PD-L1 regulates CD8 T cell/Th1 immune responses. PD-L1 expressed in the tumor microenvironment can bind to PD-1 or B7.1 on activated T cells and mediate cancer immune evasion. MPDL3280A is a human mAb containing an engineered Fc-domain designed to optimize efficacy and safety that targets PD-L1 and blocks it from binding to its receptors.Immunologic pharmacodynamics effects were evaluated in tumors and bloods from patients treated with MPDL3280A. MPDL3280A was administered IV q3w in >300 pts with locally advanced or metastatic solid tumors. PD-L1 and CD8 were measured by IHC. PD-L1 expression was evaluated in tumor and intra-tumoral immune cells. CD8 was assessed in the tumor center, periphery and invasive margin. The expression of ≈90 immune-related markers was evaluated at baseline (BL) and on-treatment using a custom-designed immunochip. BL tumor samples were available for 125 pts, and matched on-treatment samples were available for 31 pts. Further, blood-based biomarkers and circulating immune subsets were serially measured in 114 patients by modified ELISA and FACS, respectively.On treatment, responding tumors showed increase in expression of tumor cell PD-L1 and infiltration of CD8+ T-cells and a Th1-dominant immune infiltrate, providing evidence for adaptive PD-L1 up-regulation. Non-responders showed minimal tumor CD8+ T-cell infiltration and an absence of T-cell activation . We also profiled circulating biomarkers for their association with clinical outcomes. A sub-population of patients, including those with RCC and melanoma, exhibited elevated PD-L1 expression on circulating T-cells at BL that was associated with response to MPDL3280A. On treatment, we observed a delayed increase in frequency of CD4+ICOS+ and CD4+PD1+ T-cells in patients responding to MPDL3280A monotherapy. In contrast, frequency of CD8+HLA-DR+Ki67+ T-cells increased shortly following the first dose of MPDL3280A and returned to baseline levels by the end of cycle 2 when assessed in all patients, representing a transient pharmacodynamic measurement of PD-L1 inhibition. On-treatment, increase in plasma IL-6 was associated with disease progression. Associations with other circulating markers of inflammation including CRP with clinical outcomes will be presented.Our data show that changes in pharmacodynamic immune markers are associated with clinical outcomes to MPDL3280A. These data provide mechanistic insights into immune checkpoint inhibition in cancer and identify potential biomarkers that may be monitored as on-treatment markers of clinical activity for patients treated with inhibitors of PD-L1/PD1 pathway.


Pulse generation often requires a stabilized cavity and its corresponding mode structure for initial phase-locking. Contrastingly, modeless cavity-free random lasers provide new possibilities for high quantum efficiency lasing that could potentially be widely tunable spectrally and temporally. Pulse generation in random lasers, however, has remained elusive since the discovery of modeless gain lasing. Here we report coherent pulse generation with modeless random lasers based on the unique polarization selectivity and broadband saturable absorption of monolayer graphene. Simultaneous temporal compression of cavity-free pulses are observed with such a polarization modulation, along with a broadly-tunable pulsewidth across two orders of magnitude down to 900 ps, a broadly-tunable repetition rate across three orders of magnitude up to 3 MHz, and a singly-polarized pulse train at 41 dB extinction ratio, about an order of magnitude larger than conventional pulsed fiber lasers. Moreover, our graphene-based pulse formation also demonstrates robust pulse-to-pulse stability and wide-wavelength operation due to the cavity-less feature. Such a graphene-based architecture not only provides a tunable pulsed random laser for fiber-optic sensing, speckle-free imaging, and laser-material processing, but also a new way for the non-random CW fiber lasers to generate widely tunable and singly-polarized pulses. Q-switching, are generally not applicable to random fiber lasers with random feedbacks14Random lasers have received considerable attention due to the unique properties that distinguish them from conventional resonant cavity lasers, including modeless, cavity-less, low coherence output, simple design, and good reliability operation12457891112Q-switching have been examined31Graphene is an attractive two-dimensional material with a variety of exceptional electronic and photonic properties, and has been proposed in various applications16171920222324262728303133351420213638D-shaped fiber (GDF), the continuous wave (CW) from the arbitrarily-polarized random fiber laser is transformed into coherent sub-nanosecond pulses even in the non-resonant single-pass configuration. The PR and the GDF hybrid waveguide serves simultaneously as the linearly polarized pulse generator (polarization modulation) and the pulse reshaper (saturable absorption). The pulses generated from the GDF not only inherit merits of the CW random fiber laser such as a high pump-power Stokes conversion efficiency, but also offer unique advantages such as a power-dependent singly-polarized output with polarization extinction ratios up to 41 dB, and wide tunability in both pulsewidths and repetition rates, across two and three orders of magnitude respectively.Here we report graphene-based pulse generation with cavity-free random fiber lasers, for the first time. By dynamically modulating the polarization via a polarization rotator (PR) and then transmitting through a designed graphene-coated x-polarization transmission filter. With the x-polarization periodically modulated by the PR, the transmission is a sinusoidal-like temporal wave after passing the GDF. Simultaneously, with Pauli blocking and self-phase modulation at high input powers212228Ex-field distribution is shown in D-shaped fiber without graphene coverage, the x-polarized component and the y-polarized component have almost equal intensity. In comparison, with graphene cladding, the electromagnetic field distribution is pulled close to the graphene film and the x-polarized component is much stronger than the y-polarized component. With the graphene-based evanescent field enhancement, the transmitted CW random lasing field interacts slightly with the graphene film (detailed in With the finite element method (FEM), an example modeled D-shaped fiber was carefully polished with good uniformity and small surface roughness, with an insertion loss less than 10 dB over a polished length of 20 mm. Detailed fabrication process is shown in D-shaped fiber, under 633 nm transmission for clarity. In order to prepare high-quality graphene film and transfer it to the D-shaped fiber properly, monolayer CVD graphene was usedD-shaped fiber surface with good flatness and uniformity, even when the fiber was polished down with 58 μm depth (details in The A high-power CW laser at 1455 nm serves as the pump for the random fiber laser. After the accumulation of Rayleigh scatterings and Raman effects in a 50 km single mode fiber (SMF), a CW random fiber laser at 1550 nm is obtained. Random fiber lasers with other wavelengths are also achievable from their stimulated Raman scattering naturex-polarization was approximately −22 dBm, while the transmission in the y-polarization was approximately −57 dBm for a PER in excess of 34 dB. When the launched power of the GDF increases gradually, saturable absorption begins to occur along the GDF. x-polarized laser launched in the GDF increased from 1 mW to 2.9 W, the transmittance of the GDF increases from ~0.05% to ~3.4%. Contrastingly, for the y-polarized laser with launched power of 2.9 W, the transmission stays predominantly constant with only a 0.0002% increase, within the noise level and negligible. This indicates that much higher power is needed to use the y-polarized light to saturate the GDF. Such a polarization-dependent saturable absorption is determined compositely by the polarization dependent transmission of the waveguide, and the polarization dependent nonlinear response of graphene.The polarization output of the random fiber laser is next modulated periodically using the PR, with a rotation speed tunable from 1 kHz to 3 MHz, prior to launching into the GDF. The power-dependent polarization selectivity and saturable absorption of the GDF are shown in The polarization properties of the graphene based random fiber laser are shown in The intensity dynamics after the PR are measured by a real-time oscilloscope, comparing before and after the GDF. Under 2.8 MHz rotation, The correspondingly pulse spectra is shown in x-polarization. The PER reaches ~41 dB at ~2.88 W, one of the highest values observed for high power polarizers.QP = EFWHM/E0, here EFWHM is the energy in the full width at half maximum (FWHM) of the pulse, E0 is the energy of the pulse integrated over its periodQP > 70%. However, limited by the background noise and instability of the PR, the measured pulse quality is relatively lower. In the experiment, the maximum measured QP is 50.9%, with launched power 2.88 W and repetition rate 3 MHz.In addition, as the pulses are gradually compressed from the initial sinusoidal-like wave, a weak sinusoidal-like envelope is always persistent at the pulse pedestal, distinct from conventional cavity solitons or cavity mode-locking. Such a residual pedestal would deteriorate the pulse quality, which could be described as Distinct from conventional mode-locking lasers, pulse generated by the single-pass GDF configuration is compressed from sinusoidal-like envelope. To achieve short pulsewidth and acceptable pulse quality, relatively high launched power and fast PR is necessary. However, to generate pulses with cavity-free random fiber lasers, external methods are necessary. Fortunately, the GDF based pulse generation has exceptional properties, and could be further improved in the future, by optimizing the smoothness/uniformity of the GDF, the performance of the PR, and increasing the launched power or the modulation speed of polarization.In addition, beyond the random fiber lasers, the graphene based pulse generator could also be widely applied on typical non-random CW lasers in common fiber systems, as the polarization selectivity and the saturable absorption of the GDF is unique and universal. Hence, it provides a new way for the non-random CW lasers to generate widely tunable and singly-polarized pulses.In summary, we have demonstrated a graphene based 900 ps and widely-tunable pulse generation, with up to 41 dB polarization extinction for random fiber lasers. The single-pass configuration affords unprecedented widely-tunable pulses over two orders of magnitude in pulsewidth and three orders of magnitude in repetition rate, drawing from the synergistic advantages of graphene photonics and random fiber lasers. This not only inspires exploration of the full potential of graphene-based fiber devices for ultrafast photonics, but also the realization of pulsed random fiber lasers towards sensing, imaging and laser-material processing applications.D-shaped fiber (GDF), the polarization-dependent transmission, and fast pulse generation were theoretically investigated and numerically simulated. To estimate the power density in the GDF, the field distribution of the graphene-coated D-shaped fiber was simulated by COMSOL (considering the index of graphene to be determined by its conductivity) by applying εg,eq = −σg,i/ωΔ + iσg,r/ωΔ and ω2μ0ε = n2k02; see D-shaped fiber, the CW light from the random fiber laser would be transformed into highly polarized pulses with power that varies with time.The electric field distribution of the graphene-coated 3 solution, a soft and flexible PMMA/graphene was prepared. Thirdly, the PMMA/graphene hybrid was covered on the polished surface of a D-shaped fiber, which was carefully fabricated with the polished depth of 58 μm and polished length ~20 mm. Finally, the PMMA was removed by acetone vapor. The D-shaped fiber and graphene were clearly identified and characterized using optical microscopes (OPM), scanning electronic microscopes (SEM), and a Raman spectrum analyzer. More details are shown in The GDF samples were fabricated by following steps. Firstly, graphene was grown by using the CVD method on Cu foil. Secondly, after removing Cu by FeClD-shaped fiber can reach 3.5 W. The power meter with resolution of 0.1 dBm was used to detect the output power at 1550 nm. The transmittance could be calculated as PB/PA. Figure S5 in A polarization controller (PC) is used to adjust and fix the input polarization. The power launched into the graphene-coated To verify the broadband polarization selectivity of the GDF, an experimental setup was built as shown in How to cite this article: Yao, B. C. et al. Graphene based widely-tunable and singly-polarized pulse generation with random fiber lasers. Sci. Rep.5, 18526; doi: 10.1038/srep18526 (2015).


