In the central nervous system regions of the sporadic and familial FTLD and ALS patients, TDP-43 has been identified as the major component of UBIs inclusions which is abnormally hyperphosphorylated, ubiquitinated, and cleaved into C-terminal fragments to form detergent-insoluble aggregates. So far, the effective drugs for FTLD and ALS neurodegenerative diseases are yet to be developed. Autophagy has been demonstrated as the major metabolism route of the pathological TDP-43 inclusions, hence activation of autophagy is a potential therapeutic strategy for TDP-43 pathogenesis in FTLD and ALS. Berberine, a traditional herbal medicine, is an inhibitor of mTOR signal and an activator for autophagy. Berberine has been implicated in several kinds of diseases, including the neuronal-related pathogenesis, such as Parkinson’s, Huntington’s and Alzheimer’s diseases. However, the therapeutic effect of berberine on FTLD or ALS pathology has never been investigated.Here we studied the molecular mechanism of berberine in cell culture model with TDP-43 proteinopathies, and found that berberine is able to reverse the processing of insoluble TDP-43 aggregates formation through deregulation of mTOR/p70S6K signal and activation of autophagic degradation pathway. And inhibition of autophagy by specific autophagosome inhibitor, 3-MA, reverses the effect of berberine on reducing the accumulation of insoluble TDP-43 and aggregates formation. These results gave us the notion that inhibition of autophagy by 3-MA reverses the effect of berberine on TDP-43 pathogenesis, and activation of mTOR-regulated autophagy plays an important role in berberine-mediated therapeutic effect on TDP-43 proteinopathies.We supported an important notion that the traditional herb berberine is a potential alternative therapy for TDP-43-related neuropathology. Here we demonstrated that berberine is able to reverse the processing of insoluble TDP-43 aggregates formation through deregulation of mTOR/p70S6K signal and activation of autophagic degradation pathway. mTOR-autophagy signals plays an important role in berberine-mediated autophagic clearance of TDP-43 aggregates. Exploring the detailed mechanism of berberine on TDP-43 proteinopathy provides a better understanding for the therapeutic development in FTLD and ALS. TARDBP gene on chromosome 1p36.2 and plays important roles in gene regulation at RNA transcription levels involving in transcriptional repression and alternative splicing [Deposition of the pathological signature proteins in the brain is the hallmark in various kinds of neurodegenerative diseases. The pathological signature in numerous neurodegenerative diseases are characterized by the accumulation of intracellular or extracellular protein aggregates composed of amyloid fibrils , such assplicing , 5. TDP-splicing , 6 and usplicing , 7. In asplicing . The patsplicing , 10. ThuInduction of autophagy is a potential therapeutic target for accelerating the removal of aggregation-prone proteins . Most ofThe traditional herb medicine, berberine, is a potent autophagy activator through inhibition of the mTOR signal pathways , 25. RecNeuro 2a (N2a) cells were cultured in Eagle’s minimum essential medium , supplemented with 10 % (vol/vol) fetal calf serum and penicillin/streptomycin (Invitrogen). DNA transfection of N2a cells was carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.GFP-TDP-25: The cDNA of human hTDP-43 (NM_007375.3) was cloned into the HindIII/KpnI restriction sites of pEGFP-C1 vector (Invitrogen). For generation of the truncated C-terminal hTDP-43 fragments (pGFPC1-hTDP-25) (amino acids 175–414), the DNA primers used for PCR amplification were as follows: forward primer 5’-CGGAAGCTTCGAATTCTAAGCAAAGCCAA-3’; and reverse primer 5’-CAGGTACCCTACATTCCCCAGCCAGA-3’. The PCR product was subcloned into the pEGFP-C1 plasmid (Invitogen) using restriction sites HindIII and KpnI to generate pGFP-C1-hTDP-25 (GFP-TDP-25). Antibodies: Rabbit polyclonal anti-GFP (Invitrogen). Rabbit polyclonal anti-LC3 . Rabbit polyclonal anti-p-mTOR, anti-mTOR, anti-p-p70S6K and anti-p70S6K . Mouse monoclonal anti-α-tubulin (Sigma-Aldrich). Mouse monoclonal anti-GAPDH (Invitrogen).Berberine is a kind gift from Dr. Kuan-Hau Lee. 3-MA (3-Methyladenine) was purchased from Sigma Aldrich . 3VO4) (Sigma-Aldrich). The protein concentrations of the lysates were determined by Bio-rad protein assay  and then the lysates were centrifuged at 4 °C for 15 min at 12,000 rpm. The supernatants containing the RIPA soluble material were transferred into new tubes and boiled in SDS–PAGE sample buffer. The RIPA-insoluble pellets were washed twice in the lysis buffer, re-sonicated and re-centrifuged. The washed pellets were finally dissolved in the urea buffer, sonicated and supplemented with SDS–PAGE sample buffer without boiling. The soluble proteins and insoluble proteins were analyzed on SDS–PAGE gels and transferred onto polyvinylidene difluoride membranes . After blocking for 1 h in the blocking buffer , the membranes were stained with the primary antibodies at 4 °C overnight and then the secondary antibodies at room temperature for 1 h. The bound antibodies were detected by using the chemiluminescence Western blotting detection reagent ECL .Cultured cell were washed twice in PBS and pelleted at 1,000 g for 5 min. To prepare the total cell extracts, the cell pellets were directly lysed in the urea buffer (Sigma-Aldrich). For preparation of RIPA-soluble and insoluble materials , the celt-test.N2a cells grown on glass coverslips were transfected with indicated plasmids. At 48 h post-transfection, the cells were treated with different inhibitors or drugs for different periods of time. The cells were fixed with 4 % ice-cold paraformaldehyde at 4 °C for 20 min and then permeabilized with PBS–0.5 % Triton X-100 for 7 min at RT. After blocking with 10 % donkey serum for 1 h at RT, the cells were incubated overnight at 4 °C with specific primary antibodies. After washing for three times with PBS, the cells were incubated at RT for 1.5 h with DAPI (1:500) (Invitrogen) plus Alexa Fluor 488-conjugated goat anti-rabbit or Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1:500), or plus Alexa Fluora 546-conjugated goat anti-rabbit or Alexa Fluor 546-conjugated goat anti-mouse IgG secondary antibody (1:500) (Invitrogen). The images were examined on a LEICA DM6000B fluorescence microscope and quantified by MetaMorph software . For quantitative analyses of aggregates, five representative fields per sample were taken and analyzed by MetaMorph software. GFP signal was gated to exclude non-transfected cells, and the images were then superimposed with corresponding DAPI images. The Metamorph was used to count the total number of transfected cells. For aggregate analyses, the GFP images were visually adjusted to determine a common threshold across all samples to eliminate diffuse or non-aggregated signals. The numbers of individual aggregates were calculated with the Integrated Morphometry Analysis of Metamorph. The percentages of aggregated cells were calculated as the following formula: total number of GFP-positive aggregated cells/total number of transfected cells. Statistical significance was analyzed with the student First, we were interested in investigating the effect of the autophagy activator berberine on TDP-43 insoluble deposition formation. To observe whether berberine can prevent or dissolve TDP-43 aggregates, we overexpressed GFP-tagged aggregation-prone C-terminal TDP-43 fragments, GFP-TDP-25 , 34, andTo further monitor the potential therapeutic role of berberine on dissolving the insoluble aggregated TDP-43, we treated cells with berberine in GFP-TDP-25-transfected N2a cells after TDP-43 aggregates formation. After 24 h transfection, the GFP-TDP-25-transfected cells were treated with different doses of berberine for another 24 h and the insoluble TDP-43 fractions were extracted by sequential extraction method and analyzed by Western blotting. Comparing to control group, the insoluble fraction of TDP-43 (urea fraction) showed a dose-dependent decrease in cells treated with berberine after 24 h transfection, Fig. , suggestIt has been demonstrated that berberine is a potent autophagy activator through inhibiting mTOR signaling pathways , 25. AutTo further confirm that the effect of berberine on decreasing TDP-43 aggregates is mediated by autophagy activation, we inhibited autophagy by specific autophagosome inhibitor, 3-MA (3-Methyladenine). 3-MA inhibits autophagy by blocking autophagosome formation via inhibiting the mTOR key regulator, class III Phosphatidylinositol 3-kinases (PI-3 K), and is broadly used to study autophagy-related mechanism under various kinds of cellular condition . We examMost of the short-lived proteins are degraded by UPS through the 26S proteasome , whereasRecent studies have demonstrated that the traditional herb medicine, berberine, is an isoquinoline alkaloid found in a number of important medicinal plant species and is a potent autophagy activator through inhibition of the mTOR signal pathways , 30. Ber2O2 generation would cause oxidative stress and reactive oxygen species (ROS)-mediated damage [Berberine also exhibits multifaceted pharmacological effects on neuroprotection or againsting neurodegeneration through shared signaling pathways. In both Alzheimer’s and Parkinson’s patients, monoamine oxidase B (MAO-B) activity is elevated and MAO-B-mediated dopamine metabolism pathway leading to Hd damage . Berberid damage , 52, 53.d damage –58. GLP-d damage –62. Furtd damage . Berberid damage . Furtherd damage . Recentld damage . To bettBerberine has been identified as a potent autophagy activator through inhibiting mTOR signaling pathways , 25. AutWe supported an important notion that the traditional herb berberine is a potential alternative therapy for TDP-43-related neuropathology, Here we demonstrated that berberine is able to reverse the processing of insoluble TDP-43 aggregates formation through deregulation of mTOR/p70S6K signal and activation of autophagic degradation pathway. mTOR-autophagy signals plays an important role in berberine-mediated autophagic clearance of TDP-43 aggregates. Exploring the detailed mechanism of berberine on TDP-43 proteinopathy provides a better understanding for the therapeutic development in FTLD and ALS.


