with other countries, should lead to an improvement in IPF patient care in Brazil.ASB is on the advisory board and a speaker for Boehringer Ingelheim. He has also received funding for clinical research from Boehringer Ingelheim and Roche.Sergey Avdeev Fig. Fig. 3SerIPF is currently considered by Russian physicians as a disease that is difficult to diagnose and particularly difficult to treat . To dateEpidemiological studies on the prevalence and incidence of IPF in Russia have not been conducted and therefore only rough estimates based on a survey conducted in Moscow are available . ExtrapoThe data indicates that the average number of IPF patients attending a given participating center over 1 year was 49 . The participating centers were large multispecialty hospitals (attended 38 % of cases), outpatient centers (28 % of cases), and specialized pulmonology centers (50 % of cases). According to interviewed experts, the average age of patients was more than 60 years (67 % of responses). IPF patients in Russia are characterized by male predominance (66 %) with a high prevalence of smokers (61 %). At the time of initial diagnosis of IPF, the average level of forced vital capacity was 50–80 % of predicted values in 78 % of patients and less than 50 % of predicted values in 22 % of patients. The usual duration of symptoms before the diagnosis of IPF was 6–12 months (44 % of responses), 12–18 months (17 % of responses), or more than 24 months , cough (56 %), weight loss (11 %), and general weakness (5 %). The initial findings that most frequently revealed IPF were incidental findings on chest X-ray or chest CT (78 %), dyspnea (56 %), “velcro” crackles at lung auscultation (39 %), and cough (28 %). According to the interviewed experts, the most frequent comorbidities and complications in IPF patients were gastroesophageal reflux disease (57 %), cardiovascular disease (54 %), pulmonary hypertension (38 %), emphysema (33 %), sleep apnea (14 %), and lung cancer (3 %).More than 90 % of IPF patients in Russia receive pharmacological therapy. The most commonly initiated therapies are oral corticosteroids 56 %), immunosuppressants , N-acetylcysteine (83 %), long-term oxygen therapy (89 %), anti-reflux agents (72 %), and pirfenidone  reported that they used, for the management of IPF patients, the joint recommendations of the American Thoracic Society, the European Respiratory Society, the Japanese Respiratory Society, and the Latin American Thoracic Association (2011), although some also stated that they rely on other recommendations.The author declares that he has no competing interests.Zarir F. Udwadia Fig. Fig. 6ZarMost pulmonologists in India will admit to seeing a large number of patients with IPF in their practice and concede that the number seems to be increasing over the years. Nevertheless, this statement cannot be backed up by objective evidence in the form of epidemiological studies of incidence or prevalence since none are available. This section will discuss what is known about IPF in the context of a country of 1.2 billion people and focus on its distinctive features in the Indian setting.There has been a recent surge in the recognition of IPF in India. The initial Indian report of IPF occurred in 1979 by Jindal et al. , from thThus, available epidemiological data is limited to a few studies, all of which were retrospective. Nevertheless, these studies focused on different parts of India and showed no regional variations, and they all indicated that IPF is the most common cause of DPLD, being more common than other DPLDs such as sarcoidosis, hypersensitivity pneumonia, and ILD secondary to collagen vascular disease.Unfortunately, these studies do not provide information on the prevalence of IPF. If one extrapolates even the more conservative prevalence of 10 cases per 100,000 from Gribbin’s IPF data from the UK , this trOverlap with tuberculosis (TB):TB still dominates respiratory medicine in India and there is a tendency to initially mislabel patients with IPF as TB. These patients are subjected to long and unfruitful anti-TB drug trials before the correct diagnosis is finally considered . Whilst Over reliance on chest radiography:In a resource-limited setting like India, a chest radiograph is always the initial, and sometimes only, diagnostic test a patient from the rural interiors undergoes. Whilst chest radiography has a low sensitivity at the best of times, the quality of some of these radiographs is so poor that IPF is often never suspected and further work up never initiated.Poor HRCT penetrance: The first attempts at HRCT of the lungs began in centers in Mumbai and Delhi in 1991. The currently installed CT scanner