A so called all-carbon based graphene field effect transistor (GFET) in which the electrodes are composed of graphite-like nano-sheets instead of metals in the traditional devices is fabricated by one-step e-beam direct writing (EBDW). It is also found that the graphite-like nano-sheets in electrodes are perpendicular to the channel graphene, which is confirmed by the transmission electron microscopy (HRTEM). The one-step fabrication of the carbonaceous electrodes is more convenient and lower-cost comparing to the preparation of traditional metal electrodes and can be applied to many other nano-electronic devices. The resistivity of the carbonaceous electrode is measured to be about 4.8 × 10−3 Ω · cm [Supporting Information], which is much smaller than that of a heavily doped silicon  was employed to analyze the negative PMMA. The sample for the HRTEM was directly prepared on a Cu grid via the same processes and conditions in the et al.40The graphitization of the negative PMMA may originate from two concurrent processes: the lost of oxygen and hydrogen atoms (i.e. the deoxygenation and dehydrogenation) and the self-assembling of carbon-carbon (C-C) covalent bonds. Hydrocarbon gas can be decomposed into amorphous carbon by losing oxygen and hydrogen atoms under high-intensity electron irradiation in electron-beam-induced deposition (EBID) process3840et al.An interesting and important phenomenon revealed by the HRTEM images is that the graphitic nano-sheets in the annealed PMMA film are “standing” rather than “lying” on the substrate. Since the HRTEM gives a top-down view of the sample, the presence of the layer-by-layer structures with the spacing of about 0.33 nm in the HRTEM images taken at different areas of the cross bar on the Cu grid indicates that the graphite sheets in the negative PMMA are perpendicular to the substrate, which is consistence with the report by Duan It is convinced that the electrode/graphene contact could be fabricated as the vertical contact between graphite sheet and graphene if the negative PMMA was used as the electrodes in GFET. Based on the analysis above, the microstructure of the all-carbon based GFET can be sketched diagrammatically, as shown in 2 and etched to a ribbon with the width of 3 μm using O2 plasma with the assistant of EBL. The conductive silicon substrate is used as a back-gate of the GFET. From the scanning electron microscope (SEM) image of the To characterize the graphene in the channel after annealing at the temperature of 800 °C, the Raman spectra of the graphene in the channel before and after the annealing were measured, as shown in 44−4 Pa by a Keithley 4200 analyzer at room temperature, and the carrier mobility of ~3700 cm2/V/s of this GFET was obtained from the fitting of the transfer characteristic curve. The Dirac point (the minimum of the transfer characteristic curve) was at about −2 V, indicating that the device was slightly n-type doped. In our previous work2/V/s to the electrons and ~1750 cm2/V/s to the holes, which is however not so obvious in the all-carbon based GFET as shown in the Any adjacent pair of electrodes in the In summary, we have fabricated a kind of all-carbon based GFET with the carbonaceous electrodes and the channel graphene. The fabrication method of the all-carbon based GFET is simple, convenient and accurate because the electrodes are directly written out by e-beam on a PMMA film. In spite of the simpler and lower cost of the fabrication, the performance of the all-carbon based GFET is totally comparable to the metal contacted GFET from the I–V and field-effect characteristics. It is found that the carbonaceous electrodes are composed by graphite nano-sheets perpendicular to the graphene in the channel. Such vertical contacts between the graphite and graphene supplies a potential choice for the electrode/graphene contact in high-performance GFET.2 for actual) to improve the conductivity of the negative PMMA parts.First, a PMMA  film was spin-coated on a SiO2/Si substrate followed by 180 °C baking on a hot-plate for 1 minute. Second, the PMMA film was irradiated by a focused e-beam (FEB) in an EBL system (FEI 600i) to define the patterns for electrodes. During the irradiation process, the accelerating voltage and the current of the FEB were 10 kV and 1.4 nA, respectively. In order to transform the PMMA film to a negative resist, the irraidaiton dose should be exceeded 50 times more than the normal dose for a positive PMMAHow to cite this article: Chen, W. et al. All-carbon based graphene field effect transistor with graphitic electrodes fabricated by e-beam direct writing on PMMA. Sci. Rep.5, 12198; doi: 10.1038/srep12198 (2015).


The motivation behind this work is to study the gas phase chemical sensing characteristics of optical (plasmonic) nano-antennas (ONA) and graphene/graphene oxide-covered versions of these structures. ONA are devices that have their resonating frequency in the visible range. The basic principle governing the detection mechanism for ONA is refractive index sensing. The change in the concentration of the analyte results in a differing amount of adsorbate and correlated shifts in the resonance wavelength of the device. In this work, bare and graphene or graphene oxide covered ONA have been evaluated for gas sensing performance. Four different analytes  were used in testing. ONA response behavior to different analytes was modified by adsorption within the graphene and graphene oxide overlayers. This work is a preliminary study to understand resonance wavelength shift caused by different analytes. Results imply that the combination of well-structured ONA functionalized by graphene-based adsorbers can give sensitive and selective sensors but baseline drift effects identified in this work must be addressed for applied measurements. Presently, solid-state sensors are most prominently used for gas phase chemical detection1Plasmonic structures have been widely used for biological sensing810In this work, an array of dipole structures is designed as an ONA with resonance frequency in the visible spectrum. The array structure enhances the spectrum response of the ONA. The ONA was chosen with a bulk sensitivity of 450 nm/RIU. Response of the bare ONA for four different analytes  was studied. A shift of 0.5 nm to 3.5 nm in the resonance peak was observed. Motivated by a desire to attain greater sensitivity and selectivity than the bare ONA, we tested the same four analytes on graphene coated and graphene oxide coated ONA structures. We observed a much larger frequency shift with graphene and graphene oxide covered ONA. Bare ONA exhibits only a red shift when exposed to all the analytes. Graphene and graphene oxide coated ONA exhibited a blue shift when exposed to toluene and a red shift when exposed to the other three analytes. In this work, there was no baseline drift observed for the bare ONA sensor. However, graphene and graphene oxide coated ONA exhibited baseline drift apparently arising from residual adsorbed gas molecules. We propose a suggested mechanism for the frequency shifts observed for graphene and graphene oxide coated ONA for different analytes.μm as shown in ONA were prepared using e-beam lithography, metal deposition and lift-off as mentioned in the literatureThe testing setup is as shown in μmol/mol, and toluene and nitrogen dioxide with concentrations of  μmol/mol were examined. Analytes were delivered from cylinders, pre-diluted with zero air, and then further diluted to testing levels using our gas-flow manifold  were tested on the three different ONA configurations. Three configurations of ONAs were tested: (i) bare ONA, (ii) graphene covered ONA and (iii) graphene oxide covered ONA. Acetone and ethanol with concentrations of  manifold . While mThe intensity was determined by the following Equation Itransmission is the transmission spectrum of the device. Idevice is the spectrum when ONA are illuminated. Isource is the spectrum of the light passing through the fused silica without the ONA. Idark is the spectrum collected when the light source is turned off. The data processing was done using MATLAB (Disclaimer). A linear fit was used to determine the slope of concentration-wavelength shift curve for a given device. The linear fit was used only for those readings for which the response was not saturated. We don’t observe any variation in the resonance wavelength beyond the measurement capabilities of the spectrometer . In these feasibility studies each sample was tested once for all the analytes.where λ, is given by Equation 2In this work, we study the change in the resonance frequency of ONA when exposed to gas-phase analytes. We note that this proof-of-concept study uses three different ONA arrays to demonstrate the feasibility of using them for gas-phase sensing. Furthermore, we explored the possible utility of employing modifying layers to affect the response phenomena for two of the ONA arrays. The shift in the resonance wavelength, Δnanalyte and nair are the refractive indices of the analyte and air. d is the thickness of the analyte layer on top of the dipole structure. ld is the exponential field decay length. m is the bulk refractive index sensitivity of the bare ONA. For graphene/graphene oxide covered ONA, m is the bulk refractive index sensitivity of the combined device (ONA + graphene or graphene oxide). Using Lumerical’s FDTD (Finite Difference Time Domain) tool (Disclaimer) we determined that m is similar for bare ONA and graphene/graphene oxide covered ONA (which lies in the range of 450 nm/RIU to 730 nm/RIU). The values in where, λ > 0) is predicted for all the analytes used. The responses of the bare ONA are shown in μmol/mol and 100 μmol/mol for acetone and ethanol, and 10 μmol/mol and 20 μmol/mol for nitrogen dioxide and toluene). Bare ONA also has a fast response to different analytes. Bare ONA exhibits the highest sensitivity to toluene, which can be attributed to the high refractive index of toluene amongst all four analytes.Based on Equation Motivated by a desire to attain greater sensitivity and selectivity, we also tested two more sensor types with the same four analytes. We characterized the modifying layers with Raman spectroscopy to estimate the coverage level. From the Raman spectra of the covered ONA, we estimate that there are more than 4 layers of graphene oxide stacked on top of each other, while we observe evidence for only a monolayer of graphene covering the ONA See .λ > 0) for the graphene-covered ONA, in response to all the analytes, but a blue shift (Δλ < 0) is observed for toluene as shown in The response of graphene covered ONA for four different analytes is as shown in The responses of graphene oxide covered ONA are shown in μmol/mol) and detection limit is calculated as the smallest concentration that can give a spectral shift detected by the spectrometer. The slope was calculated using a linear fit on each sensitivity curve shown in Graphene, though apparently one atom thick when placed on top of the bare ONA, results in a shift of 13 nm, and graphene oxide having multiple layers, results in a shift of 45 nm. Response strengths of the three types of sensors to the four analytes are plotted as a function of analyte concentration in λ > 0). For ONA covered with graphene or graphene oxide depending on the analyte, the shift is either blue (Δλ < 0) or red (Δλ > 0). However, there may be an adsorbate history contribution to this effect (as discussed below).From The bare ONA resonates at 746.2 nm. As indicated above, when a monolayer of graphene is placed on top of the bare ONA to form graphene covered ONA, the resonance frequency is red shifted to 759 nm. The shift of around 13 nm was expected due to graphene as seen beforeThe baseline values before and after analyte adsorption for the bare ONA, graphene covered ONA and graphene oxide covered ONA are given in s and Rs. The gap between the two arms of the dipole is modeled by Cs. Cp and Rp denote capacitance and resistance connected to the ambient conditions1215s, and more dominantly Cp. Bare ONA has a fast response and is reversible, as the response comes back to its baseline value once the analyte flow is stopped. We also observe that the response saturates at higher concentrations. In the equivalent circuit diagram this corresponds to the larger values reached by capacitor Cs and Cp. The bare ONA response has no obvious selectivity towards any analyte. The bare ONA gives a red shift to all the analytes, making it hard to differentiate between analytes. However, among all the four analytes tested, bare ONA exhibited its maximum sensitivity towards toluene and nitrogen dioxide.The red shifts observed in the responses of the bare ONA can also be explained with the help of the left portion of the equivalent circuit diagram in Fig. 8p and Rp, and would yield a red shift in the peak resonance since all the analytes have a refractive index larger than air  or red shifted . A possible mechanism underlying the wavelength shift for graphene and graphene oxide coated ONA is discussed. Baseline drift was observed in the response of the graphene and graphene oxide coated ONA. Graphene and graphene oxide coated ONA have a slower response as compared to bare ONA.How to cite this article: Mehta, B. et al. Gas Sensing with Bare and Graphene-covered Optical Nano-Antenna Structures. Sci. Rep.6, 21287; doi: 10.1038/srep21287 (2016).


