This was achieved utilizing electron-beam bombardment and following developing processes, prior to the use of conventional organic solvents. Field-effect transistors were fabricated on the transferred graphene in order to explore their Dirac points and carrier motilities in the ambient condition - the results were then compared with those from the conventional wet chemical treatment. It was found that the Dirac points were located close to the zero gate bias when compared to those from the acetone and the acetic acid treatments. Most significantly, the field-effect mobility reached as high as 6770 cm Graphene was first obtained by mechanical exfoliation from graphite, and, latterly, by chemical vapor deposition (CVD) using a variety of metals such as Copper (Cu), Nickel (Ni)13 - CVD is the most reliable synthesis method and has the advantage of high quality and high productivity on an industrial scale. In particular, the graphene grown by CVD on a Cu foil is widely used because it is cheap and the growth of a single layer graphene is relatively easy13.Two-dimensional materials, such as graphene, have attracted significant interest for their unique electrical, mechanical, and optical properties, and therefore, have a potential use in various fields such as field-effect transistors (FETs)18. This is typically performed using a supporting layer such as poly methyl methacrylate (PMMA) and after the transfer process, the PMMA on the graphene surface will be cleaned using an organic solvent such as acetone19. However, because of the strong interaction between the PMMA and the graphene, its residues will inevitably be left at the graphene surface, even after the cleaning processes21. Alternative transfer techniques have been proposed to achieve high-quality graphene as summarized in the recent literature22; however, most of them suffer from residues or degradation during the removal procedures of the supporting layers. Graphene is extremely sensitive to adsorbates and molecules in contact with its surface, hence, the residues tend to act as a dominant source of doping and scattering of charge carriers, degrading the electrical properties of graphene23. Therefore, the thorough removal of the PMMA residues is crucial for improving the electrical and optoelectronic characteristics of graphene devices. Various wet chemical treatments such as acetic acid, chloroform, and formamide solution have been proposed to reduce these residues, as an alternative to the conventional acetone solution26. More recently, advanced techniques have been introduced, including thermal annealing27, current induced annealing28, laser cleaning treatment29, Oxygen (O2) plasma treatment31, and ultraviolet (UV) ozone treatment34, However, they contain rather complicated processes and some of them suffer from the lack of reproducibility. Moreover, their field-effect mobility remains mostly at 3000–4000 cm2/Vs which has been the upper limit of the devices fabricated from the CVD-grown graphene so far35.In order to fabricate the FETs, graphene grown on Cu foil, using the CVD method, is required to be transferred to the Silicon (Si) substrateThis research proposes a new approach for the effective removal of the PMMA that has been used for transferring the graphene onto silicon substrates, based on the fact that electron bombardment is best suited for removing the PMMA by its nature. The FET devices based on the transferred graphene were fabricated, with which the Dirac points and the field-effect motilities were measured and then compared with the devices from the conventional removal techniques. It was found that the e-beam treatment (ET) provided the superior device performance relative to those found in the literatures.38. After the Cu foil was fully dissolved in an ammonium persulfate solution, the PMMA/graphene film was transferred onto the silicon substrate.A schematic of the experimental approach used in this research is illustrated in Fig. 2 and current of 500 pA at 25 kV, was irradiated for 20 s through the region (50 × 200 μm2) that encompasses more than 6 devices. The exposed region of the PMMA was then removed by the developing process, which featured methyl isobutyl ketone (MIBK) 3:1 solution and lasted for 3 minutes , followed by the developing processes. The condition for the e-beam irradiation was same as conventional lithographic procedures, except that the exposure area was relatively large, with a large beam current, enabling us to remove the PMMA residues over many devices, in a relatively short time. An electron-beam, with the areal dose of 100 μC/cm24. These methods have proven to be very effective in removing the PMMA residue. For the AO process, the sample was submerged in the acetone solution for 24 hours at room temperature. Conversely, during the AA process, the sample was dipped in acetone solution for 1 hour and then immediately immersed in acetic acid for 24 hours at room temperature.To elucidate the effect of the ET proposed in this research, two different processes were added for comparison – acetone overnight (AO) and acetic acid (AA) methods19. The spectra exhibit two distinct peaks at 1597 cm−1 (G) and 2695 cm−1 (2D), whereas the D peak at 1352 cm−1 is suppressed. Clearly, the intensity of 2D peak varied with different PMMA removal techniques, whereas we could not observe a noticeable frequency shift relative to each other. As summarized in Fig. I2D/IG is highest for ET process, yielding 2.05 on average, as compared to the AO (1.66) and AA (1.78) processes. In general, 2D intensity is suppressed in the presence of PMMA residues at the graphene surface (Supplementary Information 39 and I2D/IG depends strongly on the carrier density (or the Fermi