base of 5,000 machines in the country is woefully inadequate to meet the needs of the population. India’s CT penetrance of one scanner per million population is much less than that of most of its BRIC neighbors. In addition, the majority of scanners (outside of the country’s main metropolitan areas) are of poor quality and incapable of imaging at 1-mm slice thickness. Countrywide, there are only a few radiographers with a special interest in chest imaging who will provide good quality images and their interpretation.Pulmonary function test unavailability:In smaller rural centers, though spirometry is widely performed, diffusion capacity is seldom available and the 6-minute walk testing rarely performed.Surgical biopsies and their interpretation: Although surgical biopsies are currently less commonly required in IPF since HRCT scanning has assumed a pivotal position in the diagnostic algorithm, on the occasions in which they do need to be performed, there are very few surgeons across the country capable of performing video-assisted surgical biopsies and even fewer lung pathologists capable of interpreting them accurately.Treatment of IPF in India has followed global trends: initial treatment revolved around high dose steroids and was followed by triple therapy with steroids, azathioprine, and N-acetyl cysteine; current treatment revolves around the use of pirfenidone. Interestingly, this novel anti-fibrotic drug has been available in generic form through two Indian pharmaceutical companies since 2010, several years before it received European Union and National Institutes of Health approval. Steroids are still used freely and in large doses, despite a large body of evidence suggesting they do more harm than good. Steroids, if used at all in IPF, must be used with great caution in the Indian context – the world’s largest diabetic (65 million) and TB populations (2.2 million cases) reside in this country and the potential for harm to these patients is great. India also has a huge population of patients with latent TB infection and the risk of activation of TB in these patients when given steroids for IPF is high.Lung transplantation for IPF has yet to make a foothold in this country. The first lung transplantation for IPF was performed at the Hinduja Hospital as recently as 2012. This patient survived only 3 months and the handful of cases subsequently transplanted from a few other centers across the country have also met limited success.IPF in India is widely prevalent but countrywide epidemiological studies are needed to map its exact incidence and prevalence. It is often misdiagnosed or diagnosed late (the previously described “TB effect”) and is often treated with traditional immunosuppression despite the more widespread availability of pirfenidone. Thus, centers of excellence where expert pulmonologists, radiologists, and pathologists can see these patients in consensus are required. Lung transplantation must also be expanded with the major hospitals in the larger cities offering this much needed option to IPF patients. A nationwide electronic ILD registry has recently been established and has already enrolled 700 patients from at least 20 different centers across the country ; this wiThe author declares that he has no competing interests.Zuo Jun Xu Fig. Fig. 7ZuoILD, also referred to as DPLD, is a heterogeneous group of diseases that mostly involves the pulmonary interstitium. IPF is a specific form of chronic, progressive fibrosing idiopathic interstitial pneumonia . To dateTo date, China has not conducted any national population-based epidemiological surveys on IPF and, therefore, no epidemiological data of IPF in the Chinese population are currently available. Only a few analyses have been conducted on the diagnosis and treatment data of hospitalized ILD patients in several local areas –36. A 20Two of the studies mentioned above, including 3,568 patients with ILD, found that idiopathic interstitial pneumonias were seen in 53 % of patients , IPF in 25.1 % (894 cases), connective tissue disease-associated ILD in 14 % (499 cases), sarcoidosis in 4 % (144 cases), and extrinsic allergic alveolitis in 3.8 % (100 cases) , 36. A rThe various hospital categories exhibit substantial differences in the diagnosis of IPF. At most community- or county-level hospitals, IPF is often missed or misdiagnosed. Many IPF patients are often first mistakenly diagnosed with pneumonia or chronic obstructive pulmonary disease. In addition, due to insufficient awareness of ILD, physicians sometimes misdiagnose alveolar protein deposition disease, certain secondary ILDs, and certain infectious pneumonias as idiopathic interstitial pneumonitis