This generic technique paves the way for using a whole new range of eutectic substrates for the large-area synthesis of 2D materials.Large-area synthesis of high-quality graphene by chemical vapour deposition on metallic substrates requires polishing or substrate grain enlargement followed by a lengthy growth period. Here we demonstrate a novel substrate processing method for facile synthesis of mm-sized, single-crystal graphene by coating polycrystalline platinum foils with a silicon-containing film. The film reacts with platinum on heating, resulting in the formation of a liquid platinum silicide layer that screens the platinum lattice and fills topographic defects. This reduces the dependence on the surface properties of the catalytic substrate, improving the crystallinity, uniformity and size of graphene domains. At elevated temperatures growth rates of more than an order of magnitude higher (120 μm min Innovative substrate engineering is necessary to improve the quality of CVD-synthesized graphene. Here the authors demonstrate in situ fabrication of an eutectic Pt-Si alloy that forms a wetting liquid surface on polycrystalline Pt foils, allowing millimetre-sized graphene crystals to grow in minutes. Graphene is a two-dimensional material that has great potential for use in a wide variety of applications such as transparent flexible electrodes, photonic and electronic devices, energy storage, sensors and coatings to name a fewAtmospheric pressure chemical vapour deposition (APCVD) has received much attention due to its simplicity, improved safety and lower cost over alternative procedures. When a solid substrate is used with APCVD, graphene growth is strongly influenced by the crystallographic lattice of the substrate, its defects, roughness and grain boundaries2347in situ for graphene growth when heated, over the entire substrate surface. This significantly reduces the influence of the defects, roughness and the crystallographic orientations of the substrate on the quality of graphene. Substrates covered with the film exhibit increased growth rates of up to 120 μm min−1 at elevated temperatures, one of the fastest reported to date. While metal alloys121314In this investigation we combined the advantages of both approaches by using widely available, scalable, solid polycrystalline substrates in conjunction with a thin, liquid layer on the surface of these substrates. The layer is formed by coating polycrystalline Pt foils with a silicon-containing film, which forms a wetting liquid silicide surface 2 film was deposited on pristine Pt with a tetraethyl orthosilicate (TEOS) precursor in a CVD-based process, where TEOS was introduced by an Ar carrier gas (Methods). Other techniques such as evaporation and sol-gel deposition of Si/SiO/SiO2 were also investigated and showed similar trends after the optimisation of the procedure. CVD of TEOS is fast, cheap and can be performed in a single CVD system before synthesis. Upon annealing the SiO2 layer disappeared resulting in the formation of silicidated Pt (discussed later in the paper). To synthesize graphene, pristine Pt or the Pt/SiO2 stack was subjected to a CVD procedure that consisted of an annealing stage, synthesis stage and a cooling stage at atmospheric pressure (Methods). A compromise had to be made between the length of an experiment versus the size and quality of graphene flakes because the ratio of H2 to CH4 needed to be kept high2:CH4 ratio of 100:1. The ratio of H2 to CH4 on silicidated Pt compared with pristine Pt had to be increased by as much as 50%  to reduce the carbon deposition and achieve flakes of comparable size to those on pristine Pt. The comparison of the synthesis parameters on pristine and silicidated Pt is presented in −1  is high −1 ref. ), but stl−1 ref. ) present2 to CH4 ratios on Cu is usually such that experiments often need to be conducted for hours or days−1(S/2)−2, is also shown in Interestingly, the high melting point of Pt allowed us to probe unexplored temperatures above 1,100 °C in CVD, at which single crystal graphene flakes of up to 1.8 mm were obtained in 15 min on silicidated Pt. For comparison, the window of useful Het al.et al.A number of techniques were used to extensively assess the properties of the synthesized graphene, such as size, shape, crystallinity, number of layers and its electronics properties. 2 substrates for Raman measurements (Graphene was transferred to Si/SiOurements . The D-purements  providedurements . In addiurements . The rou2/Si substrates (methods) from silicidated Pt. The carrier density n and mobility μ of the device were determined from the low-field Hall measurements with n=(e·∂ρxy/∂B)−1 and μ=(enρxx)−1. Small p-type doping was observed in our device, which is also commonly seen in transferred graphene on SiO2/Si substrates and considered to be due to the adsorption of water molecules between the interfaces27ρxx and ρxy as functions of magnetic field from −19 to +19 T for a typical device. We observed a very high hole mobility of 5,525 cm2 V−1 s−1 at a hole density of 5.86 × 1011 cm−2. Recently, graphene has been shown to be an exceptional candidate for quantum Hall metrologyB|>12 T, well-defined ν=2 quantum Hall states was clearly observed with ρxy=h/2e2 and ρxx=0, consistent with its carrier density 2/Si substrates, and are comparable to those in epitaxial graphene grown on SiC , fused silica or quartz may be described similarly due to the strong Si—O bond, which can be broken by hydrofluoric acid or some strong bases. However, when these compounds are in contact in a reducing H2 atmosphere at elevated temperatures silica may reduce to silicon, catalysed by Pt, and react with Pt to form various phases of eutectic Pt silicides as reported by numerous studies333435It is commonly known that Pt is a noble metal that is very inert; only hot aqua regia can react with it. Silicon dioxide  film was deposited on Pt to get the material in excess, resulting in the formation of a Pt3Si phase that matched well with the literatureCharacterization of the annealed and cooled substrate before graphene synthesis was performed. The cooling was rapid (quenched) to obtain the closest possible condition to that during the synthesis. 2 film before and after annealing we propose a simple mechanism of the liquid silicide formation that explains why the morphologies of graphene are so different on pristine and silicidated Pt. Most Pt silicide phases have reduced (eutectic) melting points3Si phase melts at around 830 °C  effects. For example, the silicide route may produce silicon carbide as an intermediate state. Precise in situ studies would be needed to confirm the correct chemical mechanism. We also suggest that such eutectic materials may be suitable for use as liquid-surface substrates at low synthesis temperatures, bringing all the associated benefits.On the basis of experimental observations of the SiO °C ref. . Figure  surface . The silGood crystallinity is an important quality factor of CVD-grown graphene, as it offers superior electronic and tensile properties, chemical resistivity and impermeabilityAlthough there is as yet no confirmed theory that explains the poor crystallinity of graphene on pristine Pt via CVD that we have observed, it is well known that the face-centred cubic (FCC) lattice of Pt has the largest lattice constant compared with the commonly used transition metals for graphene synthesis. On Cu(111) the lattice constant mismatch with graphene is 3.6%, whereas on Pt(111) this is significantly higher at 12.5%. This may result in incommensurable growth and the observed poor crystallinity.−2 and enhanced growth rates of up to 120 μm min−1 at elevated temperatures, allowing millimetre-sized flakes to grow easily in minutes. The silicide film formation on the substrates is an important parameter for graphene growth and may have beneficial effects if controlled. To our knowledge no current reports focusing on graphene synthesis via CVD consider the effects of the formation of the silicides, though commonly used reaction tubes are made of fused silica. It is likely that previous results were influenced by these silicides but were overlooked by other reports. In particular, we explicitly verify the dependence of the amount of silica on the crystallinity of graphene flakes on Pt. All of these beneficial effects may make the silicidation feasible commercially, especially if the procedure is extended to other metals. There is significant potential for further use of the method, such as a liquid surface for low temperature graphene synthesis or the possibility to oxidize the silicide layer to achieve transfer-free graphene. This approach is generally applicable and paves the way for a whole new field of eutectic substrates for the growth of superior quality 2D materials.In this study, we demonstrate a novel, low-cost substrate processing procedure to achieve rapid, efficient synthesis of millimetre-sized single crystal graphene. Silicidation of polycrystalline foils may be used as a replacement for expensive single-crystal substrates or as an alternative to polishing and grain enlargement. Large topographic defects, such as grain boundaries, are locally smoothed and the lattice of the grains is screened by a liquid surface, making silicidated polycrystalline foils superior to single crystals due to epitaxy-free synthesis. This ensures that graphene grows as a single crystal in its undistorted hexagonal shape. Silicidated polycrystalline Pt also exhibits low graphene flake nucleation density of up to 0.3 mm2 stack, a film of SiO2 of specified thickness (2 film of about 1 μm thickness can be deposited on pristine Pt with CVD by passing Ar gas (350 sccm) through a TEOS precursor (in a ‘bubbler') that is carried to a furnace with the substrates heated to 700 °C for 30 min. Other methods, such as Si, SiO, SiO2 evaporation, sputter coating, sol-gel processes or ion implantation are also applicable.Pristine Pt substrates  were used as received from the supplier after cleaning in acetone and deionized water. To form a Pt/SiOhickness  was depo2 stack was placed on a boron nitride holder inside a fused silica tube (28 mm inner diameter) at sufficient distance from the walls of the tube. The tube was then purged with Ar (99.999% purity) and shifted into a pre-heated furnace, followed by annealing in H2  to allow Pt silicide formation and to stabilize the temperature of the furnace. The useful annealing time to form silicidated Pt strongly depended on the temperature and the silica thickness. For example, at 1,070 °C with 1-μm-thick silica layer, 30 min was suitable . However, at 1,050 °C with a 1 μm silica film after 30 min, 10% of the SiO2 film still remained. In the experiments where reusing silicidated Pt was tested, we shortened the annealing time to 5 min to simply re-heat the solidified silicide. To synthesize graphene, a mixture of CH4 (99.5%) and H2 was introduced. The gas ratio and the duration of the synthesis depended greatly on the temperature and the silica layer thickness and are given in the 4 and 600 sccm H2 for 20 min, resulting in ∼0.5-mm-sized graphene flakes. Directly after this step, the tube was shifted from the furnace and purged with Ar to quench graphene growth.Pristine Pt or the Pt/SiOet al.2 wafer, followed by heating at 140 °C for 5 min to improve the adhesion of graphene to SiO2. The stack was then placed in acetone  for 6 h and after dried with a N2 gun. For the TEM transfers, the PMMA layer was burnt in air at 350 °C after the transfer to a gold TEM grid, as outlined in Huang et al.41The ‘bubbling transfer' method outlined in Gao Raman spectra and maps were recorded with a Horiba LabRAM Aramis microscope equipped with a 532 nm laser. Routine SEM characterization was done with a JEOL JSM-6500F microscope at 5 kV. TEM images and SAED patterns were recorded with a JEOL JEM-2010 microscope at 80 kV. AFM measurements were performed using a Cypher AFM (Asylum Research). The scan was done in contact mode by looking at the lateral motion due to changes in friction as the probe scanned over the surface. To eliminate the effects of the direction of probe movement and also any topographic effects the trace scan was subtracted from retrace for the lattice-resolved image in real space. 