Z0 = 377 Ω). Near this point, the interaction is maximized, and the conductor absorbs strongly. Here we show that monolayer graphene, a tunable conductor, can be electrically modified to reach this transition, thereby achieving the maximum absorptive coupling across a broad range of frequencies in terahertz (THz) band. This property to be transparent or absorbing of an electromagnetic wave based on tunable electronic properties (rather than geometric structure) is expected to have numerous applications in mm wave and THz components and systems.The coupling of an electromagnetic plane wave to a thin conductor depends on the sheet conductance of the material: a poor conductor interacts weakly with the incoming light, allowing the majority of the radiation to pass; a good conductor also does not absorb, reflecting the wave almost entirely. For suspended films, the transition from transmitter to reflector occurs when the sheet resistance is approximately the characteristic impedance of free space ( Efficient coupling of an electromagnetic wave to a thin conductor relies on the sheet conductance of the given material. Here, the authors demonstrate that engineering the monolayer graphene sheet resistance enables electrical or chemical tuning from the transmission to the absorption regime up to THz frequencies. To date, unpatterned monolayer graphene devices have operated exclusively in the weakly coupled, highly transmissive regime18.Two-dimensional materials provide a class of atomically thin conductors20. In free space, the maximum absorption is 50%, whereas on a substrate it can be greater than 90% depending on the direction of normal incidence, discussed in detail below. Regardless of the case (free space or on dielectric), the absorption is much larger than the 2.3% optical value21, and thus represents a milestone of coupling electromagnetic waves to an atomically thin nanomaterial. This is measured over an extremely broad range, from mm wave to THz frequencies. Strong EM absorption using a single atomic layer exemplifies the fundamental relationship between nanoscale electronics and classical electromagnetism.Here, by deliberate engineering of the sheet conductance using large-domain graphene films in combination with chemically modified substrates (to decrease interface scattering), as well as chemical and electrical doping, we fabricate monolayer graphene devices with sheet resistance crossing the characteristic impedance of free space, 377 Ω/□ Achieving this, we show that monolayer graphene can be tuned to behave as a strong absorber over a broadband frequency range, approaching the theoretical impedance at which absorption is maximizedGg = (π/4)·G0 = (e2/4ħ)21, where G0 is the conductance quantum. This results in its low absorbance, approximated by the product of Gg and the characteristic impedance of free-space, Z0 = 377 Ω. This product can be written π·(e2/ħc) ≡ π·α, where α is the fine structure = 1/137. Thus, only ≈ 2.3% of the light is absorbed, and the majority of the light is transmitted.The zero-gap band structure of graphene leads to a non-trivial frequency-dependent AC conductance, which governs the absorption of the EM radiation. As depicted in Fig. EF/h (where EF is the Fermi energy), as is the case in the THz range, the photon energy is too low to excite electron–hole pairs, and the electromagnetic sheet conductance is expected to follow the DC sheet conductance23. This should occur for frequencies up to ~ 1/τ, where τ is the scattering time, at which point the conductance is expected to undergo a Drude-like roll-off9, dropping towards the low optical value, . This prospect is indicated schematically within the THz frequency range in Fig. EF and τ values are plotted. Figure EF and τ affect the AC conductance; the absorption-dominating regime begins when the sheet resistance ≤ 377 Ω/□ . Previous investigations of single-layer graphene THz absorption fail to surpass the Z0 = 377 Ω threshold18 . Instead, as demonstrated in this work and emphasized as an arrow in Fig. τ, the graphene conductance can be large  and air (n = 1), there is an etalon effect present in the frequency-dependent transmission (even in the absence of graphene). With graphene present, we can model the effect of varying the graphene sheet resistance to calculate the net transmittance using the transmission line model , reflected (R) or absorbed (A). Electromagnetics states that a perfect conductor is a mirror  and a perfect insulator is a transmitter . What is the quantitative relationship between T, R, A and the sheet resistance? The three are not completely independent, as T + R + A must equal unity due to conservation of energy. The relationship for the transmittance, reflectance, and absorptance of a suspended thin metallic film was derived in 193419, and is given by,A plane wave can be transmitted  and substrate thickness govern T, R and A. Furthermore, interference effects from the substrate cause Fabry–Perot behavior depending on the THz frequency (f), substrate thickness (L) and index of refraction (n). When incident on the graphene side first, the general analytical expressions for T, R and A are given by,When mounted on a substrate of index of refraction, Similar expressions for incidence on the substrate side are given in Supplementary Note kL = Nπ, where k, is the wave vector, L is the substrate thickness, and N is integer values, or when the THz frequency is at half-wave resonance with the graphene–silicon etalon, which occurs at even integer multiples of T, R and A reduce to exactly the same as the Woltersdorff equations19 . Although the transmittance is low and equivalent for both incidence directions (graphene or substrate first), the sheet resistance-dependent reflection and absorption indeed depend on the incidence direction. When the THz beam is incident on the graphene side first, interference effects cause the device to behave as mostly reflecting , resulting in low absorption. On the other hand, when the THz beam is incident on the substrate first (with graphene on the backside), the absorption, transmission and reflection can be affectedly tuned by the sheet resistance. This allows the absorption maximum to increase to > 50% for low sheet resistance values  shows contrasting behavior, but like the suspended film, there still exists certain impedance values that determine the peak absorption, and the onset of the absorption-dominating threshold Fig. . The abs18. This includes our previous work9, where the graphene–etalon structures were fabricated using chemical vapor-deposited graphene films25 transferred26 onto a 90 nm gate oxide layer on high-resistivity silicon substrates. To improve τ, so as to increase the conductance of the devices, we synthesized large-domain (mm-sized) graphene films (to minimize graphene grain boundaries)27, in addition to depositing octadecyltrichlorosilane (ODTS) self-assembled monolayers (SAM) on the wafer (to decrease scattering)28 prior to graphene transfer  as shown in Fig. In order to de-convolve the etalon effect, the broadband transmission through a bare high-resistivity silicon substrate (without graphene) is first measured as a control. The multiple interference peaks of transmission around ≈ 1.0 Fig.  confirm I–V curve  was observed at voltages beyond this range, and hence gate voltages were restricted to −7 V to +45 V. Across the broadband frequency range measured, the transmittance variation at the peaks can be used to define a depth of modulation (DoM) , defined as (THigh−TLow)/THigh. The DoM is substantial, with a maximum value of ≈ 52% at the ~ 330 GHz peak  allows for useful THz modulation over 1 THz of frequency range with polarization independence. This result was independently confirmed in two different labs −1 −1), to approximately equal to (Z0)−1 , and clearly demonstrates electrical tuning from the transmission to absorption-dominating regimes. In order to achieve the necessary sheet conductance values near maximum absorption, and beyond (Z0)−1, we utilized chemical  tuning. We intentionally dope graphene films to have low sheet resistance in the zero-bias state  by using benzimidazole (BI) dissolved into the copper etching solution during graphene transfer31 . For devices covered by a graphene film, each transmittance peak is fitted to yield a AC admittance  value; the measured DC conductance and the estimated THz conductance are then fitted according to Eqs. EF and τ for each device . Supplementary Note EF and τ, for measured devices. Notably, the values of EF are moderate, while the values of τ can be large (> 100 fs), and stress the importance of engineering τ, as initially outlined in the device design plots in Fig. The measured transmission spectra can be used to determine the AC sheet conductance, using the bare silicon control sample as nearly lossless “calibration”kL = Nπ. At half-wave resonance, the free-space and etalon effect give the same predicted T, R and A vs. sheet resistance .We plot in Fig. nce Fig. . We alsonce Fig. . The 37735, or by suppressing the transmission channel36 using double-pass/reflection/“Salisbury screen” geometries14. Other thin film (multilayer) materials have also demonstrated remarkable control of absorption by controlling film thickness, such as niobium nitride (NbN)39 and indium tin oxide (ITO)40. Here, our experiments show that exceptionally large, near maximum, absorption can be reached in the thickness limit of one single layer of graphene.Instead of designing the single-layer sheet resistance to cross into the absorption-dominating regime, previous experiments for unpatterned graphene devices boosted low absorbance by the addition of multilayers41, and may further enhance the single-layer reflection DoM42. Pertaining to designing devices that operate in transmission (such as the work performed here), the suspended film and Woltersdorff equations19 stand as the ideal situation for broadband absorption. Using the transmission line model for a film on a substrate, as the substrate index of refraction value approaches n = 1, or the substrate thickness approaches, L = 0, the amplitudes of the Fabry–Perot fringes