and treat these diseases with corticosteroids with/without immunosuppressive therapy. However, in hospitals that specialize in the diagnosis and treatment of ILD, the procedures for IPF diagnosis and treatment are highly standardized. For patients with pathological data, the diagnostic guideline is to establish a final diagnosis based on discussion among clinical experts, radiologists, and pathologists who are specialized in ILD. In hospitals that do not have the capacity for C-reactive protein diagnosis, physicians sometimes transfer the related data to teaching hospitals with the appropriate capabilities for consultation.Fourteen sites in China participated in the recent phase III international clinical study using nintedanib for the treatment of IPF . A totalIn China, several problems exist in the diagnosis and treatment process of IPF patients. Firstly, most hospitals have chest radiologists, but only a few have radiologists specialized in ILD. The lack of specialized radiologists, to some extent, results in respiratory disease specialists having a better understanding of ILD chest imaging than radiologists, thus weakening the role of radiologists in the diagnosis of IPF. In addition, chest HRCT examination is not universal, and many basic primary hospitals are not able to perform these, which is one of the reasons that many primary hospitals cannot determine a correct diagnosis of IPF. Second, although some hospitals have pathologists with expertise in ILD, the vast majority of hospital pathologists in China are general pathologists, and there are almost no specialized lung pathologists in the entire country. This problem may also be shared by most other hospitals outside China. Finally, because the Chinese healthcare system does not have an established referral system, most hospitals still do not have the capacity to systematically manage the relevant clinical data of outpatients, and the information of IPF patients who only receive outpatient treatment cannot be systematically organized/managed.The majority of ILD treatment centers in China are able to conform to international standards in terms of IPF treatment regimens by studying the related literature, updating treatment programs for IPF patients, and standardizing the treatment of these patients. Further, many new drugs can enter the Chinese market in a timely manner given the increased opening of trade. With the improvement of basic pharmaceutical research capacities, China also has several new IPF drugs that were originally developed domestically, such as pirfenidone. In addition, traditional Chinese medicine and therapy are unique to China, and attempts to treat IPF using these traditional methods, including certain results from basic and clinical research, suggest that some Chinese medicines may improve the symptoms and prognosis of the disease –42. FinaHowever, there are still some shortcomings in IPF patient care. First, due to deficiencies in the follow-up of patients in the Chinese healthcare system, the vast majority of hospitals do not conduct systematic follow-up of ILD patients. Many IPF patients seek treatment at multiple hospitals, and therefore their information is used repeatedly. Second, limited by the realities of new drug approval in China, many new drugs will take 3–5 years longer to enter the market compared to those in other countries. Third, although basic research results have suggested that traditional Chinese medicine can improve pulmonary fibrosis and has been widely used for the treatment of IPF in China, the specific mechanism of action and the identities of functional components in traditional Chinese medicine remain unclear. In addition, traditional Chinese medicine treatment requires individualization, and it is very difficult to apply a single prescription to all patients or to conduct double-blind studies. Therefore, there have been no large randomized controlled trials on IPF treatment using traditional Chinese medicine. Finally, due to the extreme lack of lung donors and the high cost of organ transplantation, as well as traditional resistance to organ transplantation in China, only a few IPF patients receive lung transplants each year.In summary, ILD incidence has shown a gradual increase in China. Teaching hospitals with ILD treatment centers or specialized in ILDs are able to keep pace with the development of international IPF treatments and standardize the treatment of IPF patients. However, given the condition of China’s healthcare system, further improvements are required for IPF diagnosis and treatment.The author declares that he has no competing interests.


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.