2D fast Fourier transforms were taken of the AFM scans, the absolute values of which were then calculated and plotted on log scale; the contrast of the final images was adjusted to reduce the background.B were applied perpendicular to the sample surface. A small AC current with peak amplitude of 40 nA was applied in the longitudinal direction (Ixx). The longitudinal voltage Vxx and the Hall voltage Vxy ˙Vxx/Ixx and Vxy/Ixx, respectively, where L and W are the length and the width of the device.Eight-leg Hall bars (15 μm × 130 μm) were fabricated using e-beam lithography followed by oxygen plasma etching. A two-step ohmic contacting methodtage Vxy  were sim2 films was measured with a Nanometrics Nanospec AFT interferometer on co-deposited Si tiles. Some samples were also analysed with ellipsometry directly on a Beaglehole Instruments Picometer Ellipsometer. The XPS measurements were performed at base pressure of 5 × 10−10 torr on a VG nine channel CLAM4 electron energy analyser with a 250 W Al X-ray excitation. XRD analysis was performed on the Bruker D5000 diffractometer.The thickness of the SiOP) squared to area (A) was calculated for each geometric shape. This unit-less number is usually indicative of specific geometric shapes. For example, for a circle it is 4π≈12.57 , for an octagon it is ≈13, for a hexagon √192≈13.86, for a pentagon ≈15, for a square it is 16 and so on. Concave polygons have higher P2/A values. If the calculated ratio (P2/A) for a shape is X, then we define the hexagonality as ≈1−|X−√192|/√192 . This means that a shape with a value close to 100% is close to a perfect hexagon. The s.d. of the % hexagonality for a sample indicates how non-uniform the shape distribution is. It is understood that rounded convex or concave polygons may introduce an uncertainty in the measurement, since, for example, a rounded cube may have the ratio of the perimeter squared to area similar to a hexagon. However, we usually have either hexagonal shapes (silicidated Pt) or concave polygons (pristine Pt); thus, for a large number of graphene domains the approach characterizes the samples sufficiently.MATLAB was used to find an automatic threshold value of SEM micrographs, which were then converted into binary images. The ratio of the perimeter .Supplementary Figures 1-3


These data were related to the hydraulic data as well as the sample type, location, and time. Temporal and spatial variation was observed between and within wastewater communities and biofilm communities. The bacterial communities in biofilm were distinctly different from the communities in wastewater and exhibited greater spatial variation, while the wastewater communities exhibited variability between different months of sampling. This study highlights the variation of bacterial communities between biofilm and wastewater, and has shown both spatial and temporal variations in bacterial communities in combined sewers. The temporal variation is of interest for in‐sewer processes, for example, sewer odor generation, as field measurements for these processes are often carried out over short durations and may therefore not capture the influence of this temporal variation of the bacterial communities.This study describes the temporal and spatial variability of bacterial communities within a combined sewer system in England. Sampling was conducted over 9 months in a sewer system with intensive monitoring of hydraulic conditions. The bacterial communities were characterized by 16S  Combined sewers are a key part of the urban infrastructure and play a vital role in safeguarding public health and reducing water pollution in the environment. Sewage released into the urban environment can potentially expose people to waterborne pathogens, and sewer overflows impact upon the natural aquatic environment as well as producing esthetic pollution including noxious odors. During the transport of wastewater within a combined sewer network, it undergoes physicochemical and biological changes  which release excess sewage into a small watercourse when the sewer network is overloaded during intense rainfall events; five of these are located on the main intercepting sewer Fig. . The catThe sites for the sampling of material for bacterial community analysis were chosen based on an analysis of the flow data from the seven flow monitors  located on the intercepting sewer, as well as excluding sites where the access manhole was located in the main road as this would limit safe accessibility. Table The measured wastewater velocity from these sites was chosen as the parameter defining the sampling program as it can be related to the shear stress on the biofilms covering the submerged pipe wall in the sewer, with the assumption that the pipe geometry and wall roughness is fixed. Based on the dry weather data, the average daily velocity profile for each measuring station was calculated and is shown in Figure Based on the velocity profiles in Figure am). Wastewater samples were collected in November 2009, March 2010, and June 2010. For each manhole, 1 L of wastewater was collected in triplicate at each time point in sterile glass bottles, and biofilm samples were collected in March 2010 and June 2010. The biofilm samples were collected using a biofilm sampler constructed from a sponge attached to a piece of PE plastic. These were sterilized by autoclaving prior to use and kept in sterile 50 mL falcon tubes. The biofilm samples were collected by scraping the sponge over the pipe wall immediately above the water line to avoid mixing with the wastewater. The samples were collected in the upstream sewer pipe discharging into the manhole rather than in the actual manhole. Triplicate biofilm samples were again collected at each location. The samples were transported to the laboratory within 12 h of sampling in a cool bag to ensure stable temperature following sampling.Samples of wastewater and biofilms were collected by entering the manholes at the same time as the routine maintenance of the flow monitors, and the samples were collected during the day  for the biofilm samples and eluted in a final volume of 50 μL nuclease‐free water.Upon return to the laboratory, 50 mL samples of wastewater were filtered through a 0.22‐μm, 47 mm diameter, polycarbonate membrane filter . The filters were then stored at −20°C until the DNA was extracted. The biofilm samples were frozen at −20°C upon return to the laboratory. DNA was extracted using the MoBio Ultra Clean Soil DNA isolation kit  following the manufacturer's instructions. DNA was extracted from half a filter for the wastewater samples, corresponding to 25 mL of the original 50 mL sample volume, and from a quarter of a sponge .After DNA extraction, bacterial 16S rRNA genes were amplified using the polymerase chain reaction (PCR) with primers FAM63f and HEX1389r  test with a 5% confidence level  for FM004, 0.6 m/sec (standard deviation 0.01 m/sec) for FM012, and 3.1 m/sec (standard deviation 0.05 m/sec) for FM019. These average velocities are based on dry weather data defined as described in There were two sampling times where samples of wastewater and biofilms were collected at all three sites, that is, the March and June samples. Figure When comparing bacterial community structure between the three individual sampling locations in the network Fig. , the wasSeparating all of the biofilm communities from the wastewater communities shows that the biofilm communities overall formed three separate clusters Fig. B with seThe catchment where the sampling was carried out is located in an area where infiltration of groundwater into the sewer networks is typically observed. Groundwater infiltration is a seasonal process, and is much more influential during certain months in which the local ground water levels are elevated due to previous rainfall (Kracht et al. Figure In this study, variations in the structure of bacterial communities in sewer wastewater were investigated at three locations along the same intercepting sewer and at three separate points in time. Variation in biofilm community structure was also investigated at the same locations, but at only two time points. In addition to the samples for microbial analysis, an extensive dataset of flow data and rain data were available for the catchment. From the bacterial community profiling it was clear that the biofilm communities were different from the wastewater communities at each sampling point and at each time Figs. , 4. TherLooking only at the wastewater communities in Figure In addition to the general difference between the communities in the wastewater and the biofilms, the communities also indicate a higher variability overall in the biofilms than is found in the wastewater samples. This is particularly seen for samples collected at the same time in March and June Fig. . The higThe variability in microbial communities in sewers is of interest mainly due to their influence on in‐sewer processes which contributes to problems such as odor generation in the urban environment or corrosion of the sewer pipe walls. In addition, the vision of enhancing the naturally occurring in‐sewer processes to provide safe in‐sewer treatment of wastewater without the need for large energy inputs at large end of pipes wastewater treatment plants (Hvitved‐Jacobsen None declared.