decrease, and the widths of the fringes increase . Although it is shown by calculation43 that the (quarter-wave resonance) absorption can be greater than 50% if the THz beam is incident on the substrate side first 49, NbN39, ITO40, metal thin films or even multilayer graphite, the conductance of monolayer graphene can be easily tuned via external voltages due to the extremely high mobility. For high-frequency applications, the mobility of single-layer graphene is superior to bilayer (multilayer) graphene50, and other semiconducting materials51, but fabrication remains a challenge for large-area devices which precludes electronically tunable devices operating at very low sheet resistance (even in the simple case of EM incidence on the substrate side first). A combination of substrate modification to improve mobility with techniques such as electrolyte gating33 or device integration17 could yield electrically controlled devices with high modulation frequency and low operation voltage (see Supplementary Note 2 V−1 s−1) with sheet resistance spanning ~ (e2/4ħ) to 500(e2/4ħ) at DC applying only ~ 5 Vg52. Our studies reveal the opportunity for a device based on the tunable electronic properties, without complicated fabrication steps required for other THz modulation schemes such as metamaterials53, nanophotonic resonators54, thin film thickness tuning47 or materials with switchable metal-to-insulator phase transitions49.We were able to achieve a large transmission DoM by improving the transconductance  of the graphene on ODTS SAM layer. The high-quality, large-domain graphene films on SAM used here were confirmed via Raman mappingA > T, R), we have not pushed well below the inflection point between poor to excellent conductor . Future work with increased conductivity  may enable complete tuning from strong transmitter, to strong absorber, to strong reflector, and would enable new technologies to manipulate EM radiation in powerful ways.We have demonstrated that the control of the large-area graphene DC sheet resistance enables the tuning, either electrically or chemically, from the transmission to absorption-dominating regimes at mm wave to THz frequencies. This could have wide-ranging application in the tunable and controllable manipulation of mm wave and THz light, including, for example, gate tunable THz modulators, electronically steerable phased array antennas and radar and tunable variable focus lenses and cones. However, while we have clearly demonstrated a single layer tuned from the transmission to absorption regimes  via chemical vapor deposition (CVD) in a 5-inch-diameter quartz tube. Large-domain  graphene films are synthesized using a fast, oxygen-assisted growth using two stages of methane flow (0.8 and 2.4 sccm flow rates) to separate nucleation and edge growth as presented in ref. 2 layer on top are cleaned in hot piranha solution for 1 h followed by cleaning in deionized (DI) water. The wafers are placed in a vacuum chamber with 50 µL of ODTS solution in a glass vial. The chamber is then pumped down and the ODTS is allowed to self-assemble on the SiO2 surface for 6 h.High-resistivity Si wafers with a 90-nm-thick, gate-quality SiO2) is coated with poly(methyl methacrylate) (PMMA) solution and etched in 5% ammonium persulfate (APS) solution. The floating PMMA/graphene stack is washed in DI water and wet transferred onto the substrate. For BI doping experiments, BI (0.03 M) was dissolved in a solution of APS, H2SO4 and H2O2 for copper etching. This process simultaneously etches the copper and dopes the graphene as described in ref. 31. Following transfer, the wafer is dried overnight. Before PMMA removal, the wafer is heated on a hotplate at 130 C for 30 min. After cooling to room temperature, the wafer is placed in an acetone bath for 24 h for PMMA removal. The wafers are finally rinsed with isopropyl alcohol, and dried with N2 gas.Graphene covering a copper foil . Sheet resistance measurements were performed using a lock-in amplifier and a 4-point van der Pauw method.To probe the electrical properties of the transferred graphene films, four Cr/Au (20/50 nm) electrodes were deposited by electron beam deposition using a shadow mask. All devices use the same shadow mask to maintain a consistent area to be measured. The electrodes were deposited in a square geometry with ≈ 5 mm gap separation between each electrode. The current between two diagonally opposed electrodes, representing the drain and source electrodes, was measured with 100 mV bias applied. Gate voltages were applied through the SiO24. An aperture was placed between the sample and the THz beam to ensure that the THz beam area (≈ 3 mm diameter) was consistent from sample to sample, and less in area than the graphene film. The THz beam was then aligned at the geometric center of the four electrodes on the graphene film.An Emcore PB7200 broadband frequency-domain photomixing spectrometer was used to perform THz transmission measurements through the graphene-on-Si etalon structures. As shown in Supplementary Fig. τ, and EF from 50 fs, and 50 meV, to 200 fs and 200 meV (with a step size of 25 fs and 25 meV), respectively. Figure τ, and EF from 0.5 fs, and 0.5 meV, to 200 fs and 600 meV (with a step size of 0.5 fs and 0.5 meV), respectively.The trend lines according to Eqs. 40. The model assumes quasi-plane wave radiation, substrate index of refraction n = 3.41 and the graphene sheet is treated as a shunt, lumped impedance, Zg, where the real part of the graphene impedance (admittance) is equal to the measured DC resistance (conductance), assuming negligible imaginary contributions. The substrate thickness was inputted accordingly during calculation, and variations from device to device are listed in Supplementary Note The transmittance vs. frequency plots for various sheet resistances (dotted lines) in Fig. 9. To calculate the AC conductance data points , all data fitting consisted of both real and imaginary contributions.The fitting for trend lines in Fig. The data that support the findings of this study are available from the corresponding author upon request.Supplementary Information


Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with high mortality, uncertain cause, and few treatment options. Studies have identified a significant genetic risk associated with the development of IPF; however, mechanisms by which genetic risk factors promote IPF remain unclear. We aimed to identify genetic variants associated with IPF susceptibility and provide mechanistic insight using gene and protein expression analyses.We used a two-stage approach: a genome-wide association study in patients with IPF of European ancestry recruited from nine different centres in the UK and controls selected from UK Biobank (stage 1) matched for age, sex, and smoking status; and a follow-up of associated genetic variants in independent datasets of patients with IPF and controls from two independent US samples from the Chicago consortium and the Colorado consortium (stage 2). We investigated the effect of novel signals on gene expression in large transcriptomic and genomic data resources, and examined expression using lung tissue samples from patients with IPF and controls.AKAP13; rs62025270, odds ratio [OR] 1·27 [95% CI 1·18–1·37], p=1·32 × 10−9) and confirmed previously reported signals, including in mucin 5B  and desmoplakin . For rs62025270, the allele A associated with increased susceptibility to IPF was also associated with increased expression of AKAP13 mRNA in lung tissue from patients who had lung resection procedures (n=1111). We showed that AKAP13 is expressed in the alveolar epithelium and lymphoid follicles from patients with IPF, and AKAP13 mRNA expression was 1·42-times higher in lung tissue from patients with IPF (n=46) than that in lung tissue from controls (n=51).602 patients with IPF and 3366 controls were selected for stage 1. For stage 2, 2158 patients with IPF and 5195 controls were selected. We identified a novel genome-wide significant signal of association with IPF susceptibility near A-kinase anchoring protein 13 (AKAP13 is a Rho guanine nucleotide exchange factor regulating activation of RhoA, which is known to be involved in profibrotic signalling pathways. The identification of AKAP13 as a susceptibility gene for IPF increases the prospect of successfully targeting RhoA pathway inhibitors in patients with IPF.UK Medical Research Council, National Heart, Lung, and Blood Institute of the US National Institutes of Health, Agencia Canaria de Investigación, Innovación y Sociedad de la Información, Spain, UK National Institute for Health Research, and the British Lung Foundation. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic lung disease of unknown cause with a poor prognosis. The incidence of IPF in the UK is about 4·5–7·5 per 100 000 and is increasing,MUC5B]), telomere maintenance (such as telomerase reverse transcriptase [TERT] and CST complex subunit [STN1]) and cell–cell adhesion (such as desmoplakin [DSP] and dipeptidyl peptidase 9 [DPP9]).MUC5B and Toll interacting protein (TOLLIP) variants are associated with reduced disease progression.The current theory suggests that IPF is characterised by initial damage to the alveolar epithelium, which then signals to various cell types, predominantly fibroblasts and macrophages, promoting tissue damage and extracellular matrix synthesis that leads to parenchymal destruction and alveolar replacement by dense fibrotic tissue.Evidence before this studyWe searched Web of Science between Aug 21, 2015, and June 7, 2017, with the search terms “pulmonary fibrosis” and “genome wide” with no restrictions on publication date or language. Previous genome-wide association studies (GWAS) have reported 16 independent signals associated, at genome-wide significance, with susceptibility to idiopathic pulmonary fibrosis (IPF). Although previous studies have shown that epithelial cell function, lung defence, cell–cell adhesion, and telomere maintenance might play important roles in IPF, the precise mechanisms through which the genes identified in previous GWAS promote IPF are still poorly understood.Added value of this studyAKAP13) as an IPF susceptibility gene. AKAP13 is a Rho guanine nucleotide exchange factor (RhoGEF) that regulates activation of Rho A. RhoA is a molecule with a known role in profibrotic signalling pathways; however, AKAP13 has not previously been implicated in the pathogenesis of IPF. These studies provide genetic veracity for targeting Rho signalling in IPF.To our knowledge, this study is the largest genetic study of IPF done to date, bringing together 2760 patients with IPF and 8561 controls. We present a novel genome-wide significant genetic association signal and novel gene expression data that implicate A-kinase anchoring protein 13  in stage 1 were then further analysed in independent samples of patients with IPF and controls from two US samples, the Colorado consortium and the Chicago consortium (stage 2). We defined statistically significant associations with susceptibility to IPF as variants that met genome-wide significance (p<5 × 10−8) after meta-analysis of stages 1 and 2 together. Variants that became less significant in the meta-analysis than in stage 1 alone were not reported as showing an association.This GWAS used a two-stage approach to identify novel genome-wide significant signals associated with susceptibility to IPF. In stage 1, a GWAS was done using patients with IPF from nine different centres in the UK and controls from UK Biobank. Variants showing a significant association . The remainder of the controls were genotyped using the Affymetrix Axiom UK Biobank array . Genotyping and imputation procedures for studies contributing to stage 1 and 2 are described in the −3, the analysis was rerun using the Firth test using EPACTSThe genome-wide association analysis of IPF susceptibility was done assuming an additive genetic effect and conditioning on age, sex, and the first ten principal components to adjust for ancestry.−6 in association testing in stage 1 were followed up in stage 2. Conditional analyses were used to identify additional independent signals in the same genomic region  association with IPF risk in stage 1, available association test statistics from stages 1 and 2 were meta-analysed using a fixed-effects model. Signals that had genome-wide significance (p<5 × 10−8) when meta-analysing stages 1 and 2 were reported as significantly associated with susceptibility to IPF. A Bayesian approach was used to fine-map those signals to create 95% credible sets  were investigated in three expression quantitative trait locus (eQTL) datasets: a lung eQTL database comprising individuals from three cohorts , a blood eQTL database, and in the Genotype-Tissue Expression project (GTEx) cohort  expression in controls and patients with IPF while also controlling for age, smoking (ever or never and pack-years), and sex.RT-PCR gene expression analysis was done on RNA extracted from human lung tissues of 46 patients with IPF and 51 controls from the Lung Tissue Research Consortium using standard methods . InitialFormalin-fixed paraffin-embedded human lung tissue sections were obtained from tissue distant from the tumour that was obtained during lung resection from controls and tissue from patients with IPF taken at either post-mortem examination or lung transplantation. Immunohistochemistry was done using standard methods .We performed in-silico analyses to establish whether known drugs target the proteins identified in the genome-wide and expression analyses or the proteins that interact with them .The funders had no role in the study design. GlaxoSmithKline Research and Design participated in collection of data and had access to the raw data from a subset of the UK IPF patient data. LVW and RGJ were involved in all stages of study development and delivery, had full access to all data in the study, and had final responsibility for the decision to submit for publication.−6) and were followed up in stage 2  were included in the stage 1 analysis of 13 076 821 variants . The pat stage 2 .Figure 1−3, after Bonferroni correction for 27 tests) in stage 2, and had genome-wide significance (p<5 × 10−8) in the meta-analysis of stage 1 and 2 together . This SNP was also significant in stage 2 alone after Bonferroni adjustment for 27 tests . The 95% credible set  produced for this signal contained 113 variants (MUC5B (rs35705950) and DSP (rs2076295).12A total of 2158 patients with IPF and 5195 controls were included in stage 2 , table 1together . A novel KLHL25; , had genvariants . The othce p<5 × −8 in the95% CI 1·–1·33, p=vs 1231 controls); the results were consistent with the original analysis . We reimputed the region with only variants genotyped on both arrays using the new Haplotype Reference Consortium imputation reference panel;−6; meta-analysis OR 1·27 [95% CI 1·19–1·35], p=1·55 × 10−9).We explored whether genotyping array differences could have influenced the association test result for SNP rs62025270. In the control–control comparison , rs62025−3) with a consistent direction of effect to previous GWAS; namely variants in MUC5B, DSP, family with sequence similarity 13 member A (FAM13A), TERT, mucin 2 (MUC2), DPP9, and TOLLIP . In non-diseased whole-lung tissue from the lung eQTL databases , the minor allele A of rs62025270, associated with increased susceptibility to IPF, was associated with increased expression of AKAP13 (r2=0·60) with rs17636666, the variant that was most significantly associated with expression of AKAP13 in lung tissue. This co-localisation of IPF susceptibility association signal and strongest AKAP13 gene expression association signal (eQTL) in lung tissue  was also associated with expression of AKAP13 in whole blood  with the SNP most strongly associated with AKAP13 expression in blood (rs870689). This finding suggests differences in regulation of AKAP13 expression between lung and whole blood, which have different cell-type compositions. Furthermore, the minor allele A of rs62025270 was associated with decreased expression of AKAP13 in blood, rather than increased expression as seen in lung tissue. The SNP rs62025270 was also found to be associated with expression of RNA genes RP11–158M2.3, RP11–158M2.4, RP11–158M2.5, RP11–815J21.3, and RP11–815J21.4 in a range of tissues in GTEx .Having identified that the minor allele A of rs62025270 was associated with increased susceptibility to IPF and increased expression of ithelium  and alveithelium  in contrithelium . High AKithelium  than in ithelium . Real-tiWe tested the association of rs62025270 with survival time in all patients from stage 1 for whom survival data were available . The median survival time after diagnosis of IPF was 1621 days . rs62025270 showed no association with survival time .11 proteins that interact with AKAP13 were identified as known targets for existing drugs or compounds in development, including aspirin, dextromethophan, and GSK-690693 .AKAP13 as a potential driver of IPF pathogenesis. We showed that the minor allele (A) of rs62025270 , associated with increased susceptibility to IPF, was associated with increased expression of AKAP13 in lung tissue. Morphological analysis of control and diseased lung tissue identified AKAP13 expression primarily in epithelial cells, with some additional expression in lymphoid tissue in patients with IPF. Furthermore, we showed increased expression of AKAP13 in fibrotic lung tissue from patients with IPF compared with samples from healthy controls.We studied 2760 patients with IPF and 8561 controls and identified a novel genome-wide significant association of variant rs62025270 that implicates AKAP13-null mice are embryonic lethal because of arrested cardiac development at embryonic day 10.NCT01371305).The high AKAP13 expression in epithelial cells further supports the hypothesis that IPF is a disease characterised by epithelial susceptibility to injury, consistent with findings of other genetic risk factors associated with epithelial cell function.We also identified AKAP13 in lymphoid follicles within fibrotic regions of lung tissue from patients with IPF. Lymphoid follicles without germinal centres are well described in IPF and are thought to be composed primarily of B cells and dendritic cells.Because of the known function of AKAP13, it probably has some potential downstream molecular interactions that could be targeted therapeutically, most notably RhoA and Rho kinase.AKAP13 at genome-wide significance in IPF susceptibility.MUC5B promoter (stage 1 p=1·86 × 10−37) was the strongest genetic risk factor for susceptibility to IPF with an OR of 4·11,DSP (stage 1 p=4·14 × 10−12), MUC2 (stage 1 p=1·33 × 10−5), TOLLIP (stage 1 p=1·60 × 10−5), TERT (stage 1 p=8·25 × 10−5), DPP9 (stage 1 p=4·17 × 10−4), and FAM13A (stage 1 p=0·002). 