Adrenocortical carcinoma (ACC) is a rare tumor in which prognostic factors are still not well established. Programmed Death Ligand-1 (PD-L1) expression in ACC and its association with clinico-pathological features and survival outcomes are unknown.Formalin-fixed paraffin-embedded (FFPE) specimens were obtained from 28 patients with ACC. PD-L1 expression was evaluated by immunohistochemistry (IHC) in both tumor cell membrane and tumor infiltrating mononuclear cells (TIMC). PD-L1 positivity on tumor cells was defined as ≥5% tumor cell membrane staining. TIMC were evaluated by IHC using a CD45 monoclonal antibody. For PD-L1 expression in TIMC, a combined score based on the extent of infiltrates and percentage of positive cells was developed. Any score greater that zero was considered PD-L1 positive. Baseline clinico-pathological characteristics and follow up data were retrospectively collected. Comparisons between PD-L1 expression and clinico-pathological features were evaluated using unpaired t-test and Fisher’s exact test. Kaplan-Meier method and log-rank test were used to assess association between PD-L1 expression and 5-year overall survival (OS).Among 28 patients with surgically treated ACC, 3 (10.7%) were considered PD-L1 positive on tumor cell membrane. On the other hand, PD-L1 expression in TIMC was performed in 27 specimens and PD-L1 positive staining was observed in 19 (70.4%) patients. PD-L1 positivity in either tumor cell membrane or TIMC was not significantly associated with higher stage at diagnosis, higher tumor grade, excessive hormone secretion, or OS.PD-L1 expression can exist in ACC in both tumor cell membrane and TIMC with no relationship to clinico-pathologic parameters or survival.The online version of this article (doi:10.1186/s40425-015-0047-3) contains supplementary material, which is available to authorized users. Adrenocortical carcinoma (ACC) is a rare and highly lethal malignancy arising from the adrenal cortex. In the United States, around 0.7-2 new cases per million are estimated every year ,2. OveraComplete surgical resection remains the only chance of cure for patients with early-stage disease (stage I and II). When feasible, resection is the mainstay of therapy, even for patients with locally advanced disease. Not without controversy, some studies suggest that adjuvant mitotane may improve clinical outcomes . HoweverThe management of metastatic disease is challenging and disappointing results have been reported with the few available systemic therapeutic options . DespiteThe biology underlying ACC is poorly understood . So far,The current progress in understanding how the immune system can modulate tumor progression or effective responses against cancer is unfolding . Immune In this study, our goal is to characterize PD-L1 expression in ACC tissues and to correlate levels of PD-L1 expression with clinico-pathological features as well as survival outcomes.A total of 28 patients with ACC were included in this study. Patient characteristics are summarized in Table In this cohort, 8 specimens were from metastasis and 20 from primary tumors. Among the metastatic specimens, only one had metastatic disease at diagnosis. Others had specimens collected at the time of relapse.Overall, PD-L1 expression on tumor cell membrane was considered positive in 3 of 28 patients (10.7%) . The extent of TIMC were recorded as focal in 7 patients (26%), mild in 9 patients 33.3%), moderate in 9 patients (33.3%), and high in 2 patients (7.4%). For PD-L1 expression in TIMC, scores greater than zero were identified in 19 patients (70.4%). There was no significant correlation between PD-L1 expression in TIMC and stage (UICC and/or ENSAT), grade, or excessive hormone secretion .p = 0.65)  Figure .Figure 2Multiple retrospective analyses described the correlation between levels of PD-L1 expression and prognosis in several malignancies . Some ofTP53 mutations, as predictors of poor prognosis and its value still needs to be confirmed [Tumor stage at diagnosis is the most important prognostic factor in ACC . Howeveronfirmed . From a Some tumors are infiltrated by immune cells and it can dynamically influence the host immune response against tumor . InteresWith the goal of restoring effective T cell responses, the inhibition of immune checkpoints such as PD-1 or PD-L1 has been considered attractive therapeutic targets using monoclonal antibodies. A set of well conducted clinical trials have reported encouraging clinical activity on PD-1/PD-L1 blockade across multiple tumor types. The first phase I clinical trial of nivolumab, an anti-PD-1 monoclonal antibody, showed significant clinical activity in RCC, melanoma, and NSCLC, leading to deeper investigations . Other aNo biomarkers have been established to precisely select patients for therapeutic strategies blocking the PD-1/PD-L1 axis. Moreover, while several studies have reported that PD-L1 expression in both tumor cell or tumor infiltrating immune cells is a potential predictor of response to immunomodulatory agents, the meaning and significance of PD-L1 expression in tumor cells or immune cells is still being investigated . Prelimi. Finally, we focused on the clinical significance of PD-L1 expression in either, tumor cells and immune cells. However, the criteria defining PD-L1 positivity (cut-offs) as well as the antibodies used to stain for PD-L1 have varied among different studies. Therefore, comparisons with other studies should be done with caution and a more specific definition of immune cells subtypes should be focus