The optical characteristics of graphene oxide (GO) were explored to design and fabricate a GO-based optical humidity sensor. GO film was coated onto a SU8 polymer channel waveguide using the drop-casting technique. The proposed sensor shows a high TE-mode absorption at 1550 nm. Due to the dependence of the dielectric properties of the GO film on water content, this high TE-mode absorption decreases when the ambient relative humidity increases. The proposed sensor shows a rapid response (<1 s) to periodically interrupted humid air flow. The transmission of the proposed sensor shows a linear response of 0.553 dB/% RH in the range of 60% to 100% RH. These functional groups include hydroxyl, epoxy and carboxylic acid moieties [3 hybridized carbon atoms turn GO into an insulator by decreasing its conductivity, and enhance its hydrophilic properties, [Aside from the basic needs of high sensitivity and wide detection range additional demands for fast response and short recovery time exist for humidity sensors to meet industry requirements. Consequently, research on humidity-sensitive nanomaterials such as silicon nanostructures, ceramic nanomaterials, semiconductor nanoparticles and metal oxide nanowires have attracted much attention due to its high surface to volume ratio ,2. Latelmoieties ,3. The sperties, ,6. Besidperties, , which eCapacitive-based sensors having high response time, low temperature dependence, low hysteresis, low drift with good performance in harsh environment are desirable in various applications –14. Duri2.The experimental setup of the humidity sensor is shown in On the other hand, GO solution was prepared by an improved version of Hummer's method . The GO A gas tube providing controlled humidity vapour was placed about 1 cm above the GO-coated waveguide (sensing region) of the proposed sensor. Humid nitrogen gas was produced by bubbling dry nitrogen gas through water before flowing out from the gas tube. The response time of the proposed sensor to humid air (∼90% RH) was measured using a photodiode and by placing a mechanical shutter in between the gas source and the sensing region to control the exposure intervals. Measurement of the change in transmitted power to different relative humidity values was carried out by placing the proposed sensor in a humidity control box. Relative humidity in the box was controlled by mixing dry nitrogen gas and humid nitrogen gas with different mixing flow rates before flowing into the box. Before each optical measurement, the relative humidity of the gas flow or inside the humidity control box was measured using a HI 8562 hygrometer from Hanna Instruments. A long working distance optical microscope  was used to monitor the sensing region during the experiment. Throughout the optical measurement, the polarization of the incident light was set to be TE-polarized using a polarization controller (PC). Extra care in the handling of the launch fibre was taken to ensure that the linear polarization state of the incident light was not scrambled by minor disturbances on the fibre. Also, the polarization dependent loss between TE- and TM-polarized modes of the incident light for another uncoated polymer waveguide was measured and found to be lower than 0.5 dB, limited only by the performance of the PC.3.Prior to the introduction of humid air (up to the 6th second), no water condensation was observed on either the GO film or the substrate, as shown in To study the response time of the proposed sensor, the output of the sensor was connected to a photodiode and the transmitted power was observed using an oscilloscope, and the results are shown in The polarization state of the transmitted light was measured when the proposed sensor was exposed to a relative humidity of 100% RH, and was TE-polarized. In fact, rotating the PC only rotates the polarization state and does not affect the transmitted power. The water saturated GO film seems to lose its polarization capability and becomes transparent to both TE- and TM-polarized light. There are reports showing that, at high relative humidity, water molecules can easily permeate in and out of the GO film of up to 10 μm in thickness . The per4.The humidity sensing ability of a GO-coated optical waveguide has been studied. The optical transmission characteristics of the proposed sensor are found to be affected by the relative humidity of its surroundings. The increase in transmitted power at higher humidity is due to a change in the dielectric properties of the GO film where the conductivity of the GO film is reduced in the presence of water molecules. The proposed sensor exhibits fast response of less than 1 s to humid air breezes. In addition, it shows a good linear response of 0.553 dB/% RH in the humidity range from 60% to 100% RH. The all optical nature of the proposed sensor enables its application in long distance humidity sensing with fast response.


Processes of selective allocation of visual attention play a prominent role for survival, but also for development and maintenance of clinically relevant symptoms such as in anxiety or depression. Previous research provided evidence for increased attentional orienting and preoccupation with biologically relevant and mood-congruent stimuli, indicating tendencies of attentional biases. For instance, in anxiety, the visual-attentional system may be overly sensitive toward threat- and avoidant of reward cues.Behavioral and physiological bases of attentional biases: paradigms, participants, and stimuli cover these topics and give a comprehensive overview on current directions and challenges in attentional bias research. Our driving motivation was to critically evaluate parameters that may directly or indirectly influence attentional biases and may thus be important for our understanding of attentional biases.The research articles appearing in the E-book dot-probe task (MacLeod et al., Our first aim was to demonstrate the variety of experimental paradigms and outcome measures used. So far, the Our second aim was to demonstrate the variety of populations and stimuli, which may show, or elicit, differing attentional biases. Wittekind et al.  examinedInvestigating the impact of participant sex on attentional biases, Pfabigan et al.  applied Kappenman et al.  investigChallenges in attentional bias research become quite clear in the course of the articles in this E-book. There is no common agreement as to whether particular stimuli or experimental set-ups are more reliable than others. For example, only a few studies addressed the topic of different presentation durations and their impact on attentional bias measures so far (e.g., Koster et al., Nevertheless, the current research topic also points toward future directions of attentional bias research. In particular, Kappenman et al.  emphasizThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In immunocompetent mouse models, blocking PD-L1 induced a longer tumour growth delay following irradiation. The inhibition of T cell functions including proliferation and cytotoxicity against tumor cells was responsible to the effects of PD-L1 on radiation response. In conclusion, PD-L1 could be a significant clinical predictor for clinical stage and treatment response of bladder cancer.Identification of potential factors that can stratify a tumor’s response to specific therapies will aid in the selection of cancer therapy. The aim was to highlight the role of programmed cell death 1 ligand 1 (PD-L1) in bladder cancer. In this study, 92 of muscle-invasive bladder cancers and 28 of non- muscle invasive bladder cancers were selected for immunohistochemical staining analysis. Furthermore, human and murine bladder cancer cell lines were used to examine the correlation between PD-L1 and radiation response. Our data revealed that PD-L1 was overexpressed in the bladder tumor specimens compared with adjacent non-malignant specimens. Furthermore, the staining of PD-L1 was significantly linked to higher clinical stage, lower complete response rates and reduced disease-free survival rates. By  Bladder cancer is a significant public health issue worldwide and manifests itself in two distinct forms with different clinical and biological behaviors3Tumor-induced immune suppression in cancer patients is a major issue that not only promotes tumor progression but also inhibits the efficiency of anti-cancer treatment579111213141617Bladder tissue specimens retrospectively collected from 65 patients with MIBC  were constructed into TMA. IHC staining of TMA slides demonstrated that PD-L1 was overexpressed in the tumour tissues of 40 patients (61%) compared with adjacent non-malignant epithelial tissues . Figure in vitro. For human bladder cancer cell line HT1197, RT increased the expression of PD-L1 in tumour cells compared with nontreated cells, especially at 48h, and the increased levels of this protein were positively associated with the irradiation dose  with or without anti–PD-L1. The results shown in As shown in We analysed the recruitment of CD8+ T cells in tumours using irradiated mice treated with or without anti-PD-L1. The data in Malignant tumors possess mechanisms for evading host immune responses. A novel mechanism that tumor may evade host immune response through the expression of PD-L1. PD-L1 expression has been observed in a wide variety of solid malignancies, and might be correlated with the higher malignant grade of tumours, increased tumour growth, and the patients’ prognoses101920in vitro. The upregulation of PD-L1 was also observed in irradiated tumours in mice, suggesting that the alteration of PD-L1 levels in tumour microenvironments might play a role in the radiation response of bladder cancer. To address this issue, we combined irradiation with anti-PD-L1 antibody therapy to treat murine MB49 bladder cancer in immunocompetent animal models. The data revealed that the anti-PD-L1 antibody sensitized bladder cancer to radiation, as demonstrated by a longer tumour growth delay for ectopic tumors. Furthermore, by mouse orthotopic models, blocking PD-L1 augmented the RT-induced tumour cell death associated with decreased tumor cell proliferation compared with tumours after radiation therapy alone.Emerging evidence suggests that the generation of antitumor immune responses might play an important role in the effectiveness of radiotherapy22911in vitro. The data revealed that tumor cells’ PD-L1 expression was triggered by radiation. Blockade of PD-L1 effectively inhibit CD8+ T cells cytotoxicity against irradiated bladder cancer cells. Furthermore, we demonstrated that the increase in PD-L1 expression on irradiated bladder cancer cells was associated with a decreased T cell proliferation upon co-culture experiments, and anti-PD-L1 had reduced the suppressive ability for T cell proliferation. Thus, these results showed that PD-L1 expression negatively correlated with the numbers and function of cytotoxic T cells, which linked to tumor irradiation response demonstrated by the immunocompetent animal model. Therefore, we suggested that PD-L1 affects the radiation response of bladder cancers mediated by regulating the tumor microenvironment in the immunocompetent host.Radiation increased the T cell recognition of irradiated tumour cells, making the cells vulnerable to cytotoxic T lymphocyte-mediated clearance242628For the interpretation of our results, different limitations have to be taken into account. First, we examined PD-L1 expression mainly in biopsy tissue specimen from TURBT to evaluate its prognostic value. Second, the patient population enrolled in the retrospective study are heterogeneous with relatively small group sizes. Thus, further investigations that included more patients in a prospective trial are needed.The ultimate cancer treatment would leverage different cytotoxic pathways for the complete eradication of tumours. The data indicated that PD-L1is a predictor of treatment response and loco-regional recurrence for bladder cancer. Therefore, measured PD-L1 expression levels in bladder cancer excision tissues provides further data stratifying patients to higher risk disease and allow selection for