17 variants were identified in stage 1 that have not been previously reported to be associated with IPF (of which 14 were low frequency or rare), but we could not investigate all of these in the stage 2 analyses because not all data were available. These could be additional true positive signals of association with susceptibility to IPF that will reach genome-wide significance in larger and more densely imputed studies.To our knowledge this is the first GWAS implicating A strength of our study is that we were able to bring together the largest sample size of patients with IPF and controls available to date and apply a robust and widely used two-stage study design−3), and two additional signals that reached genome-wide significance (MUC5B p=1·86 × 10−37 and DSP p=4·14 × 10−12). In addition, the diagnostic practices between the UK and USA might be different but the concordant results from the three case-control studies reassures us that these observations are robust.This study also has some limitations. Although clinical investigators were careful to exclude other fibrotic lung diseases, a small number of non-IPF cases of pulmonary fibrosis might have been included. However, the advantage of collating a large dataset is that the effects of misclassification on the analysis are reduced. Furthermore, we expect that misclassification would lead to attenuation of signals rather than produce false-positive findings, which was supported by our replication of nine previously reported signals of association with IPF in our previously unreported stage 1 data. This included five signals that reached a Bonferroni-adjusted significance threshold for 16 tests (p<3·13 × 10AKAP13 expression in patients with IPF compared with controls. The discordance between these results could be due to a number of factors, including the difference in normalisation strategies between the two techniques and sample heterogeneity. The eQTL data that we present from control lung tissue shows that the minor allele A of rs62025270, associated with susceptibility to IPF, is also associated with increased expression of AKAP13 in three independent studies.AKAP13 mRNA. Therefore, these data provide strong independent evidence that increased AKAP13 expression in alveolar cells has a role in IPF, and the cellular heterogeneity within the fibrotic lung samples used for RNA analysis is likely to explain this discordance.A previous study of 119 patients with IPF and 50 donor lung controls from the Lung Tissue Resource Consortium reported a small decrease of AKAP13 expression in patients with IPF compared with controls using gene expression data from the Affymetrix Gene ST1·0 array;AKAP13. AKAP13 is a RhoGEF that is known to interact with a central fibrogenic pathway—G protein-coupled receptor activation of RhoA—and expression is increased in fibrotic regions of lungs from patients with IPF. Expression of AKAP13 occurs primarily in the epithelium thereby reinforcing the idea that epithelial processes are central to IPF pathogenesis. The identification of a RhoGEF that regulates a pathway known to be involved in the pathogenesis of IPF, a pathway for which drugs are in development, could potentially provide a novel target for antifibrotic therapy for IPF.In summary, we report a novel genome-wide significant association for IPF susceptibility with SNP rs62025270 and present evidence that this SNP might exert its effect via expression of the nearby gene


Benefit from exceptional electrical transport properties, graphene receives worldwide attentions, especially in the domain of high frequency electronics. Due to absence of effective bandgap causing off-state the device, graphene material is extraordinarily suitable for analog circuits rather than digital applications. With this unique ambipolar behavior, graphene can be exploited and utilized to achieve high performance for frequency multipliers. Here, dual-gated graphene field-effect transistors have been firstly used to achieve frequency quadrupling. Two Dirac points in the transfer curves of the designed GFETs can be observed by tuning top-gate voltages, which is essential to generate the fourth harmonic. By applying 200 kHz sinusoid input, arround 50% of the output signal radio frequency power is concentrated at the desired frequency of 800 kHz. Additionally, in suitable operation areas, our devices can work as high performance frequency doublers and frequency triplers. Considered both simple device structure and potential superhigh carrier mobility of graphene material, graphene-based frequency quadruplers may have lots of superiorities in regards to ultrahigh frequency electronic applications in near future. Moreover, versatility of carbon material system is far-reaching for realization of complementary metal-oxide-semiconductor compatible electrically active devices. Frequency multipliers, cooperated with low frequency local oscillators, can provide high frequency electromagnetic signal for radio astronomy, radar technology and other communication applications1245ds–Vgs) curves of GFETs2425et al. developed the new concept and reported the first graphene-based frequency tripler constructed by two GFETs connected in seriesOn the other side, graphene, the first discovered two-dimension monolayer material7913141517182021222427total) can be written asThe V-shaped transfer curves can be obtained easily in GFETs if homogeneous graphene material is used as the channel materialC is the metal/graphene contact resistance (nearly a constant)0 is the electron-hole puddleG) dependent charge carrier concentration which can be described by:where RDirac is the gate voltage of charge neutrality point. Hence n equals to zero with VG reaching VDirac, and the minimal drain current of the V-shaped transfer curve can be obtained as Rtotal reaches the peak value. However, if inhomogeneous graphene, such as a graphene PN junctionwhere, ε and t are the permittivity and thickness of the dielectric layer and V1 and L2 are the length of the N-type doping graphene and P-type doping graphene, respectively. μ1 and μ2 are the carrier mobility in the two regions. As as two graphene parts are connected in series. Here, LDirac1 and VDirac2 are the Dirac voltage of N-type region and P-type region, respectively. With different values of VDirac1 and VDirac2, two maxima will be discovered in Rtotal at gate voltages of VDirac1 and VDirac2.where Vout with one-cycle input Vin. Therefore, a frequency quadrupler can be realized with only one GFET if two different graphene parts with different doping levels, such as graphene PN junctions, are formed in the channel.Although no effective bandgap, graphene-based PN junctions find their applications in electronic384042444742TG is the length of the TG, and the charge carrier concentrations in graphene under the TG and away from the TG can be given by:where L is the channel length, L1, ε2 and t1, t2 are the permittivities and thicknesses of the back gate (BG) dielectric layer and TG dielectric layer, respectively, VDiarc BG is the Dirac voltage of the channel graphene without TG and VBG is the BG voltage. The peak value of Rtotal can be obtained by setting either where εBG1 is tunable with variable TG voltages and a linear relation can be observed from Hence the newly arising Dirac voltage V++ silicon with the SiO2 cover layer works as global BG. The narrow Ti/Au finger with Al2O3 bottom layer is utilized as local TG. With suitable TG voltage, the one-cycle input electronic signals applying to global BG can result in the four-cycle output signals at the drain electrode. The circuit implementation of the frequency quadrupler is displayed in TG is applied to the TG to achieve electrostatic doping in the underneath graphene. Direct-current (DC) voltage VBG is applied to BG to bias the working point of the frequency quadrupler, while an alternating current (AC) sinusoidal input signal Vin is added to VBG. VS is connected to the ground. High performance bias tee is utilized to apply DC bias and extract the output RF power.The three-dimensional schematic of the dual-gated GFET-based frequency quadrupler is shown in 2 layer working as the dielectric layer of BG was formed on the surface of the silicon substrate (2 layer after the copper foil being etched away. The photolithography and O2 plasma etching were successively used to fabricate the patterned graphene channel (50 μm * 20 μm) that works as the active region (2O3 dielectric was generated on the graphene films successfully by atomic layer deposition (ALD) /Au (200 nm) films working as TG were finished by lift-off in acetone after the photolithography and thermal evaporated processes  . Here, irocesses . As the 2O3 dielectric layer, Raman spectrum (2 layer and became invisible after deposition of 40 nm Al2O3 layer. Scanning electron microscopy (SEM) image of the device surface is displayed in Before deposition of the Alspectrum  of graphBGS (VBG-VS) swept from −30 V to 0 V and bias voltage Vds (VD-VS) was set at 1 V. By decreasing TG voltage VTGS (VTG-VS) from −1 V to −8 V, the second obvious Dirac point is arising. When VTGS was set at −1 V, only one Dirac point at about −21 V is discovered, indicating homogenization of the whole graphene channel and heavy n-type doping of the original graphene. When VTGS was decreased to −8 V, two clear conductance minima can be found at the BG voltages of about −21 V and −4 V, respectively. Occurrence of the second Dirac point at −4 V indicates formation of soft n-doped graphene underneath TG. The inset shows relationship between location of the newly formed Dirac point and TG voltages and illustrates that new Dirac voltage is approximately a linear function of TG voltage, which agrees well with TGS. In other words, the total area of the soft n-doped graphene increases with decrease of VTGS. Therefore, resistance of this part graphene plays a growing important role in the total channel resistance. The reason is that TG can dope the graphene near the TG edges due to edge effect of the capacitor structure and the total doping range increases when VTGS decreases . Nonlinear behaviour in the inset at lower VTGS is another result of edge effect proposed above. Therefore, with narrow TG for underneath graphene electrostatic doping, obvious inhomogeneous graphene could be formed in the FET channel and a W-shaped transfer curve was obtained with dual-gated GFET.TGS was set at −8 V and the Vds was set to 1 V. The capacitor C from a bias tee was used to block the DC signal from the output Vout. A 200 kHz sinusoidal input (VBGS ranges from −22 V to −2 V) was applied to the BG to initiate the frequency quadrupling. In principle, a pure frequency quadrupler (nearly 100% spectrum purity) can be achieved when two symmetric conductance minima arise in the operation area simultaneously, which is sketchily shown in TGS = −6 V and VBGS ranged from −10 V to −2 V) and about 78% of output RF power is concentrated at useful 400 kHz, with an input of 200 kHz. TGS was set to −8 V and VBGS swept from −15 V to −1 V. About 79% of total RF power is concentrated at 600 kHz with 200 kHz input. Therefore, dual-gated GFET can work as high performance frequency doublers, frequency triplers and frequency quadruplers by choosing suitable operation areas. This is the first time that a multi-mode frequency multiplier is reported.A frequency multiplier with simple structure and tunable multiplication factor may have lots of superiorities for RF electronic applications due to low cost and high integration. In our case, dual-gated GFET can also work as high performance