energy) in graphene40. Therefore, the increase in I2D/IG is a strong indication that the PMMA residues have been removed more effectively though our novel technique. We also note that ID did not change significantly with the ET procedure, indicating that it did not induce the noticeable defect in graphene. This was also consistent with the root-mean-square surface roughness (Rq) characterized from the AFM images41. As shown in Fig. Rq for ET sample is 0.336 nm, which is lower than AO (0.448 nm) and AA (0.476 nm) cases. We also tested with more than 10 samples, yielding the average Rq values of 0.380, 0.421, and 0.446 nm, respectively for ET, AA, and AO samples.In Fig. I-VG characteristics of the devices were measured in the back-gate geometry for the three different PMMA removal procedures as shown in Fig. VDS = 10 mV. For devices with AO and AA processes, the gate voltage (VG) was swept from −80 V to 80 V, with sweep speed of 5 V/s, and for ET processes, the gate voltage was swept from −40 V to 40 V, again with a sweep speed of 5 V/s. All measurements were taken in the ambient condition. For all of the devices, the switching behavior with the clear Dirac point located in the region of −80 V < VG < 80 V could be identified. The Dirac point of the AA sample was found at VG = 44.5 V, which is closer to VG = 0 V in comparison to that of the AO treated device, which was located at VG = 68.0 V. At this point, an average value was evaluated for the two Dirac points obtained with different sweeping directions. The shift in Dirac points for AA devices, in comparison to AO devices, is likely to be due to the reduction in PMMA-induced doping effects, considering that PMMA residue causes p-type doping in the graphene as mentioned above39. Importantly, the Dirac point approaches closer to VG = 0 V for ET samples, as shown in Fig. VG = 11.3 V, on average. This is a strong indication that a greater amount of PMMA was removed using e-beam bombardment and the following developing procedures, ultimately proving the usefulness of the approach proposed in this research.The μFE is graphene field-effect mobility, Wch are channel length and width, respectively, and μh = 1000 cm2/Vs for the AO device, and, in the case of the AA device, the hole and electron motilities reached as high as μh = 2580 cm2/Vs and μe = 1590 cm2/Vs, respectively. However, those in the ET sample in Fig. μh = 12440 cm2/Vs and μe = 12640 cm2/Vs, for the hole and electron carriers, respectively. The significant increase in the mobility upon the removal of the PMMA can be attributed to the decrease in carrier scattering23.More significantly, the transconductance increased dramatically for the ET devices, strongly suggesting that the device mobility is much higher in the ET samples. The mobility can be extracted from the following relationship,qeff) responsible for the voltage shift  and seven times that of the AO devices . Similarly, electron mobility is shown to be as large as 7350 cm2/Vs for the ET devices, four times that of AA devices (1850 cm2/Vs). Surprisingly, the maximum values obtained in this research yields were as large as ~17000 cm2/Vs (similar for both electron and hole), which is unprecedentedly high for the CVD-grown graphene, among those measured in the ambient condition.The increase in field-effect mobility is illustrated in Fig. μh ~ 4700 cm2/Vs), however, these techniques could either induce defects to the graphene or suffer from the lack of reproducibility33. Conversely, the techniques proposed in this research are based on the common knowledge that electron bombardment is best suited for removing the PMMA by its nature. It has been suggested that the entangled polymer chain on the graphene cannot be easily removed by the conventional solvents26; however, electron beam irradiation will induce the scission of the polymer chains, resulting in the effective removal of the PMMA residues26. In particular, our method has the advantage of removing the interaction between PMMA and the graphene surface, which is free from creating additional defects and contaminants. Thus, it was beneficial in helping to obtain a higher carrier mobility and move the Dirac point closer to a zero-gate bias even in the ambient condition. In addition, our technique requires relatively short periods of time and provides high mobility values.Finally, we summarize in Table 2/Vs and 7350 cm2/Vs on average for holes and electrons, respectively, with the maximum values of up to 17000 cm2/Vs for hole mobility. This is greater than a seven time improvement in comparison to conventional acetone treatment, in terms of the average value, which is superior to other approaches reported for CVD grown graphene devices. Our work will open the door to the development of optimal procedures for fabricating future functional devices based on various two-dimensional materials.In conclusion, an efficient technique was developed for removing PMMA layers at the graphene surface using electron beam bombardment. In this research, the FETs were fabricated with improved electrical properties on the transferred graphene after e-beam treatment. For instance, the Dirac point of the devices fabricated from this method was located very close to the zero-gate bias, in comparison to those from the acetone and the acetic acid treatments. Increased 2D peak in Raman spectra for the e-beam processes confirms the effective removal of the PMMA residues. More significantly, the field-effect mobility reached as high as 6770 cmGraphene was prepared utilizing the CVD method on a 25 µm Cu foil  using methane and hydrogen gases. PMMA was employed as a graphene carrier from the Cu foil onto a Si substrate with an oxide layer of thickness 220 