personalized treatment.This study was approved by the Institutional Review Board of Chang Gung Memorial hospital, and performed in accordance with the approved guidelines. Informed written consent for the acquisition and storage of medical information and tissue specimens was obtained from all of the patients. There were 72 patients with histopathological proven muscle invasive urothelial cell carcinoma who completed definite chemoradiotherapy (CCRT) treatment enrolled in the study. On completion of definite CCRT, patients underwent abdomen CT, cystoscopy and urine cytology to assess the treatment response and disease status every 3 to 4 months for the first 2 years, and then every 6 months during follow-up. The median follow-up time was 3.8 years. Clinical CR was defined as no tumor visible on cystoscopy, negative tumor site biopsy, and negative urine cytology after the completion of CCRT. The identification of clinical lymph node (LN) involvement with CT was based on measurements of node size, with greater than 1 cm being the criterion. Specimens were retrospectively collected from the 72 patients for immunochemical analysis. The end points were overall survival (OS), disease-free survival (DFS) and the response to definite CCRT. The loco-regional recurrence was defined by tumor progression in bladder or pelvic nodes assessed with CT and cystoscopy. Survival probabilities were analyzed using the Kaplan-Meier method.Formalin-fixed, paraffin-embedded bladder tissues collected from TURBT at diagnosis and/or radical cystectomy for bladder cancer were subjected to immunochemical analyses. These 120 urothelial cell carcinoma patients in HT1197, a human bladder cancer cell line, was obtained from the American Type Culture Collection (ATCC). We maintained the bladder cancer cells in DMEM supplemented with 10% foetal bovine serum. Mouse bladder carcinoma MB49 cells were kindly gifted from Dr Yi-Wen Liuin vivo. In the ectopic tumour implantation model, 1 × 106 tumour cells were subcutaneously implanted by injection into the dorsal gluteal region . In the orthotopic tumour implantation model, we performed intravesical instillation of cancer cells as previously describedin vivo, local irradiation to 12 Gy was performed when the ectopic tumours reached 0.5 cm3 or at 2 weeks after orthotopic tumour implantation. Radiosensitivity was indicated as growth delay . Duplicate experiments were performed for growth delay analyses. To evaluate the effect of PD-L1 blockade on the radiation response and immune cells in vivo, the intraperitoneal (i.p.) injection of a PD-L1 neutralization antibody  was initiated at 1 day prior to irradiation and continued for 2 weeks.This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals as promulgated by the Institutes of Laboratory Animal Resources, National Research Council, USA. The protocol was approved by the Committee on the Ethics of Animal Experiments of Chang Gung Memorial Hospital. Eight-week-old female C57 mice were used as the tumour implantation model. To investigate the link between PD-L1 expression and the radiation response in immunocompetent hostTo obtain single-cell suspensions, the tumour tissues were digested using 1 mg/ml collagenase IV (Sigma-Aldrich) and 0.2 mg/ml DNase I (Sigma-Aldrich) for 45 minutes at 37 °C. The cells were blocked and subsequently stained with antibodies against CD8, CD3, and PD-L1. For cytotoxicity assays, tumor cells were stained with PI after the removal of CD8+ cells. The murine cytotoxic T cells from fresh tumour specimens were characterized and sorted as CD3+CD8+.5 cells/well), and tumors were previously isolated at a 2:1 ratio. T cell proliferation was induced using anti-CD3/CD28 stimulation beads . After three days, the suppression assay was analysed for T cell proliferation using flow cytometry.The negative regulation of tumour-infiltrating T cells through PD-L1/PD-1 might be an important host-mediated mechanism of acquired radioresistance in tumours33The significance of the differences between the samples was determined using Student’s t-tests. The data are presented as the means ± standard error of the mean (SEM). All experiments, comprising three replicates, were performed at least twice independently. A probability level of p < 0.05 was considered statistically significant, unless otherwise stated.How to cite this article: Wu, C.-T. et al. The role of PD-L1 in the radiation response and clinical outcome for bladder cancer. Sci. Rep.6, 19740; doi: 10.1038/srep19740 (2016).


Fourier transformed infrared spectroscopy analysis indicates that graphene oxide is reduced into graphene. Transmission electron microscope observation shows that SrTiO3 nanoparticles are well assembled onto graphene sheets. The photocatalytic activity of as-prepared SrTiO3-graphene composites was evaluated by the degradation of acid orange 7 (AO7) under a 254-nm UV irradiation, revealing that the composites exhibit significantly enhanced photocatalytic activity compared to the bare SrTiO3 nanoparticles. This can be explained by the fact that photogenerated electrons are captured by graphene, leading to an increased separation and availability of electrons and holes for the photocatalytic reaction. Hydroxyl (·OH) radicals were detected by the photoluminescence technique using terephthalic acid as a probe molecule and were found to be produced over the irradiated SrTiO3 nanoparticles and SrTiO3-graphene composites; especially, an enhanced yield is observed for the latter. The influence of ethanol, KI, and N2 on the photocatalytic efficiency was also investigated. Based on the experimental results, ·OH, h+, and H2O2 are suggested to be the main active species in the photocatalytic degradation of AO7 by SrTiO3-graphene composites.SrTiO61.46. + w; 78.67.Bf; 78.66.Sq In these processes, the high recombination rate of the photogenerated carries greatly limits the photocatalytic activity of catalysts. Therefore, the effective separation of photogenerated electron–hole pairs is very important in improving the photocatalytic efficiency.Semiconductor photocatalysts have attracted considerable attention over the past decades due to their potential applications in solar energy conversion and environmental purification ,2. Amongllutants . Furthernductors -10. It isp2-hybridized carbon atoms, possesses unique properties including high electrical conductivity, electron mobility, thermal conductivity, mechanical strength, and chemical stability [3-graphene nanocomposites has been rarely reported.Graphene, being a two-dimensional (2D) sheet of tability -13. On atability -21, supetability , field-etability , and fuetability . Particutability -21. In t4[Eg of the photocatalyst, during which graphene oxide receives electrons from the excited photocatalyst and is thus reduced to graphene. During the photocatalytic reduction process, photocatalyst nanoparticles are assembled onto graphene sheets to form photocatalyst-graphene composites. Herein, we report the synthesis of SrTiO3-graphene nanocomposites via the photocatalytic reduction method. The photocatalytic activity of the composites was evaluated by the degradation of acid orange 7 (AO7) under ultraviolet (UV) light irradiation, and the photocatalytic mechanism involved was discussed.Up to now, semiconductor-graphene nanocomposites have been generally prepared using graphene oxide as the precursor, followed by its reduction to graphene. To reduce the graphene oxide, several methods have been employed including chemical reduction using hydrazine or NaBH4, high-te4, hydroth4, green c4, and pho4-21. Amon3 nanoparticles were synthesized via a polyacrylamide gel route as described in the literature [3-graphene composites were prepared via a photocatalytic reduction route. A certain amount of graphene oxide was dispersed in 50 mL distilled water, followed by ultrasonic treatment of the suspension for 30 min. Then, 0.1 g SrTiO3 nanoparticles and 0.0125 g ammonium oxalate (AO) were added to the suspension under magnetic stirring. After stirring for 10 min, the mixture was purged with nitrogen and exposed to UV light irradiation from a 15-W low-pressure mercury lamp for 5 h under mild stirring. During the irradiation, the color of the mixture changed from brown to black, indicating the reduction of the graphene oxide. After that, the product was separated from the reaction solution by centrifugation at 4,000 rpm for 10 min, washed several times with distilled water and absolute ethanol, and then dried in a thermostat drying oven at 60°C for 4 h to obtain SrTiO3-graphene composites. A series of samples were prepared by varying the weight fraction of graphene oxide from 2.5% to 10%.SrTiOterature . The graλ = 254 nm). The initial AO7 concentration was 5 mg L-1 with a photocatalyst loading of 0.5 g L-1. Prior to irradiation, the mixed solution was ultrasonically treated in the dark to make the photocatalyst uniformly dispersed. The concentration of AO7 after the photocatalytic degradation was determined by measuring the absorbance of the solution at a fixed wavelength of 484 nm. Before the absorbance measurements, the reaction solution was centrifuged for 10 min at 4,000 rpm to remove the photocatalyst. The degradation percentage is defined as (C0 - Ct) / C0 × 100%, where C0 and Ct are the AO7 concentrations before and after irradiation, respectively. To investigate the photocatalytic stability of the SrTiO3-graphene composites, the recycling tests for the degradation of AO7 using the composite were carried out. After the first cycle, the photocatalyst was collected by centrifugation, washed with water, and dried. The recovered photocatalyst was introduced to the fresh AO7 solution for the next cycle of the photocatalysis experiment under the same conditions. The process was repeated four times.The photocatalytic activity of the samples was evaluated by the degradation of AO7 under UV light irradiation of a 15-W low-pressure mercury lamp . By measuring the photoluminescence (PL) intensity of TAOH that is pronounced around 429 nm, the information about · OH can be obtained. TA was dissolved in a NaOH solution (1.0 mmol L-1) to make a 0.25-mmol L-1 TA solution and then to the solution was added 0.5 g L-1 SrTiO3-graphene composites. The mixed solution, after several minutes of ultrasound treatment in the dark, was illuminated under a 15-W low-pressure mercury lamp. The reacted solution was centrifuged for 10 min at 4,000 rpm to remove the photocatalyst and was then used for the PL measurements through a fluorescence spectrophotometer with the excitation wavelength of 315 nm.Terephthalic acid (TA) was used as a probe molecule to examine hydroxyl (·OH) radicals produced over the irradiated SrTiOThe phase purity of the samples was examined by means of X-ray powder diffraction (XRD) with Cu Kα radiation. Fourier transform infrared spectroscopy (FTIR) measurements were carried out on a Bruker IFS 66v/S spectrometer . The morphology of the samples was observed by a field emission transmission electron microscope (TEM). The UV-visible diffuse reflectance spectra were measured using a UV-visible spectrophotometer with an integrating sphere attachment.3 nanoparticles. It is noted that the SrTiO3 particles have an isoelectric point at pH 8.5 [3 particles are added to the graphene oxide suspension, the pH value of the mixture is measured to be approximately 6.5, implying that the SrTiO3 particle surface is positively charged. On the other hand, the oxygen-containing functional groups of graphene oxide (such as carboxylic acid -COOH and hydroxyl -OH) are deprotonated when it immersed in water, which leads to negative charges created on graphene oxide [3 particles are expected to be adsorbed onto the graphene oxide sheets through electrostatic interactions. Upon UV-light irradiation, electrons and holes are produced on the conduction band (CB) and valence band (VB) of the SrTiO3 particles, respectively. The photogenerated holes are captured by ammonium oxalate that is a hole scavenger [3 particles. The electrons are injected from the SrTiO3 particles into the graphene oxide and react with its oxygen-containing functional groups to