of further studies. Although the same methodology used in this analysis has been applied to other studies, the methodology for PD-L1 staining should be standardized to allow for reliable evaluation of PD-L1 expression and comparison among differing cohorts [Though this is the first study to date to evaluate the prevalence and prognostic significance of PD-L1 expression in ACC, there are several limitations. First, the retrospective nature of this analysis has led to missing data which may result in selection bias. In addition, considering that ACC incidence is low, it is difficult to perform studies with large and homogeneous cohorts. In our study, the sample size and the number of events in each group according to PD-L1 positivity were very small, limiting our ability to detect statistically significant differences between groups. At the same time, there may be a missing-variable bias, since other clinico-pathological features such as Ki67 were not available for the majority of patients in this cohort. Furthermore, tumor heterogeneity is a confounding factor and differences between of primary and metastasis still need to be investigated. In 2 patients from our series, we had the opportunity to review primary and metastatic sites of disease. In one case, the primary tumor and one met were evaluated; in the other case the primary tumor plus 3 independent metastases were compared. Interestingly, in both cases the same results regarding PD-L1 expression (positive or negative) were observed in primary and metastatic samples stained for PD-L1. In this study, we tried to minimize the selection bias due to tumor heterogeneity by analyzing whole tissue sections from surgical resections and excluding needle biopsies cohorts .In summary, ACC can express PD-L1 on both tumor cell membrane and immune cells and it may represent a potential target for therapeutic interventions. The current progress in cancer immunotherapy warrants prospective validation of our findings and further investigation of agents blocking the PD-1/PD-L1 pathway in this aggressive disease.Twenty-eight patients with ACC treated surgically at Dana-Farber Cancer Institute or Brigham and Women’s Hospital were retrospectively selected. Formalin fixed paraffin-embedded (FFPE) blocks from primary tumors or metastases were retrieved, and for each patient one representative tumor block was selected for analysis by a genitourinary pathologist. Baseline clinico-pathological characteristics such as age, gender, tumor size, grade, stage using UICC and ENSAT staging systems, hormone-related symptoms at clinical presentation, as well as follow up data were retrospectively collected for all patients. Patients who presented with Cushing’s syndrome, virilization, or feminilization were classified as functional tumors. This study received Institutional Review Board approval before tumor staining and data acquisition.PD-L1 expression was evaluated by immunohistochemistry (IHC) using a mouse monoclonal anti-PD-L1 antibody (405.9A11) developed in Dr. Gordon Freeman’s laboratory at Dana-Farber Cancer Institute. The immunohistochemical assay was validated using FFPE cell line controls known to be positive or negative for PD-L1 expression by flow cytometry . Four miCD45 immunostaining was performed on adjacent four micron-thick tumor sections, which were initially deparaffinized, rehydrated and heated with a pressure cooker to 125°C for 30 seconds in citrate buffer for antigen retrieval and then incubated with peroxidase  and protein blocking reagents (Dako #X0909) each for 5 minutes. Sections were then incubated with anti-CD45  antibody for 1 hour at room temperature followed by incubation with the Dako EnVision + System HRP labeled polymer anti-mouse (Dako #K4001) for 30 minutes. All sections were developed using the DAB chromogen kit (Dako K3468) for 2 minutes and then lightly counterstained with hematoxylin.For each sample, the percentage of tumor cells with PD-L1 expression on the cell membrane was estimated by two independent genitourinary pathologists who were blinded to clinical outcomes.TIMC were identified on the basis of IHC positivity for CD45, a pan-leukocyte marker expressed in lymphocytes, macrophages and dendritic cells ,43. The In this exploratory biomarker study, the pre-defined primary objective of this study was to characterize levels of PD-L1 expression on tumor cell membrane and TIMC in patients with ACC. Secondary endpoints were the correlation of PD-L1 expression with clinico-pathological features as well as 5-year survival rates. Five-year survival rate was defined as the time period between date of diagnosis and the date of death, or censored at 5 years after diagnosis. PD-L1 tumor positivity on tumor cells was defined as ≥5% tumor cell membrane staining. For PD-L1 expression in TIMC, any score greater than zero was considered positive.Statistical analyses were performed using SAS . Descriptive data are presented as mean and standard deviation (SD), or percentage. Comparisons between PD-L1 expression and clinico-pathological features were evaluated using Fisher’s exact test for categorical variables and unpaired t-test for continuous variables. Kaplan-Meier method estimated the distribution of 5-year survival rates by PD-L1 positivity. The association of 5-year survival rates with PD-L1 expression (negative vs. positive) was assessed by log-rank test and univariate Cox proportional regression analysis. Multivariate analysis were to be performed only if p-values were <0.05, considered statistically significant.