nm and heavily doped p-type Si layers of thicknesses 550 µm (the resistivity was 0.001–0.003 Ω/cm). The Si substrate contained the drain and source electrodes (Cr/Au) fabricated previously with conventional lithography techniques. After the Cu foil was fully dissolved in an ammonium persulfate solution, the PMMA/graphene film was transferred onto the silicon substrate.2 and current of 500 pA at 25 kV, was irradiated for 20 s through the region (50 × 200 μm2) that encompasses more than 6 devices. The exposed region of the PMMA was then removed by the developing process, which featured methyl isobutyl ketone (MIBK) 3:1 solution and lasted for 3 minutes. After the developing, PMMA residues were removed thoroughly by dipping the sample in acetone solution for 2 hours. Besides from the ET process, two conventional processes (AO and AA processes) were added for comparison. For the AO process, the sample was submerged in the acetone solution for 24 hours at room temperature. Conversely, during the AA process, the sample was dipped in acetone solution for 1 hour and then immediately immersed in acetic acid for 24 hours at room temperature. After the PMMA removal processes , graphene patterns were generated (width ~1 µm and channel length of 7–10 µm) using the electron-beam lithography technique with negative e-beam resist (Ma-N 2401), followed by reactive ion etching.After the transfer process, the PMMA layer was removed using electron-beam bombardment (ET process), followed by the developing processes. An electron-beam, with the areal dose of 100 μC/cmOur homemade micro-Raman spectroscopy equipment consists of a 532 nm DPSS laser, spectrometer (Andor Shamrock 303i), and CCD (Andor iDus401A). An objective lens  was used to focus the laser with resolution of ~1 μm with the laser intensity at 10 mW.Supplementary Information


In this work, we developed an interface modified by graphene to simultaneously improve the mechanical and frictional properties of carbon/graphene/carbon (C/G/C) composite. Results indicated that the C/G/C composite exhibits remarkably improved interfacial bonding mode, static and dynamic mechanical performance, thermal conductivity, and frictional properties in comparison with those of the C/C composite. The weight contents of carbon fibers, graphene and pyrolytic carbon are 31.6, 0.3 and 68.1 wt %, respectively. The matrix of the C/G/C composite was mainly composed of rough laminar (RL) pyrocarbon. The average hardness by nanoindentation of the C/G/C and C/C composite matrices were 0.473 and 0.751 GPa, respectively. The flexural strength (three point bending), interlaminar shear strength (ILSS), interfacial debonding strength (IDS), internal friction and storage modulus of the C/C composite were 106, 10.3, 7.6, 0.038 and 12.7 GPa, respectively. Those properties of the C/G/C composite increased by 76.4%, 44.6%, 168.4% and 22.8%, respectively, and their internal friction decreased by 42.1% in comparison with those of the C/C composite. Owing to the lower hardness of the matrix, improved fiber/matrix interface bonding strength, and self-lubricating properties of graphene, a complete friction film was easily formed on the friction surface of the modified composite. Compared with the C/C composite, the C/G/C composite exhibited stable friction coefficients and lower wear losses at simulating air-plane normal landing (NL) and rejected take-off (RTO). The method appears to be a competitive approach to improve the mechanical and frictional properties of C/C composites simultaneously. Carbon/carbon (C/C) composites have been widely used as structural and braking materials in the aerospace industry ,2,3,4,5.Graphene, a recently discovered carbon material, possesses a two-dimensional structure and exhibits extraordinary mechanical , electriIn the work reported here, we take advantage of the excellent weaving properties of polyacrylonitrile-based carbon fibers (PANCFs) to improve the preform of C/C composites ,18. We d3. The size of the preforms was Φ = 100 mm × 15 mm.Polyacrylonitrile carbon fiber (12k PANCF)  preforms were constructed from layers (0° and 90°) of non-woven long fiber cloth and short-cut fiber web and then needle-punched. The preforms showed a density of approximately 0.55 g/cm3, and 200 mL of H2SO4 were mixed together in an ice bath for 30 min. Then, 12 g of KMnO4 was slowly added into the mixture while stirring, and the rate of addition was controlled in the ice bath. The mixture was then transferred to a 30 °C water bath and stirred for about 30 min. Subsequently, 200 mL deionized water was added dropwise into the mixture with stirring. After 15 min, the mixture was further treated with 700 mL of deionized water and 60 mL of 30% H2O2 solution. The solution was then filtered and subjected to wash with deionized water until the pH value of reached 6. GO suspension was collected in a seal vessel.Graphene oxide (GO) was synthesized according to the literature methods ,20. In av/v) concentrated H2SO4 and HNO3 and then sonicated for 4 h at 60 °C. Then, the carbon fibers were taken out and washed several times with deionized water until the pH of the wash water was neutral and then dried under vacuum; (2) The GO (1 g) was placed in a round-bottomed flask (500 mL) and dispersed in deionized water for 1 h; (3) The preforms were placed in the round-bottomed flask for 12 h at 25 °C under sonication and then dried at 80 °C under vacuum; (4) The preforms were heat-treated at 1100 °C for 2 h; (5) After grafting of the graphene sheets on the surface of the carbon fibers, the preforms were densified using natural gas (mainly contain CH4) and C3H8 through CVI at 1080–1100 °C with total pressure of 1–5 kPa. The mass ratio of natural