reduce graphene oxide.Figure t pH 8.5 ; that isne oxide . As a recavenger , leaving3 particles, and SrTiO3-graphene(10%) composites. In the spectrum of graphene oxide, the absorption peak at 1,726 cm-1 is caused by the C = O stretching vibration of the COOH group. The peak at 1,620 cm-1 is attributed to the C = C skeletal vibration of the graphene sheets. The absorption peak of O-H deformation vibrations in C-OH can be seen at 1,396 cm-1. The absorption bands at around 1,224 and 1,050 cm-1 are assigned to the C-O stretching vibration. For the SrTiO3 particles, the broad absorption bands at around 447 and 625 cm-1 correspond to TiO6 octahedron bending and stretching vibration, respectively [-1 is due to the bending vibration of H-O-H from the adsorbed H2O. In the spectrum of the SrTiO3-graphene composites, the characteristic peaks of SrTiO3 are detected. The absorption peak at 1,630 cm-1 is the overlay of the vibration peak of H-O-H from H2O and C = C skeletal vibration peak in the graphene sheets. However, the absorption peaks of oxygen-containing functional groups, being characteristic for graphene oxide, disappear. The results demonstrate that graphene oxide is completely reduced to graphene during the photocatalytic reduction process.Figure ectively . The abs3 particles and the SrTiO3-graphene (10%) composites. It is seen that all the diffraction peaks for the bare SrTiO3 particles and the composites can be index to the cubic structure of SrTiO3, and no traces of impurity phases are detected. This indicates that the SrTiO3 particles undergo no structural change after the photocatalytic reduction of graphene oxide. In addition, no apparent diffraction peaks of graphene in the composites are observed, which is due to the low content and relatively weak diffraction intensity of the graphene.Figure 3 particles, revealing that the particles are nearly spherical in shape with an average size of about 55 nm. The TEM image of the SrTiO3-graphene(10%) composites is presented in Figure 3 particles are well assembled onto the graphene sheet.Figure 3 particles and SrTiO3-graphene composites. The composites display continuously enhanced light absorbance over the whole wavelength range with increasing graphene content. This can be attributed to the strong light absorption of graphene in the UV-visible light region [R) with respect to wavelength λ , where the peak wavelength is characterized to be the absorption edge of the samples. It is seen that the SrTiO3 particles and composites present two absorption peaks in the derivative spectra. The strong and sharp absorption edge at approximately 370 nm is suggested to be attributed to the electron transition from valence band to conduction band. In comparison to the SrTiO3 particles, the SrTiO3-graphene composites show almost no shift in this absorption edge, indicating that the effect of graphene on the band structure of SrTiO3 can be neglected. From this absorption edge, the Eg of the samples is obtained to be approximately 3.35 eV. In addition, the relatively weak absorption edge at approximately 335 nm may be ascribed to the surface effects.Figure t region . Figure 3-graphene composites was evaluated by the degradation of AO7 under UV light irradiation. Figure 3-graphene composites as a function of irradiation time (t). The blank experiment result is also shown in Figure 3 particles, about 51% of AO7 is observed to be degraded. When the SrTiO3 particles assembled on the graphene sheets, the obtained samples exhibit higher photocatalytic activity than the bare SrTiO3 particles. In these composites, the photocatalytic activity increases gradually with increasing graphene content and achieves the highest value when the content of graphene reaches 7.5%, where the degradation of AO7 is about 88% after irradiation for 6 h. Further increase in graphene content leads to the decrease of the photocatalytic activity.The photocatalytic activity of the SrTiO3 particles and SrTiO3-graphene(7.5%) composites. The blank experiment result indicates almost no PL signal at 429 nm after irradiation without photocatalyst. On irradiation in the presence of the SrTiO3 particles, the PL signal centered around 429 nm is obviously detected, revealing the generation of · OH radicals. When the SrTiO3-graphene composites are used as the photocatalyst, the PL signal becomes more intense, suggesting that the yield of the · OH radicals is enhanced over the irradiated composites.Figure +, ·OH, ·O2, and H2O2 are thought to be the main active species responsible for the dye degradation [+[+ and · OH in the photocatalysis. The role of · O2 and H2O2, which are derived from the reaction between dissolved O2 and photogenerated e-, on the dye degradation can be examined by investigating the effect of N2 on the photocatalytic efficiency since the dissolved O2 can be removed from the solution by the N2-purging procedure. Figure 2 (bubbled at a rate of 0.1 L min-1), ethanol (10% by volume), and KI (2 × 10-3 mol L-1) on the degradation percentage of AO7 after 6 h of photocatalysis. It is demonstrated that when adding ethanol to the reaction solution, the photocatalytic degradation of AO7 undergoes a substantial decrease, from approximately 88% under normal condition to approximately 40% on addition of ethanol. This suggests that · OH radical is an important active species responsible for the dye degradation. Figure 3-graphene composites. The addition of KI to the reaction solution results in a higher suppression of the photocatalytic efficiency compared to the addition of ethanol, where only 16% of AO7 is caused to be degraded, indicating that the photogenerated h+ also plays a role in the degradation of AO7. In addition, the photocatalytic efficiency decreases slightly under N2-purging condition, implying comparatively minor role of · O2 and/or H2O2 for the dye degradation.Generally, hradation . It is kdation [+,33. By i3 can be calculated using the following relation [In order to understand the photocatalytic mechanism of semiconductor-based photocatalysts, it is essential to determine their energy-band potentials since the redox ability of photogenerated carriers is associated with energy-band potentials of photocatalysts. The conduction band and valence band potentials of SrTiOrelation :X is the absolute electronegativity of SrTiO3 (defined as the arithmetic mean of the electron affinity and the first ionization of the constituent atoms) and estimated to be 5.34 eV according to the data reported in the literature [Ee is the energy of free electrons on the hydrogen scale (4.5 eV), and Eg is the bandgap energy of SrTiO3 (3.35 eV). The conduction band and valence band potentials of SrTiO3 vs. normal hydrogen electrode (NHE) are therefore calculated to be ECB = -0.84 V and EVB = +2.51 V, respectively.where terature ,36, Ee i3-graphene composites toward the degradation of AO7 is schematically shown in Figure 3 is irradiated with light of energy greater than its bandgap energy, electrons are excited to the conduction band from the valence band, thus creating electron–hole pairs (Equation 2). Generally, most of the photogenerated electrons and holes recombine rapidly, and only a few of them participate in redox reactions. It is noted that graphene, which is an excellent electron acceptor and conductor, has a Fermi level (-0.08 V vs. NHE [3 (-0.84 V). When SrTiO3 particles are assembled onto graphene sheets, the photogenerated electrons can readily transfer from the conduction band of SrTiO3 to graphene (Equation 3). Thus, the recombination of electron–hole pairs can be effectively suppressed in the composites, which leads to an increased availability of electrons and holes for the photocatalytic reactions. The Fermi level of graphene is positive to the redox potential of O2/·O2 (-0.13 V vs. NHE) but negative to that of O2/H2O2 (+0.695 vs. NHE) [- which transferred onto the graphene cannot thermodynamically react with O2 to produce · O2, but can react with O2 and H+ to produce H2O2 (Equation 4). H2O2 is an active species that can cause dye degradation, and moreover, H2O2 can also participate in the reactions as described in Equations 5 and 6 to form another active species · OH. The valence band potential of SrTiO3 (+2.51 V) is positive to the redox potential of OH-/·OH (+1.89 V vs. NHE) [+ can react with OH- to produce · OH (Equation 7). As a consequence, the active species · OH, h+, and H2O2 work together to degrade AO7 (Equation 8).Based on the obtained experimental results, a possible photocatalytic mechanism of SrTiO vs. NHE ) positivvs. NHE) ,38. Thisvs. NHE) , indicat3 particles decorated on the surface of graphene is expected to increase, thus providing more photogenerated carriers for the photocatalytic reaction. When the graphene content in the composites reaches 7.5%, the SrTiO3 particles are decorated sufficiently, consequently leading to the achievement of the highest photocatalytic activity. However, with further increasing graphene content above 7.5%, the photocatalytic efficiency begins to exhibit a decreasing trend. The possible reason is that the excessive graphene may shield the light and decrease the photon absorption by the SrTiO3 particles, and moreover, the amount of available surface active sites tends to be reduced due to an increasing coverage of graphene onto the surface of the SrTiO3 particles.From Figure 3-graphene(7.5%) composites is examined by the recycling photocatalytic experiment, as shown in Figure 3-graphene composites. Figure 3 particles are still well decorated on the graphene sheets.Besides the photocatalytic activity, the reusability of photocatalysts is another crucial factor for their practical applications. The stability of the SrTiO3-graphene nanocomposites were prepared by irradiating the mixture solution of SrTiO3 nanoparticles and graphene oxide sheets, during which graphene oxide receives electrons from the excited SrTiO3 nanoparticles to be reduced to graphene, simultaneously leading to the assembly of SrTiO3 nanoparticles onto graphene sheets. Compared to the bare SrTiO3 nanoparticles, the as-prepared SrTiO3-graphene composites exhibit an enhanced photocatalytic activity for the degradation of AO7 under irradiation of UV light. This can be attributed to the effective separation of photogenerated electron–hole pairs due to the electron transfer from SrTiO3 to graphene and, hence, increased availability of electrons and holes for the photocatalytic reaction. The enhanced generation of · OH radicals is observed over the irradiated SrTiO3-graphene composites compared to the bare SrTiO3 nanoparticles. The photocatalytic efficiency is slightly deceased by purging with N2 but is significantly suppressed by the addition of ethanol and KI . Based on the experimental results, ·OH, h+, and H2O2 are suggested to be the main active species causing the dye degradation.SrTiO-: photogeneration of electron; Eg: bandgap energy; FTIR: Fourier transform infrared spectroscopy; h+: photogeneration of hole; H2O2: hydrogen peroxide; NHE: normal hydrogen electrode; OH: hydroxyl radicals; PL: photoluminescence; TA: terephthalic acid; TAOH: 2-hydroxyterephthalic acid; TEM: transmission electron microscope; UV: ultraviolet; VB: valence band; XRD: X-ray powder diffraction.AO: ammonium oxalate; AO7: acid orange 7; CB: conduction band; eThe authors declare that they have no competing interests.HY and TX conceived the idea of experiments. TX, LD, JM, and HZ carried out the preparation and characterization of the samples. HY, TX, and JD analyzed and discussed the results of the experiments. TX drafted the manuscript. HY improved the manuscript. All authors read and approved the final manuscript.HY is a professor and a Ph.D. degree holder specializing in the investigation of photocatalytic and nanometer materials. JD is a professor and a Ph.D. degree holder specializing in the investigation of nanometer materials. JM and HZ are instructors and M.Sc. degree holders specializing in the research of nanometer materials. TX is a doctoral candidate major in the study of photocatalytic materials. LD is a graduate student major in the preparation of photocatalytic materials.