frequency doublers or frequency triplers with a suitable operation area. 2 gate dielectric layer with thickness of 90 nm is formed between BG and the graphene channel. With this structure, time constant of about 1.74 ps (R of about 200 Ω and C of about 8.7 fF) can be obtained, indicating cut-off frequency of about 92 GHz and 368 GHz generated output signal. Under a 1 V bias voltage, required carrier mobility of graphene is only 3680 cm2/Vs, which can be achieved easily due to ultrahigh intrinsic carrier mobility of graphene material. Hence graphene-based frequency quadruplers have great potential to generate ultrahigh frequency signals and could find its roles easily in ultrahigh frequency electronic applications in near future.With potential ultrahigh carrier mobility and high saturation velocity, graphene can response to high frequency signals completely. Hence large RC time constant of the device structure is major obstacle to improvement of operation bandwidth of graphene based device. Therefore, performance of graphene based devices can be enhanced significantly if an optimized fabrication process is carried out to decrease both parasitic capacitance and output resistance. More clearly, in order to decrease the parasitic capacitance of the GFET-based frequency quadrupler, a highly resistive substrate such as quartz assisting with a local BG technique is preferred. On the other hand, both contact resistance and channel resistance make contribution to output resistance of GFET. A polymeric residue-free graphene fabrication processA multi-mode frequency multiplier. The zero band gap of graphene enables tunable electronic transport polarity. In this work, a narrow top gate was utilized to dope underneath graphene with electrostatic field, and a tunable transfer curve was achieved with one GFET. With the tunable transfer curve, GFET can work as high performance frequency doubler, frequency tripler and frequency quadrupler.High multiplication factor. Without any additional filter system, it is impossible to achieve a frequency multiplier with high multiplication factor by using traditional nonlinear electronic devices because output power of the high-order harmonic is much smaller than the low-order harmonic. In this work, a graphene based frequency quadrupler with about 50% power purity was realized with dual-gated GFET. Developing idea proposed in this work, a frequency multiplier with multiplication factor of 2 * N (N > 2) can be realized by fabricating N-1 top gates in the GFET channel. With superhigh carrier mobility of graphene, this graphene-based frequency multiplier will play an important role in the ultrahigh-frequency electronic applications, such as signal generator for THz.Low cost. The cost-effective high performance CVD graphene material can be available with rapid improvement of the synthetic technology. Simplicity of the GFET structure results in low fabrication cost. Furthermore, the demand of this multi-mode frequency multiplier increasing rapidly as communications would become indispensable nowadays.Here, we summarize potential superiorities that dual-gated GFET offers as a new device for RF applications.In summary, a novel dual-gated GFET based high performance frequency quadrupler is presented. Benefit from electrostatic doping effect of the narrow TG to the underneath graphene, a W-shaped transfer curve was obtained with only one GFET. With this new type nonlinear I-V feature, a graphene-based frequency quadrupler was achieved. This device can also operate as high performance frequency doublers and frequency triplers. To the best of our knowledge, it is the first reported multi-mode graphene based frequency multiplier in the world. The potential ultrahigh carrier mobility of the graphene, together with simplicity of the device structure and CMOS compatible fabrication processes makes the dual-gated GFET based frequency quadrupler one of the most captivating candidates for future ultrahigh-frequency electronics, especially RF applications.We characterized the fabricated device at room temperature in ambient conditions. The DC characteristics of the dual-gated GFET were measured by a Keithley 1602B semiconductor analyzer. Both Agilent 33250A signal generator and Agilent MSO-X 3034A mixed signal oscilloscope were used to obtain AC characteristics of the graphene based frequency quadrupler.How to cite this article: Cheng, C. et al. A graphene based frequency quadrupler. Sci. Rep.7, 46605; doi: 10.1038/srep46605 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


PD-L1 expression on TCs in primary tumors  and metastatic sites  was associated with poor chemo-response, represented by significant shortened DSS. These results suggest that PD-L1 may be a potential target being involved in chemo-resistance mechanisms and poses potential for therapy stratification in the future.Platinum-based chemotherapy is the standard of care in metastatic bladder cancer. With the approval of various checkpoint inhibitors, immunotherapy has revolutionized the traditional treatment modalities. The aim of the study was to evaluate whether PD-L1 expression on tumor cells (TCs) and tumor-infiltrating immune cells (ICs) can be used as biomarker to predict recurrence-free survival (RFS), overall survival (OS) and disease-specific survival (DSS) in bladder cancer patients after radical cystectomy (RC) developing disease recurrence followed by first-line chemotherapy. PD-L1 was measured on formalin-fixed, paraffin-embedded tissue sections of RC specimens in all patients (n=61) and in 27 matched metastatic biopsy samples by immunohistochemistry. PD-L1 expression on TCs was defined by the percentage of PD-L1 positive tumor cells , and was considered negative or positive for ICs. On 27 paired samples, IC1/2/3 score on TCs was homogeneous distributed with 59.3% in primary tumors and metastases, but with a high discordance rate of 44.4% of PD-L1 positivity on ICs. High PD-L1 expression (IC2/3) on TCs was more frequently seen in histologic subtypes of urothelial cancer compared to pure urothelial cancers (46.2%  Moreover, a high PD-L1 expression on TCs was associated with histologic subtypes of urothelial cancer (p=0.002).Interestingly, patients with histologic variants of urothelial cancer (median [95% CI] DSS 3.1 [0.6-5.6] months) responded less to platinum-based chemotherapy with a shorter DSS than those patients with pure urothelial cancer , Figure vs. low PD-L1 score: median [95% CI] OS 32.6 [17.9-47.2] vs. 14.4 [9.7-19.1] months; p=0.134), Figure The median OS after RC was confirmed with 25.5 95% CI 10.1-40.8) months in all patients, Figure 0.1-40.8 p=0.008), Figure p=0.019], histological variants of urothelial cancer  and lower OS.Focusing on histopathological factors, patients with histological subtypes of urothelial carcinoma (median OS: 11.1 months) showed to have a significant poorer OS than patients with pure urothelial carcinoma in the RC specimens  and pelvic LN dissection in localized MIBC, approximately 50% of patients will relapse on follow-up, depending on pathological tumor stage and LN status at RC specimens . CisplatThe high accessibility of bladder cancer to immunotherapy is well known since the introduction of BCG in 1976 , still bPrognostic significance of PD-L1 protein expression has been studied in many tumor entities  and its PD-L1 expression can occur on the TCs or on TILs . Thus, wMoreover, PD-1 expression correlated between primary tumors and metastatic lymph nodes with a concordance rate of 78% , withoutIn addition to PD-L1, other potential biomarkers including state of the art molecular subtyping, in depth TCR sequencing, somatic mutational density quantification, and identification of T cell-inflamed/non-T cell-inflamed tumor microenvironments using immune gene expression profiling are currently under investigation with promising results , 36, higet al (2016) in non-small-cell lung cancer patients undergoing cisplatin-based neoadjuvant chemotherapy [in vivo and in vitro [in-vitro findings, we are able to show that those patients with histological subtypes of urothelial cancer  confirmed no significant association between PD-L1 status and response to neoadjuvant or adjuvant chemotherapy in MIBC [et al (2016) [vs. 49.2%; p<0.001), [Here, we describe that higher PD-L1 expression on TCs in chemotherapy-naive RC specimens is correlated with poor clinical outcome in terms of DSS and OS in patients who developed metastatic disease and were homogeneously treated with first line platinum-based chemotherapy. In line with our results on the prognostic role of PD-L1 positivity on TCs it has been reported that PD-L1 expression was associated with a shorter survival after RC , 38–39, otherapy . Moreovein vitro . These rin vitro . In mammin vitro . Vice vein vitro . In bladin vitro , and thuin vitro . A possiin vitro . In linell known ) had siget al 20 in non-sl (2016)  and claul (2016)  were idel (2016) . In cont<0.001), . These c<0.001), –42.Our results must be interpreted with cautions, as several limitations exist: we included a relatively small sized patient cohort from a single-center institution with retrospective and observational study design. Concerning statistical limitations, there is lack of multivariate analyses due to multicollinearity, moreover, excluding those patients who developed recurrence and were followed-up elsewhere postoperatively may cause a selection bias. However, our findings of this study are clearly hypothesis generating, identifying a pathophysiological link between PD-L1 expression and therapy response. These preliminary findings are claiming for further validation in prospective, multicenter trials as well as for experimental studies analyzing gene expression profiling of tumors as well as of tumor microenvironment to get more insights how the immune phenotype can influence response to immunotherapy and chemotherapy.This study was approved by the local ethics committee of the Medical University Innsbruck (study number 1006/2017) and was performed according to the principles of the Declaration of Helsinki and its subsequent amendments . ReviewiAll 61 cystectomy specimens