gas:C3H8 was 2:1  versus ln(σi); here, m represents the slope of the fitted straight line and σ0 is the flexural strength of the composites. The analysis results of the Weibull parameters (m and σ0) are summarized in 0) [0 values of the C/C and C/G/C composites are 110 and 188 MPa, respectively. The average flexural strength of the C/G/C composite is 187 ± 12 MPa, much higher than that of the C/C composite (106 ± 10 MPa). Compared with untreated carbon fiber, the flexural strength of functionalized carbon fiber composites was increased by 76.4%, which was higher than that of previous studies [ed in 0) ,47. The  studies ,49,50.ILSS tests were conducted on 6 samples of each composite. The ILSS results of C/C and C/G/C composites are shown in In W is the maximum stored elastic energy per unit volume.The internal friction of the material could be expressed as the following Equation (10):d is the fiber diameter, ∆σ is the fatigue stress range, EF is the elastic modulus of fiber, ME is the elastic modulus of matrix, and τ is the IDS.After assuming only defects of matrix cracks and corresponding interfacial debonding in fibers reinforced composites without broken fibers, the friction work of one load cycle can be estimated by the Equation (11) ,52(11)∆The IDS depends on the material and structure of the interface, is the only factor related to the interface. This characteristic indicates that the fiber/matrix interface bonding of the composites exerts significant effects on their properties. In general composite materials, the temperature increase not only reduces or eliminates thermal stresses in the fibers and matrix, leading to enhancements in the bonding of microcracks in the interface and matrix. In addition, the number of some debonded fiber–matrix regions around matrix cracks are reduced, and this phenomenon would increase attrition energy loss ,55.By contrast, in the C/G/C composite, graphene sheets are grafted onto the fiber surface, forming a new hierarchical structure with increased carbon fiber surface roughness. As the temperature increases, the internal friction of the C/G/C composite remains nearly unchanged because of improvements in the fiber/matrix interface bonding strength of the composite. This result indicates that graphene significantly affects the evolution of internal friction with temperature.Dynamic modulus refers to the capability of a material to resist strain under external loading and strongly depends on the integrity and continuity of materials . The temThe mechanical properties of the C/G/C composites in comparison with those of the C/C composites are listed in X–Y and Z directions. The thermal conductivities of both composites in the X–Y and Z directions initially increase and then subsequently decrease with increasing temperature. Under the same temperature level, the thermal conductivity of the C/G/C composites is higher than that of C/C composites in both directions at 50–900 °C.X–Y and Z directions are higher than those of the C/C composite by 13.3%–19.8% and 37.7%–176.3%, respectively, at 50–900 °C. This result may be attributed to the easy formation of a percolation channel of graphene sheets on the carbon fibers and the function of graphene sheets as a bridge in the interfacial area and pyrocarbon matrix.As shown in In composites, the thermal conductivity λ is proportional to the outcome of the phonon mean free path, the speed of sound and the heat capacity. Where the phonon contribution to the thermal conductivity dominates in composites ,59. And The results indicate the potential superiority of the C/G/C composite over the C/C composite in terms of thermal conduction. Therefore, research on the thermal properties of the C/G/C composites may help improve the current understanding on the heat transfer way of graphene grafted onto carbon fibers and promote the use of their prominent thermal conduction properties .In the NL and RTO braking condition, the friction coefficient curves of both composites are shown in While the temperatures of both composites considerably increase with increasing braking energy, their values are relatively different in In the NL and RTO braking condition, SEM morphologies of the worn surfaces of both composites are shown in A simple and effective strategy is developed to prepare C/G/C composites. It demonstrates that microstructure, mechanical, thermal and frictional properties of C/G/C composites are strongly influenced by fiber/matrix interface modified by graphene. PLM, SEM and Raman spectra show that graphene grafted on fiber can modify the pyrocarbon matrix by strongly affecting the deposition behavior of pyrolytic carbon during CVI. Mechanical tests verify that graphene sheets in fiber/matrix interface are more beneficial to obtain higher flexural strength, interlaminar shear strength, and storage modulus in C/G/C composite than that of C/C composites without graphene. Owing to the self-lubricating properties of graphene, the highly textured matrix of the modified composite shows lower hardness and good fiber/matrix interface bonding strength in comparison with those of the unmodified composite. A complete friction film could be easily formed on the friction surface of the modified composite, resulting in stable friction coefficients and lower wear losses at both braking levels tested. According to the surface temperature of both composites at the NL and RTO braking levels, the C/G/C composite exhibits better thermal diffusion properties than the C/C composite. This result is consistent with our analysis results of thermal conductivity. This new method of fabricating C/G/C composites proposed in this work offers new possibilities in the field of aviation materials.