The PD-1/PD-L1 axis plays a paramount role in the immune escape of tumor cells by negative regulation of T-cell functions. The aim of the present study was to characterize the PD-L1 expression pattern and its clinical implication in soft-tissue sarcomas (STS).We analyzed PD-L1 expression in 82 STS patients with 5 subtypes: rhabdomyosarcoma, synovial sarcoma, Ewing sarcoma, epithelioid sarcoma, and mesenchymal chondrosarcoma.P = 0.004); highest in epithelioid sarcoma , followed by synovial sarcoma , rhabdomyosarcoma , and Ewing sarcoma , while it was not expressed in mesenchymal chondrosarcoma . STS patients with PD-L1 expression had worse overall survival compared with those without PD-L1 expression . The Cox proportional hazard model adjusted for histologic subtype, initial metastasis, and PD-L1 expression showed that PD-L1 expression was significantly associated with shorter overall survival .The median age at diagnosis was 26 (range: 1–78) and the male to female ratio was 1.6. The majority (80 %) of patients showed locoregional disease rather than metastatic disease at diagnosis. Thirty-five cases (43 %) showed PD-L1 expression and the proportion of PD-L1 expression was significantly different according to histologic subtypes  contains supplementary material, which is available to authorized users. Sarcomas are a rare and highly heterogeneous group of neoplasms originating from the bone and soft tissue, which account for <1 % of all human malignancies . With stIt is well known that the prognosis of a malignant tumor is closely related to host immune responses. During the immune response, the priming and activation of T-cells are critical processes in the induction of adaptive immunity, and the ultimate amplitude of the immune response is regulated by a balance between co-stimulatory and inhibitory signals. In this T-cell-mediated process, cytotoxic T-lymphocyte antigen 4 (CTLA-4) provides inhibitory signals in the priming phase of the T-cell response within the lymph node. The programmed death 1 (PD-1) receptor is one of the key inhibitory signals that is induced during the chronic antigen exposure in peripheral tumor microenvironments. The interaction between PD-L1 in tumor cells and PD-1 in T-lymphocytes negatively regulates the effector function of tumor-specific T-lymphocytes and allows tumor cells to evade the host immune system. Recent studies have indicated that high expression of PD-L1 is associated with poor prognosis in non-small cell lung cancer (NSCLC), ovarian cancer, and kidney cancer . FurtherIn this study, we investigated PD-L1 expression in soft tissue sarcoma and evaluated its clinical relevance according to different subtypes of sarcoma. We thereafter analyzed the prognostic potential of PD-L1 to provide a practical guide as a diagnostic and therapeutic strategy.This study was conducted in a retrospective cohort of patients who were pathologically diagnosed with CD99 positive sarcomas such as rhabdomyosarcoma, synovial sarcoma, Ewing sarcoma, epithelioid sarcoma, and mesenchymal chondrosarcoma, between 1994 and 2013 at Yonsei Cancer Center. A total of 82 formalin-fixed, paraffin-embedded tissue blocks were available for examination of PD-L1 expression. All hematoxylin and eosin (H&E) slides were independently reviewed by two experienced pathologists (E.K.K. and S.H.K).The clinicopathologic variables such as sex, age, maximal tumor size, tumor histology and grade, tumor location, tumor stage, initial presentation of disease, and the status of the resection margin were reviewed retrospectively based on electronic medical records. Tumors were graded according to the French Federation Nationale des Centre de Lutte Contre le Cancer (FNCLCC) criteria . StagingSimple, inexpensive, and precise paraffin TMAs were constructed with a conventional microcompound table and a drill grinder. The original H&E slides were reviewed by two pathologists (E.K.K and S.H.K). One or two different tumor areas per case were selected for tissue microarray construction. Core tissue biopsies (3 mm in diameter) were taken from the individual paraffin blocks (donor blocks) and arranged in recipient paraffin blocks (tissue array blocks) using a trephine apparatus. All TMA blocks were confirmed by H&E staining.Immunohistochemistry (IHC) staining of PD-L1 was performed on the TMA blocks using a mouse monoclonal antibody for PD-L1 . Four-micrometer-thick sections were prepared and stained using a Ventana automatic immunostainer  . After dt-test for the continuous variables and the chi-square test for the discrete variables. For survival analysis, recurrence-free survival (RFS) was defined as the time interval between surgery and tumor recurrence or last follow-up. OS was defined as the time interval between the diagnosis of metastatic/recurrent disease and death or last follow-up. Survival analysis was performed using the Kaplan-Meier method with the log-rank test. Multivariate analyses for OS were performed with Cox’s regression. The accepted level of statistical significance was P < 0.05.The Statistical Package for the Social Sciences (SPSS) version 18.0  was used for statistical analyses. The correlations between PD-L1 expression and clinicopathologic variables were analyzed using the independent sample n = 32, 39 %), followed by synovial sarcoma , Ewing sarcoma , epithelioid sarcoma , and mesenchymal chondrosarcoma . One third of the tumors were located in the extremities . Most patients had no distant metastasis at the time of diagnosis (82 %) and underwent surgical resection (93 %). Adjuvant chemotherapy was administered to 47 patients (57 %), and 37 of these patients (79 %) also received adjuvant radiotherapy.The baseline clinicopathologic characteristics of the patients are summarized in Table P = 0.004). The proportion of PD-L1-expressing tumors was highest in epithelioid sarcoma , followed by synovial sarcoma , rhabdomyosarcoma , and Ewing sarcoma , whereas it was not expressed in mesenchymal chondrosarcoma . Patients with PD-L1 expression had a more negative resection margin than the PD-L1-negative group . There was no significant difference in PD-L1 expression regarding age, sex, tumor size, location, histologic grade, surgical resection, and adjuvant treatment.Among the 82 sarcoma patients, PD-L1 was expressed in 35 cases (43 %). Representative images of PD-L1-positive and -negative staining for each histologic type are shown in Fig. P = 0.015, Fig. P = 0.037, HR = 2.57, 95 % CI = 1.06–6.23).After a median follow-up duration of 33.8 months, 27 patients (33 %) had died at the time of survival analysis. The 5-year OS for all patients was 58.8 % and the 5-year OS rates of the subgroups were as follows: 64 % for rhabdomyosarcoma, 77 % for synovial sarcoma, 39 % for Ewing sarcoma, 18 % for epithelioid sarcoma, and 100 % for chondrosarcoma. There was a trend toward a worse RFS for the positive PD-L1 expression group compared to the negative group Fig. . ExpressIn this study, we evaluated the clinical relevance of PD-L1 expression in various sarcoma subtypes. PD-L1 was differently expressed according to the histologic subtypes of sarcoma and it was found to be an independent prognostic factor for OS.William Coley, a bone surgeon at New York Memorial Hospital, depicted the spontaneous tumor regression of sarcoma patients after severe bacterial infection more than 100 years ago . SubsequBased on this theoretical background, numerous clinical trials were attempted in sarcoma patients with various immunomodulatory agents such as macrophage-colony stimulating factor (GM-CSF), peptide vaccines, and anti-CTLA-4 antibody –16. InhaRecently, with the impressive and outstanding success of pembrolizumab and nivolumab in melanoma, NSCLC, and other malignancies , 18, immThe PD-1/PD-L1 expression level has been reported to be related to poor survival in other solid tumors. In a previous report of renal cell carcinoma, PD-1 expression in tumor-infiltrating lymphocytes was observed in half of the cases and was associated with poor survival . MoreoveMoreover, besides the prognostic value of PD-L1 expression in human cancers, it is becoming increasingly recognized as an important biomarker for predicting the treatment efficacy of PD-1/PD-L1 blockade. In the KEYNOTE-012 phase IB trial  , patientUnfortunately, the IHC criteria for PD-L1 expression have not yet been standardized. Indeed, different definitions of positive PD-L1 expression have been used in the clinical trials conducted to date. In the CheckMate 017 study with NSCLC patients, OS and response rate were significantly better with nivolumab than with docetaxel, regardless of the PD-L1 expression level . ConsideThe main limitations of our study include its patient selection, small sample size, and imbalance of histologic type. Because we initially intended to analyze the PD-L1 expression in CD99 positive tumors, this study mainly enrolled pediatric STS, which could result in relatively younger median age of patients and longer survival outcome. Moreover, due to the small sample size and imbalance of histologic type between PD-L1 positive and negative groups, especially epithelioid sarcoma and mesenchymal chondrosarcoma, there could be a possibility of type I error. Therefore, our findings should be validated in an independent STS cohort and according to the response to PD-1/PD-L1 inhibitors in future clinical trials.In conclusion, we have revealed PD-L1 expression in various STS of young population and demonstrated its independent negative prognostic role, thereby suggesting the PD-1/PD-L1 axis as a potential therapeutic target for the treatment of young STS patients.CTLA-4, cytotoxic T-lymphocyte antigen 4; IHC, immunohistochemistry; OS, overall survival; PD1, programmed death 1; PD-L1, programmed death ligand 1; RFS, recurrence-free survival; TMA, tissue microarray