were reviewed for diagnosis, tumor grade (according to the WHO 1973 and 2004) and tumor stage  by two experienced uropathologists (BA and SS). Further pathology parameters such as the presence of lymphovascular invasion (LVI), surgical resection margins (R status), pelvic lymph node (LN) infiltration, total number of resected LNs, number of positive LNs, and variants of urothelial carcinoma  have routinely been reported. Of every case one to three representative blocks were selected for further analysis including superficial tumor, if available, invasive tumor and invasion front.We further compared the dynamic changes in PD-L1 expression of primary tumors and metastasis, and the corresponding chemotherapy response (DSS) and outcome. We assessed PD-L1 expression in 27 patients with matched biopsy tissues from metastasis  and RC samples.For detection of PD-L1 expression a monoclonal antibody against PD-L1 , which has been validated at the Division of Pathology, Medical University of Innsbruck, was used. Human placental tissue served as positive control.Staining was performed using an automated immunostainer . Briefly, formalin-fixed, paraffin-embedded (FFPE) tissue sections were cut in widths of 1.5 μM. After deparaffinization, the slides were treated with cell conditioning reagent 1  for antigen retrieval. Primary antibodies were incubated for 32 minutes at 37°C. The Ultra View DAB Detection Kit  was used for visualization, in accordance with the manufacture’s recommendation. Finally, slides were washed in distilled water, counterstained with hematoxylin (12 minutes) and bluing reagent (4 minutes), dehydrated in a descending order of alcohols, cleared in xylene, and coverslipped with Tissue-tek mounting medium .Analysis of PD-L1 staining was done separately for expression on TCs as well as on tumor-infiltrating ICs. Expression on TCs was evaluated as positive or negative and the exact percentage (%) of positive TCs was noted; in addition, a semi-quantitative score for the PD-L1 expression status on TCs in the tumor microenvironment was applied for statistical analysis and defined by the percentage of PD-L1-positive TCs as published previously : < 1%= SInflammation was scored as weak, intermediate and strong and the composition of the inflammatory infiltrate was described  . PD-L1 es). Dynamic changes in PD-L1 expression levels between primary tumor and metastatic sites were analyzed in matched pair analyses with marginal homogeneity test and McNemar test, separately for expression levels in tumor cells and immune cells. Recurrence-free survival (RFS), disease-specific survival (DSS), and overall survival (OS) were investigated through Kaplan-Meier survival analysis, survival curves were plotted, and the influence of various factors on survival was tested using log-rank tests. For all statistical tests, a significance level of α=0.05 (two-tailed) was applied. SPSS, version 22.0  was used for statistical analysis.All patient-, clinico- and histopathologic characteristics as well as PD-L1 expression levels were analyzed descriptively by giving frequencies or means, standard deviations, and medians, as appropriate. Categorical variables were compared in bivariate analyses using Fisher’s exact test. Correlations between various PD-L1 expression levels were assessed using Spearman’s ρ correlation coefficient  and subsequent metastasis (44.4%). The most stable parameter was the PD-L1 score on TCs, with homogeneous PD-L1 positivity in both primary tumors and metastatic sites. This important pathological finding suggests that PD-L1 can either be determined in the primary tissue as well as in metastasis and implicates similar biological behavior of primary and metastatic tumor. High PD-L1 expression was associated with histologic subtypes of urothelial cancers and had a negative predictive value for response to first-line chemotherapy. Furthermore, high PD-L1 expression on TCs was also a negative prognostic marker for DSS and OS. Thus, PD-L1 may be a potential target in predicting response to chemotherapy in metastatic bladder cancer, being a possible promising strategy for individualized targeted therapy with selection of patients that mostly benefit from chemotherapy or immunotherapy. Further trials are necessary to prove these preliminary data and to elaborate mechanistic aspects of chemo-resistance and PD-L1 overexpression in bladder cancer.


Immunohistochemistry , PD-L1 mRNA in situ hybridization (ISH), microsatellite instability (MSI), and EBV ISH were performed in 392 stage II/III GCs treated with curative surgery and fluoropyrimidine-based adjuvant chemotherapy, and two public genome databases were analyzed for validation. PD-L1+ was found in 98/392 GCs (25.0%). The proportions of immune types are as follows: PD-L1+/CD8High, 22.7%; PD-L1−/CD8Low, 22.7%; PD-L1+/CD8Low, 2.3%; PD-L1−/CD8High, 52.3%. PD-L1+/CD8High type accounted for majority of EBV+ and MSI-high (MSI-H) GCs , and genome analysis from public datasets demonstrated similar pattern. PD-L1−/CD8High showed the best overall survival (OS) and PD-L1−/CD8Low the worst (P < 0.001). PD-L1 expression alone was not associated with OS, however, PD-L1−/CD8High type compared to PD-L1+/CD8High was independent favorable prognostic factor of OS by multivariate analysis (P = 0.042). Adaptation of recent molecular classification based on EBV, MSI, E-cadherin, and p53 showed no significant survival differences. These findings support the close relationship between PD-L1/CD8 status based immune types and EBV+, MSI-H GCs, and their prognostic significance in stage II/III GCs.We co-assessed PD-L1 expression and CD8 Helicobacter pylori and Epstein-Barr virus (EBV) infection has been investigated [Gastric cancer (GC) is the fifth most common cancer worldwide , the thistigated , 5, and stigated .+/TILHigh, adaptive immune resistance), type II , type III , and type IV  [CD8A expression as a surrogate marker for TILs, revealed significant association between TMIT I (HighPD-L1/HighCD8A) and features like high mutational burden and oncogenic viral infection, suggesting the clinical relevance of this classification [Clinical trials of immune checkpoint inhibitors have shown favorable outcomes in some solid tumors, including GC –9. Curreof TILs) . Though of TILs) , a comprfication .+) cytotoxic T cells, is important for the action of immune checkpoint inhibitors [+) GCs and MSI-high (MSI-H) GCs are frequently accompanied by heavy infiltration of TILs [TP53 mutations, could be used for further molecular classification [Recent studies suggest that the type of TILs, especially CD8-positive  determine the association between TMIT and clinicopathologic features, especially prognostic significance, in stage II and III GCs, as well as the molecular subtypes of GCs, specifically EBV and MSI status, (ii) validate the findings by analysis of publicly available genomic datasets, and (iii) suggest potential biomarkers for better patient selection for immune checkpoint inhibitor therapy.Considering the importance of both PD-L1 expression and CD8ironment –13, we c+ TILs ranged from 6.90 cells/mm2 to 1374.94 cells/mm2 with the median value of 195.23 cells/mm2. The number of Foxp3+ TILs ranged from 1.22 cells/mm2 to 785.88 cells/mm2 with the median value of 60.12 cells/mm2.The baseline clinicopathologic characteristics of the study population are shown in Table PD-L1 mRNA overtranscription (a PD-L1 mRNA ISH score of 4+) was detected in 14 samples (3.6%). When PD-L1 IHC and mRNA ISH were compared, all cases with mRNA ISH score of 4+ were PD-L1 IHC positive, and the correlation coefficient between the 2 tests was 0.467, which was statistically significant at the 0.01 level (PD-L1 IHC was positive in 98 samples (25.0%), and 01 level . Altered+ TIL density , 89 (22.7%); type II (PD-L1−/CD8Low), 89 (22.7%); type III (PD-L1+/CD8Low), 9 (2.3%); and type IV (PD-L1−/CD8High), 205 (52.3%). Type I showed more male predominance than the other types (P = 0.021), and AJCC 7th pT stage was significantly associated with TMIT (P = 0.004). In addition, type I was associated with Foxp3High status, and type II was associated with Foxp3Low status (P < 0.001). No other significant associations between other features and TMIT classification were observed.We categorized the study population into TMITs I–IV based on the results of PD-L1 IHC and CD8+ (group 1), MSI-H (group 2), microsatellite stable (MSS)/MSI-low (MSI-L)/EMT-like (group 3), MSS/MSI-L/p53-IHC+ (group 4), and MSS/MSI-L/p53-IHC− (group 5). Of the 392 patients, 25 were in group 1 (6.4%), and 36 were group 2 (9.2%); none of the EBV+ GCs showed an MSI-H phenotype, and vice versa. The number of patients in groups 3, 4, and 5 were 105 (26.8%), 73 (18.6%), and 153 (39.0%), respectively.Next, we modified and adapted previously described molecular classification models for GC , 18 in oHigh group had significantly better overall survival (OS) than the CD8Low group  had the best OS, and type II (PD-L1−/CD8Low) had the worst OS  were compared, type IV (PD-L1−/CD8High) had better OS, with marginal statistical significance (P = 0.070). However, according to the molecular classification, no significant survival differences were detected among the 5 groups , and TMIT IV showed the best DFS while TMIT II was associated with the worst DFS (P < 0.001).In addition, we performed survival analysis of disease free survival (DFS) , which s+ TILs, Foxp3+ TILs, and TMIT IV are the key clinicopathologic features that are significantly associated with OS  were type I (PD-L1+/CD8High); none of the EBV+ GCs were CD8Low, and only 2 (8.0%) EBV+ GCs were PD-L1−. Similarly, MSI-H GCs (group 2) also had a distinct relationship with TMIT I; 26 of 36 (72.3%) MSI-H cases were PD-L1+, and 24 cases (66.7%) were classified as TMIT I. Within group 3, only 4 of 105 (3.8%) cases were TMIT I, and the proportion of TMIT II cases was relatively high . In groups 4 and 5, the proportion of each TMIT was similar to that from the whole study population.To determine the implications of the molecular classification from an immune microenvironment perspective, we compared TMIT and molecular classification. The