Since 2004 the field of graphene research has attracted increasing interest worldwide. Especially the integration of graphene into microelectronic devices has the potential for numerous applications. Therefore, we summarize the current knowledge on this aspect. Surveys show that considerable progress was made in the field of graphene synthesis. However, the central issue consists of the availability of techniques suitable for production for the deposition of graphene on dielectric substrates. Besides, the encapsulation of graphene for further processing while maintaining its properties poses a challenge. Regarding the graphene/metal contact intensive research was done and recently substantial advancements were made towards contact resistances applicable for electronic devices. Generally speaking the crucial issues for graphene integration are identified today and the corresponding research tasks can be clearly defined. Besides1.1−6 K−1) [−6 K−1) [The CVD growth substrate can either be a copper foil, which is most commonly used, or a Cu film deposited by physical vapor deposition (PVD) on a silicon wafer substrate with a diffusion barrier between the silicon substrate and the Cu film. The first approach allows for the production of very large graphene films on a polymer carrier foil even in a roll-to-roll system –9. The s−6 K−1) –12 and t[−6 K−1)  typicall[−6 K−1) –18, but In history, the first transfer technique for graphene films from the CVD growth substrate was the ex situ transfer by reinforcing the graphene layer with a polymer film, e.g., poly(methyl methacrylate) (PMMA), and etching off the Cu growth substrate. There are several options for subsequent graphene deposition onto the final substrates discussed in  Figure .Focusing on wafer-level processes, the metal underetch times are rather long, even when the graphene film is patterned to provide distributed access for the etch medium. A technique that copes with this problem is the so-called “bubble transfer”, which uses the generation of electrochemically generated hydrogen bubbles at the graphene–metal interface to release the polymer-supported graphene film without the need of lengthy underetching –21. Neve2 (450 mJ/m2) [After release of the polymer-reinforced graphene film from the growth substrate the graphene is contacted with the dielectric device substrate, either using a liquid medium (“wet transfer”)  or a fle0 mJ/m2)  supporte0 mJ/m2)  graphene1.2The ex situ transfer approach suffers from the potential introduction of defects between the graphene film and the device substrate as well as from the generation of cracks in the graphene film itself . For thi1.3The ideal procedure includes the direct deposition of graphene on the dielectric device substrate without a catalytic metal layer in between. Two concepts can be characterized by the type of metal removal. The first approach is based on the removal of the metal layer during the CVD process by evaporation  or agglo2 substrate to graphene [By choosing the appropriate temperature profile and C/Ni ratio the number of graphene layers can be controlled. However, process control is difficult and Ni grain boundaries can lead to an inhomogeneous thickness distribution of the graphene layer. A similar approach utilizes the diffusion of carbon species from a CVD atmosphere along grain boundaries through a copper film to the underlying substrate . As thergraphene .A newer procedure is the use of a suitable growth substrate that is compatible with device fabrication. Such a suitable substrate material is germanium –33 but o2The quality of the graphene material itself is a result of the growth and transfer process and can be influenced by several intrinsic properties.2.1One property is the density of defects. In the case of good quality graphene it is mainly determined by the density of grain boundaries between the single-crystalline domains . If a gr2.213 to 1014 atoms·cm−2, which corresponds to every tenth to hundredth atom in a monolayer. The main contamination is Cu from the CVD process, but also Fe is found in remarkable amounts. Several cleaning processes have been evaluated, but no substantial contamination removal could be achieved [10 to 1011 atoms·cm−1, depending on the metal type and technology). They also are influencing the graphene properties by charge-transfer doping [Another important intrinsic property of graphene is the amount of contaminations from the synthesis and transfer process. The synthesis process mainly introduces metallic contaminations typically in the range of 10achieved . These mr doping –41. The r doping .32·V−1·s−1 for SiO2 [2·V−1·s−1 for h-BN [2 with hexamethyldisilazane (HMDS) has a strong influence on the charge carrier mobility , this issue is of enormous importance, especially for short-channel devices. Numerous studies have been published during the last few years on the understanding and improvement of graphene–metal contacts, reporting contact resistivity values in a wide range from several ohm-micrometers to several hundred thousand ohm-micrometers. Metal contacts can interact with graphene in different ways , as showMetals physisorbed on graphene cause charge-transfer-induced doping of the graphene sheet because of the difference in work function values . Metals 6Referring to the aspects mentioned above, the currently most suitable integration scheme includes the utilization of the encapsulation layer as etching hardmask for structuring the graphene layer in order to avoid a contamination of graphene by following lithography steps. One method utilizes a one-step plasma-etch process with low selectivity to the underlying dielectric substrate to etch the encapsulation/hardmask layer and the underlying graphene simultaneously. An alternative approach applies two consecutive steps to etch the hardmask by a selective plasma etch or wet etch, followed by a second highly substrate-selective oxygen-plasma etch step to remove graphene. Both methods are schematically depicted in Because graphene as a carbon allotrope can react with other materials at high temperatures forming, e.g., metal carbides or carbon dioxide, care has to be taken that the subsequent processing steps do not employ temperatures exceeding 500 °C. The favorite solution to this issue is the so-called graphene-last approach in which graphene devices are formed in the metallization levels of a CMOS device .2, have to be adapted, even if the best possible graphene performance cannot be achieved. The encapsulation of graphene by Al2O3 seems to be solvable in the near future, as well as the formation of low-resistance graphene metal contacts. From today’s perspective the main issues for wafer-scale integration of graphene are well known, and the future of graphene  in microelectronics depends on the successful solution of these problems.The integration of graphene into CMOS-compatible electronic devices poses a lot of challenges when mass-production aspects are taken into account. An appropriate graphene quality can be produced by CVD on Cu substrates. Unsolved issues are the influence of grain boundaries and the reduction of metallic contamination. However, the synthesized graphene films have to be transferred to a dielectric substrate forming the device. This transfer process is critical as it is sensitive to defects, such as particles, cracks and wrinkles that degrade the graphene quality. Direct deposition methods on dielectric substrates exist, but do not yield a sufficient quality yet. We assume that this improvement of the deposition complex is crucial for commercializing graphene for microelectronics. Additionally, substrate interactions are strongly degrading the graphene quality. Hexagonal boron nitride has been identified as the ideal substrate material, but it is not available yet in the required quality for producing wafers. In order to obtain manufacturable graphene devices in the near future, established materials, such as SiO


In this work, we have designed, fabricated and experimentally characterized a printed graphene nano-flakes enabled flexible and conformable wideband radar absorber. The absorber covers both X (8–12 GHz) and Ku (12–18 GHz) bands and is printed on flexible substrate using graphene nano-flakes conductive ink through stencil printing method. The measured results show that an effective absorption (above 90%) bandwidth spans from 10.4 GHz to 19.7 GHz, namely a 62% fraction bandwidth, with only 2 mm thickness. The flexibility of the printed graphene nano-flakes enables the absorber conformably bending and attaching to a metal cylinder. The radar cross section (RCS) of the cylinder with and without absorber attachment has been compared and excellent absorption has been obtained. Only 3.6% bandwidth reduction has been observed comparing to that of un-bended absorber. This work has demonstrated unambiguously that printed graphene can provide flexible and conformable wideband radar absorption, which extends the graphene’s application to practical RCS reductions. Radar absorbers are widely used in both civil and military applications such as EMI, EMC, antenna pattern shaping, stealth technology and etc.1236789111314Ω/sq16252627With the development of the printed electronics, many conductive inks are developed and can be used to replace conventional conductors161718192021171820161620However CVD/exfoliated graphene absorbers need clean room process and are too expensive for practically deployable radar applications. Graphene nano-flakes ink and its printed products are obviously preferable alternative due to its compatibility with mass-production printing process and low fabrication costs2930313233313233Ω/sq. The geometrical parameters in a1 = 3.75 mm, a2 = 2.8 mm, a3 = 2.1 mm, b1 = 3.2 mm, b2 = 2.25 mm, d = 1.25 mm, l1 = 5.0 mm, l2 = 4.0 mm, w1 = 0.8 mm, w2 = 0.75 mm. The whole unit cell dimension is 15 mm × 15 mm. Due to the symmetry of the structure, its responses to vertically and horizontally polarized incident waves are the same for planar applications. In this work, the vertical polarization has been chosen for simulations and measurements.A wideband radar absorber based on modified second-order Saltire cross structure and four H shape coupling additives have been designed, as shown in 11) and absorption of the absorber with various silicone thicknesses h, can be found in h = 2 mm. In this case, the optimized absorber has a wideband effective absorption (above 90% absorption) from 8 GHz to 19 GHz, which covers both X and Ku bands. The absolute bandwidth is 11 GHz and fractional bandwidth is 81.5%. It can be found that the absorption band extends to lower frequency with thicker substrate, whereas thinner substrate provides better absorption at higher frequency band. Trade-off can be achieved through optimization. In this work, the thickness of the optimized absorber is less than 0.1λo at central frequency (13.5 GHz), which is thinner in comparison with similar worksThe simulated reflection coefficient  and from 13 GHz to 20 GHz (higher band) to experimentally examine the RCS of the printed graphene wideband absorbers. In Ω/sq). As it can be seen in the insertion of Ω/sq. However, it varies in different sections due to the inconsistency of thickness in the manually sputtered graphene nano-flakes layer. To further examine the cause of this discrepancy, simulations on the influence of sheet resistance variation of graphene nano-flakes layers were carried out. As it can be seen in Ω/sq to 30 Ω/sq, the lower band absorption reduces. As the printed graphene pattern was designed for Ω/sq, the larger the differences on sheet resistance, the more deterioration on lower band performance will be. From this analysis, it can be concluded that the differences on simulation and measured results was mainly caused by the non-uniformity of sheet resistance distribution of the printed graphene patterns.The same measuring procedure for the planar absorber has been conducted in the higher frequency band from 13 GHz to 20 GHz, and the results are shown in For radar absorbers, it would be highly desirable that the absorbers can be mechanically flexible and conformable so to cover differently shaped objects. For this purpose, flexibility and conformability of the printed graphene nano-flakes radar absorber has been investigated. The measurement setup for the conformable case has been explained in We have applied printed graphene nano-flakes to radar absorbers for RCS reduction in this work. A