The Chilean National Examination of Orthopaedic Surgery (EMNOT) has been administered since 2009. It was developed to determine whether individual residents are meeting minimal knowledge standards at the end of their training programs.We performed a retrospective evaluation of the EMNOT for all years it has been administered (2009–2015). The test was analyzed for content, taxonomy of questions asked , residents’ performance, difficulty index and discrimination index.During the years of EMNOT administration, the most frequently tested areas have been pediatric orthopaedics (22.9 %), spine (13.8 %), general orthopaedics (13.8 %) and musculoskeletal trauma (9.9 %). A significant increase in questions with images was observed, as well as a significant decrease in the percentage of Type 1 and an increase in Type 3 questions. The Difficulty Index showed a medium level of difficulty for all years the examination has been administered. The Discrimination Index showed good discrimination in 2009, fair discrimination from 2010 through 2012, and excellent discrimination from 2013 through 2015.The EMNOT has evolved over several years to include better quality questions, better discrimination, and a more representative distribution of questions covering the different orthopaedic sub-specialties. This examination represents an effective instrument for quality assurance of orthopaedic residency programs in Chile. In recent decades, there has been a dramatic increase in the levels of scientific knowledge, diagnostic expertise and overall competence that orthopaedic surgery residents must master during their training. This growing amount of information creates a need for objective and standardized assessment methods to assess residents’ learning throughout their education, and especially at the end of their residency programs. In the United States of America, the Orthopaedic In-Training Examination (OITE), a national test taken annually by orthopaedic residents that evaluates knowledge of the most important subjects in orthopaedic surgery, has been assessed in several studies –4. HowevIn Chile, the rising expectations of patients, combined with the country’s economic growth and its aging population, have required that more new physicians be trained in different specialties. In response to this demand, the number of orthopaedic surgery residency programs throughout the country has grown during the last decade. As a result, the number of orthopaedic surgeons graduating each year in Chile has also increased significantly. The Chilean Society of Orthopaedic Surgery (SCHOT) plays an active role in the continuing education of orthopaedic surgeons and residents in Chile, and many of the Society’s active members serve as certifying experts to ensure the quality of training programs. This is similar to the other medical societies in Latin America, which have played an active role in quality assurance for specialty training –7.To determine whether individual residents are meeting minimal knowledge standards at the end of their training programs, the SCHOT developed the EMNOT, an acronym for “Examen Medico Nacional de Ortopedia y Traumatologia” . The EMNOT is a multiple-choice exam covering the areas of general orthopaedics (including basic sciences); musculoskeletal trauma; pediatric orthopaedics; spine; shoulder and elbow; hand; hip and pelvis; knee and sports medicine; foot and ankle; and musculoskeletal oncology. The EMNOT was first administered in 2009, and, to date, a total of 279 final-year residents have taken the test.In this study, we analyzed this educational tool by assessing the examination’s distribution of questions in the different subspecialties of orthopaedics, its question taxonomy, and its difficulty and discrimination indices.For this study, we first obtained institutional board approval from the President and the Board of Directors of the SCHOT and its ethics committee. The information we analyzed did not include the examinees’ and institutions’ identifying data.We retrospectively assessed the 7-year period during which the examination has been administered (2009–2015). The total number of residents taking the EMNOT (each year and during the entire period) was recorded. All questions were categorized using the taxonomic classification described by Buckwalter et al. . A questEach question was also classified into one of ten areas: general orthopaedics (including basic sciences); musculoskeletal trauma; pediatric orthopaedics; spine; shoulder and elbow; hand; hip and pelvis; knee and sports medicine; foot and ankle; and musculoskeletal oncology. The percentage of questions from each area as a fraction of the entire EMNOT was established. Residents’ performance on the complete examination, as well as on each taxonomic type of question, was recorded.P = R/T, where R is the number of correct responses and T is the total number of responses . Levels of P were established as proposed by Backhoff et al. [The Difficulty Index (P) was determined as