relationship between the 2 classifications is shown in Table + or MSI-H GCs, we performed analysis of the genomic dataset from TCGA and SMC cohort. As shown in Figure + stomach adenocarcinomas in both datasets were classified as TMIT I (81.1% in TCGA and 88.9% in SMC). Genomic analysis according to MSI status showed that, in accordance with the findings from our tissue samples, most of the MSI-H cases were TMIT I (70.5% in TCGA and 76.5% in SMC), followed by type IV, II, and III . We found that TMIT I (PD-L1+/CD8High) is closely correlated with EBV infection and MSI-H phenotype than TMIT IV (PD-L1−/CD8High). Additionally, to validate our results, we analysed datasets from TCGA [+ and MSI-H cases in the both datasets were likely to be type I (PD-L1High/CD8AHigh).In this study, we classified a large cohort of stage II and III GC patients who were managed with standard treatment into one of four TMITs, using immunohistochemical assessment of PD-L1 expression and CD8rom TCGA  and the rom TCGA . The res+ and MSI-H GCs [+ GCs and MSI-H GCs are associated with heavy lymphocytic infiltration [+ T cell-infiltrated GCs are associated with PD-L1 expression [+/CD8High) with EBV+ and MSI-H, compared to type IV (PD-L1−/CD8High), using both tissue samples and genomic analysis. TMIT I status (PD-L1+/CD8High) implies the adaptive immune escape responses, and based on many previous studies, there is a good chance that GCs with this signature can be reversed by immune checkpoint blockade [+/CD8High) TMIT could serve as a biomarker for a good response to immune checkpoint inhibitors, and that PD-L1 and CD8 TIL status should be evaluated in patients with EBV+ or MSI-H GC.Numerous studies have shown that PD-L1 expression is increased in both EBVSI-H GCs –22. Likeltration , 24. Howpression . Here, wblockade , 25. TheHigh group differs according to the differential expression of PD-L1; type I (PD-L1+/CD8High) showed significantly poorer OS than type IV (PD-L1−/CD8High) by multivariate analysis. From this we could infer that although heavy immune cell infiltration might play the favorable anti-tumor effect in gastric cancer, effective immune evading occurs by expression of PD-L1, possibly resulting in decreased OS. Since PD-L1 expression alone failed to discriminate survival in the total study population, the significant survival difference elucidated by differential PD-L1 expression in the CD8High group strongly suggests that the clinical implication of PD-L1 expression could become more meaningful when interpreted in combination with other components of the TME. Therefore, we suggest co-assessment of both PD-L1 and CD8+ TILs as a useful way of defining the TME, which also has a significant prognostic role in stage II and III GC.In addition, we also found that the TMIT has prognostic value. TMIT II, which implies the immune ignorant state of tumor microenvironment, shows worse survival outcome compared to highly inflamed status (types I and IV), and this finding is consistent with previous studies from diverse tumor types including GC , 23. EvePrevious studies on the prognostic role of PD-L1 expression in GC showed conflicting results. For example, the most recent study of a large Caucasian cohort of GC showed that PD-L1 expression in tumor and stromal immune cells was associated with better tumor-specific and overall survival , while p−/CD8High) from type I (PD-L1+/CD8High) GCs. The characteristic immune microenvironment of type IV is activation of the immune-suppressing mechanism other than PD-L1. Therefore, we assessed the role of Tregs in type IV GCs by measuring Foxp3 expression by IHC. Contrary to our assumption, the proportions of Foxp3High and Foxp3Low within the type IV group were approximately equal, and analysis of TCGA and SMC datasets did not show discernible high expression of Foxp3 in type IV, suggesting that the immune tolerance mechanism in type IV cannot be solely explained by Tregs. Further studies on the various components of the TME in the type IV group, such as tumor-associated macrophages or myeloid-derived suppressor cells should provide a deeper understanding of this topic.Next, we assessed the characteristics that distinguished type IV  and 5 (MSS/non-EMT-like/ p53-IHC−) were compared according to classification, the proportions of the 4 TMITs within these 2 groups were similar to the proportions in the total population; thus, no distinct immuno-oncologic features according to p53 overexpression were observed.Since the introduction of molecular subtypes of GC in TCGA study, EBVsubtypes . Yet, de and p53 , which w and p53 , 33. How+ is still a matter of debate; applying different cut-off level for PD-L1 IHC results would inevitably result in different proportions among the TMIT subtypes. However, since there is no general consensus in this topic till nowadays, we have done thorough review of previous studies in pursuit of identifying an ideal cut-off criteria for PD-L1 IHC, and chose our criteria referenced from the most recent studies of GC [+ GC and MSI-H GC. This can be attributable to the limitation of IHC itself; we have interpreted p53 IHC into either positive or negative, however, this may not reflect the actual TP53 gene mutation status accurately. Thus, more precise techniques for assessing genetic mutation and gene expression levels should be warranted in future studies.This study has the limitation of being a retrospective study at a single institution. However, compared to other studies, our study population is a large, relatively homogeneous cohort with restricted confounding factors. The cut-off value for PD-L1es of GC , 25. In es of GC . Therefoes of GC  to ensur+ T cell infiltration. We found that EBV+ and MSI-H GCs are distinct subtypes that are tightly associated with TMIT I (PD-L1+/CD8High), and OS within the CD8High group differs according to PD-L1 expression. Therefore, we conclude that co-assessment of PD-L1 and CD8+ TILs is clinically relevant, has a possible prognostic role, and warrants further investigation as a predictive marker for immune checkpoint blockade.In summary, this is the first study to classify a large homogeneous cohort of stage II and III GCs into 4 immune types by co-assessment of 2 key components of TME, PD-L1 expression and CD8A total of consecutive 406 patients with stage II or III GC who were treated in Seoul National University Bundang Hospital  from 2006 to 2013 were screened for inclusion. Among them, the tumor tissue samples of 14 patients were found inadequate for immunohistochemistry, thus excluded . All 392Surgically resected GC specimens from patients were formalin-fixed and paraffin-embedded (FFPE). In all cases, one representative 2-mm core was selected from the invasive margin of the tumor by 2 experienced gastrointestinal pathologists (S.Y. and H.S.L.), and tissue microarrays (TMA) were constructed as described previously  .All human FFPE tissue samples were obtained from the archive of the Department of Pathology, Seoul National University Bundang Hospital. This study was approved by the institutional review board (IRB) of Seoul National University Bundang Hospital (IRB number: B-1606/349-308). Written patient consent and the consent process were waived by the IRB.IHC for CD8, Foxp3, E-cadherin, p53, and PD-L1 were performed with an automatic immunostainer , according to the manufacturer's instructions. The IHC antibodies used in this study were as follows: CD8 ; Foxp3 ; E-cadherin ; p53 ; and PD-L1 . EBV ISH was performed with the INFORM EBV-encoded RNA (EBER) probe .+ and Foxp3+ cell densities (positive cell counts per mm2) in each core of TMA were counted by an Aperio image analysis system . The CD8High and CD8Low groups were defined using the 25th percentile as the cut-off value, and median value was used as the cut-off for Foxp3.To interpret the CD8 and Foxp3 staining, immunostained TMA slides were scanned, and the CD8All other immunostaining was interpreted by 2 pathologists (J.K. and H.S.L.) who were blinded to patient characteristics at the time of interpretation. Membrane staining of PD-L1 on more than 5% of tumor cells was interpreted as positive , 25. ForPD-L1 mRNA on the tissue microarray, the PD-L1 RNAscope 2-plex detection kit  was used according to the manufacturer's guidelines. The results were interpreted by 2 pathologists (J.K. and H.S.L.) according to the instructions in the RNAscope FFPE Assay Kit and were scored as described previously [PD-L1 overtranscription.To detect eviously : 0, no sMSI status was assessed by comparing the allele profiles of 5 markers  in tumor cells to those in matched normal samples. The polymerase chain reaction (PCR) products from the FFPE samples were analysed with a DNA autosequencer  according to a previously described protocol .http://genome-cancer.ucsc.edu) on June 3, 2015, which included clinical information and mRNA expression data obtained by RNAseq (Illumina HiSeq V2 platform) of TCGA samples. The mRNA expression data were presented as reads per kilobase per million (RPKM) and were transformed into log 2 values for the analysis. MSI status was available for 414 stomach adenocarcinoma (STAD) samples, and EBV status was referenced from TCGA clinical data.We used the publicly available level 3 data from TCGA downloaded from the UCSC Cancer Browser  shared by Cristescu and colleagues  .For application of the TMIT classification to the genomic data, after merging the log 2-transformed RPKM values of PD-L1 mRNA ISH. Kaplan-Meier analysis of OS and DFS according to TMIT and molecular classification was performed, and the significance of survival differences was assessed by the log-rank test. A P-value less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS statistics 22.0 , with the exception of the genomic analysis and data presentation which were performed using the R statistical package 3.1.3 (http://www.r-project.org).The associations between clinicopathological characteristics and TMITs were analysed by Chi-square, linear-by-linear, Kruskal-Wallis, and Wilcoxson/Mann-Whitney tests, if appropriate. Spearman rank correlation was used for the correlation analysis between PD-L1 IHC and