wideband absorber has been designed, which consists of patterned printed graphene nano-flakes layer, silicone substrate and backed metal. The absorber is fabricated with stencil printing method and the graphene nano-flakes ink are sputtered on silicone layer to form patterns through stencil with the help of air blaster. The absorber has been measured in anechoic chamber and experimentally demonstrated to provide effective absorption from 10.4 GHz to 19.7 GHz and a fractional bandwidth of 61.8%, with thickness only around 2 mm. The printed graphene nano-flakes absorber is mechanically flexible and its performance when bended to cover a metal cylinder have been measured to characterize its conformability. The measurement results show that only 3.6% reduction of the effective absorption bandwidth due to bending. The maintained wideband absorption reveals the potential applications of the printed graphene nano-flakes radar absorber in providing RCS reduction for objects of various shapes. In conclusion, this work has demonstrated unambiguously that printed graphene nano-flakes ink can be used not only for wideband radar absorption, but also flexibility and conformability.3 g expanded graphite (EG) was added into a mixture solution of 15 ml ethylene glycol and 45 ml ethanol. Non-ionic polymer-type surfactant was added to improve the dispersion of graphene nano-flakes. After stirring for 20 min, the dispersion was treated with sonication at 820 W with a cooling system for 24 hours. After that, the dispersion was heated on a hot plate with continuous stirring to remove the ethanol, yielding a final viscous ink of about 17% w/w solid in ethylene glycol, as seen in To make patterns on silicone, a stencil was fabricated with industrial laser cutting technology . As seen in All data generated or analysed during this study are included in this published article and its How to cite this article: Huang, X. et al. Experimental Demonstration of Printed Graphene Nano-flakes Enabled Flexible and Conformable Wideband Radar Absorbers. Sci. Rep.6, 38197; doi: 10.1038/srep38197 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Characteristics of tumor microenvironment have been suggested as predictive markers of anti-EGFR or anti-HER2 treatment response. However, the effect of EGFR/HER2 signal blockade on the tumor immune microenvironment is unclear.EGFR/HER2 pathway signaling and PD-L1 expression in gastric cancer cell lines were screened by western blot analysis. PD-L1 and HER2 expressions in 251 resected gastric tumors were determined by immunohistochemistry, and changes in EFGR, HER2, and PD-L1 expression in paired specimens between pre- and post-chemotherapy were evaluated. PD-L1 expression in HER2-amplified cell lines was evaluated by western blotting, fluorescence-activated cell sorting, reverse transcription, and real-time quantitative PCR analyses before and after afatinib, lapatinib, pictilisib and trametinib treatment. Changes in cytokines were evaluated by reverse transcription, real-time quantitative PCR, and enzyme-linked immunosorbent assay after EGFR/HER2 inhibition.p=0.030). PD-L1 overexpression accompanied by increased HER2 expression was identified in a post-chemotherapy specimen from a patient with an initial HER2/PD-L1-negative tumor. In HER2-overexpressing cell lines, PD-L1 expression was decreased in a dose- and time-dependent manner after afatinib and lapatinib treatment. PI3K pathway inhibition by pictilisib, but not MEK pathway inhibition by trametinib, resulted in PD-L1 suppression. After lapatinib treatment, the release of CCL2, CCL21, VEGF and CXCL1 decreased in a dose-dependent manner.Cell lines with pEGFR or pHER2 overexpression showed higher PD-L1 expression. In resected gastric tumors, HER2 expression was significantly associated with PD-L1 expression (Inhibition of the EGFR/HER2 signaling pathway, particularly of downstream PI3K activity, suppressed PD-L1 and release of cytokines, suggesting that EGFR/HER2 inhibition may create a more favorable milieu for tumor immunotherapy. The interaction between tumors and the tumor microenvironment is complicated. Tumor cells and the components of the tumor microenvironment communicate directly through cell-to-cell contact and indirectly through paracrine signals, which predominantly involve cytokines , 2. The Targeting the PD-1/PD-L1 immune checkpoint signaling to restore cancer cell-directed immune response is a confirmed strategy for several tumor types. Although there are controversies, PD-L1 expression has been suggested as a predictive marker of PD-1/PD-L1 checkpoint inhibitor treatment response –9. RecenTo elucidate the effect of EGFR/HER2 signal blockade on the tumor immune microenvironment, we examined the association between PD-L1 expression and the EGFR/HER2 signaling pathway and the direct effect of EGFR/HER2 pathway inhibition on the cytokines release by tumor cells.To determine the association between PD-L1 and the EGFR/HER2 pathway, we examined the protein expressions of PD-L1 and molecules related to the EGFR/HER2 signaling pathway in seven gastric cancer cell lines. Western blot analysis revealed that SNU216, SNU668, and NCI-N87 had higher p-EGFR, p-HER2, and PD-L1 expression  . A total of 10 specimens from five patients were included in this analysis. In one patient with an initial HER2-negative tumor, the post-chemotherapy specimen showed increased PD-L1 and HER2 expression levels  were evaluated using afatinib and lapatinib . The protein expression of PD-L1 was suppressed in a dose-dependent manner when pHER2 was suppressed by lapatinib and afatinib Figure . The mRNThe PI3K-AKT-mTOR and RAS-RAF-MEK pathways are two of the most important downstream pathways of EGFR/HER2 signaling. We hypothesized that one of these downstream pathways could be dominantly involved in the regulation of PD-L1 by HER2 overexpression. To determine whether PD-L1 expression was dependent on active PI3K-AKT-mTOR signaling or RAS-RAF-MEK signaling, HER2-amplified cell lines with high PD-L1 expression were treated with pharmacologic inhibitors of specific components in the pathway. We treated the SNU216, NCI-N87, and SKBR3 cell lines with 0.1 μM pictilisib (a selective inhibitor of class I PI3Ks), 0.1 μM