described by Crocker and Algina . P referf et al. , with vaFinally, we calculated the Discrimination Index (D). D refers to the capacity of an item to discriminate between high-ability examinees and low-ability examinees. We first scored each examinee's test and rank-ordered the test scores; next, the top 50 % of students (high-ability examinees) and the bottom 50 % (low-ability examinees) were separated for analysis. D for each question is the number of examinees in the upper group who answered the item correctly minus the number of examinees in the lower group who answered the item correctly, divided by 50 % of the number of students taking the test each year. Levels of D were established as proposed by Ebel and Frisbie , with D Statistical analysis was conducted using Statistical Program for the Social Sciences (SPSS) version 18 . Categorical variables were expressed as percentages. Fisher’s exact test was used to analyze categorical variables. A p value less than 0.05 was considered statistically significant.A total of 279 residents finishing their orthopaedic surgery programs have taken the EMNOT since 2009; the number of examinees has increased each year, as shown in Table p < 0.01 , p < 0.05. Variations in the proportion of questions by taxonomy level are shown in Tables The proportion of Type 1 questions has decreased significantly (from 77.8 % in 2009 to 59.2 % in 2015). There has also been a significant increase in Type 3 questions (from 5.6 % in 2009 to 22.5 % in 2015), The most frequently tested areas have been pediatric orthopaedics (22.9 %), spine (13.8 %), general orthopaedics (13.8 %) and musculoskeletal trauma (9.9 %), as shown in Table P = 65.2; range 52.7 – 71.6) showed that the test has presented medium difficulty in each of the years it has been administered. The Discrimination Index  showed good discrimination in 2009 but only fair discrimination from 2010 through 2012; D values increased to excellent discrimination from 2013 through 2015, as shown in Table The Difficulty Index of the test  and an increase in the number of evaluation/decision-making questions (Type 3), the proportion of correct answers did not change during the administration period of the test Table . Therefo and an iThe EMNOT migrated from a paper-based exam to a computer-based exam in 2012. This change facilitated the inclusion of images, which resulted in an increased percentage of questions involving figures. There were no images in 2009 and 2010 and the test had a median of 34 questions with images from 2013 to 2015. This also helped with the development of type 2 and type 3 questions: the inclusion of radiographs, patient photos, and graphs has resulted in more intricate questions that require a more complex approach, requiring the examinee to provide diagnosis and treatment decisions.This examination was developed as a comprehensive examination testing core knowledge in all areas of orthopaedic surgery. In this analysis, we found that not all sub-specialties were proportionately represented by the test; pediatric orthopaedics and spine surgery were the most represented sub-specialties. Similar results were also found by Papp et al. in their report on the OITE exam . In our The test has evolved throughout the years, with an increase in the total number of questions, an increase in Type 2 and Type 3 questions, and a more homogeneous representation of the different sub-specialties. The Difficulty Index shows that the exam has had a medium difficulty level throughout all years of administration; the Discrimination Index shows that the test has increased its discrimination capacity in the last three years. While these results are encouraging, the EMNOT still faces the limitation that, as a multiple-choice examination, it only evaluates knowledge, diagnostic skills, and evaluation/decision-making skills. The assessment of surgical skills and attitudes of examinees still remains a challenge , 16.Although beyond the scope of this article, the results of these exams have also helped to identify learning needs for the development of a more relevant continuing education program. Moreover, yearly reports are given to Residency Program Directors in Chile to aid in curriculum development.In a recent review, Gurgacz et al. postulated that a socially useful credentialing process in surgical specialties should adhere to three main conditions: (a) only one institution should be responsible for the credentialing process, (b) best-practice standards for design, implementation, and monitoring of the examination should be used and (c) the organization should have a strong quality-improvement culture . The SCHThe EMNOT has evolved over several years to include higher quality questions, better discrimination, and a more representative distribution of questions covering the different orthopaedic sub-specialties. Thus, this examination developed and administered by the SCHOT represents an effective instrument for quality assurance of orthopaedic residency programs in Chile.