trametinib (a MEK inhibitor), 0.1 μM lapatinib, and 0.02 μM afatinib. By using flow cytometer, we found that the expression of PD-L1 was reduced in pictilisib-treated, lapatinib-treated, and afatinib-treated cell lines Figure . In contTo more clearly demonstrate the effect of inhibition, cell lines were treated with interferon gamma (IFNγ) to induce PD-L1 expression. Indeed, PD-L1 expression in the cell lines was stimulated after IFNγ treatment. Pictilisib but not trametinib inhibited the induction of PD-L1 through IFNγ both at the protein and mRNA level Figure . These rWe examined the direct effect of EGFR/HER2 inhibition on the cytokines release of tumor cell lines. SNU216, NCI-N87, and SKBR3 cell lines were treated with different concentrations of lapatinib . The mRNA expression of CCL2, CCL21, and CXCL1 was suppressed in a dose-dependent manner Figure . FurtherIn the present study, we demonstrated that PD-L1 expression was higher in gastric cancer cell lines with higher pEGFR and pHER2 expressions compared with those with lower pEGFR and pHER2 expressions, and inhibition of the HER2/EGFR pathway decreased PD-L1 expression as well as cytokines release in HER2-amplified cancer cell lines. Furthermore, inhibition of the PI3K but not MEK pathway reduced PD-L1 expression in these cell lines, thus suggesting that the modulation of PD-L1 expression by the EGFR/HER2 pathway is likely to be mediated via downstream PI3K-AKT-mTOR signaling.The interaction between tumors and the tumor microenvironment is complicated. Signaling pathways of the inner tumor can be associated with the tumor microenvironment of the outer tumor. The tumor microenvironment also influences tumor behavior. PD-L1 is a transmembrane surface glycoprotein expressed by lymphocytes and dendritic cells as well as aberrantly on the surface of epithelial cells in a wide range of solid tumors. In addition, PD-L1 is a ligand of PD-1, which is known to be expressed by activated T-cells in the germinal centers of lymphoid follicles as well as TILs . The intRecent studies have demonstrated that PD-L1 expression is not only a marker of response to immune checkpoint inhibitor treatment but also cytotoxic chemotherapy and targeted therapy. PD-L1 expression can predict pathologic complete response in neoadjuvant chemotherapy-treated patients with breast cancer . A studyIn our tissue microarray data, HER2 was expressed in approximately 33% of tumors with PD-L1 expression, and PD-L1 was expressed in 84% of tumors with HER2 protein expression. HER2 amplification was not associated with PD-L1 expression in our cohort, which is in contrast with a study reporting HER2 overexpression in nearly 50% of PD-L1-positive gastric cancer cases . Accordiin vivo studies are warranted to confirm the findings in our in vitro study.Our study has several limitations. The effect of EGFR/HER2 inhibition on PD-L1 and cytokine release was only evaluated in pEGFR and/or pHER2 overexpressed cell lines; thus, a possible association between the EGFR/HER2 pathway and PD-L1 expression in other cell lines could have been overlooked. To address the microenvironment impact of the treatment, 2-dimensional experiments have limitation. Although co-culture system with immune cells may be better to prove the concepts, this study could not incorporate this system. In addition, we were unable to evaluate the change in PD-L1 expression and cytokines release in tumor tissues obtained from patients treated with different inhibitors used in this study (PI3K inhibitor and MEK inhibitor) since these agents are not used in routine clinical practice. Nevertheless, paired tissues of pre-chemotherapy and post-chemotherapy were available and the association between PD-L1 and EGFR/HER2 was evaluated. Further Overall, our findings suggest that agents targeting EGFR/HER2 could inhibit the induction of PD-L1, which may be dependent on the PI3K-AKT-mTOR pathway and suppress cytokines release in HER2-amplified cancer cells. EGFR/HER2 inhibition may create a more favorable milieu for tumor immunotherapy.3, 10% fetal bovine serum , penicillin (100 U/mL), and streptomycin . The cell cultures were maintained in a humidified CO2 incubator at 37°C. The drugs including Afatinib, pictilisib, trametinib, and lapatinib were purchased from Selleck Chemicals . IFNγ was obtained from ProSpec-Tany TechnoGene Ltd. . SNU216, NCI-N87, and SKBR3 were seeded in 6-well tissue culture plates at a density of 1 × 106 cells per well. Cells were cultured in RPMI 1640 with 10% fetal bovine serum. After overnight growth, the cells were treated with specific drugs at different concentrations and time points. After each exposure time of drugs, cells were harvested for experiments. To enhance PD-L1 expression, IFNγ-induction was applied. After cell seeding and overnight growth, the cells were treated with specific drugs. And then, IFNγ (10 ng/mL) was added after 4h of drug treatment, and incubation was continued for a further 24h. At 28 h after drug treatment, cells were harvested for experiments.NCI-N87, AGS, and SKBR3 cells were obtained from the American Type Culture Collection. SNU216, SNU668, and SNU719 cells were obtained from the Korean Cell Line Bank. YCC3, YCC10, and PC9 cells were obtained from Cancer Metastasis Research Center of Yonsei University College of Medicine. All cell lines were authenticated using by short tandem repeats. All were cultured in RPMI 1640  with L-glutamine (300 mg/L), supplemented with 25 mM HEPES, 25 mM NAHCOCells were lysed in 1× lysis buffer  containing 1 mM of the protease inhibitor PMSF , and the cell extracts were centrifuged at 16000g for 15 min. All samples were denatured in buffer containing 60 mM Tris, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, and 0.1% bromophenol blue at 100°C for 5 min. SDS polyacrylamide gels  were loaded with 25 μg total protein per lane. Prestained protein molecular weight markers (Bio-Rad) were run as standards. The electrophoresed samples were transferred to PVDF membranes . After transfer, the membrane was blocked with 5% skim milk in TPBS  for 1 h at room temperature. The anti-PD-L1 was obtained from Abcam . The antibodies including anti-HER2, anti-phospho-HER2, anti-Akt, anti-phospho-Akt, anti-ERK, anti-phospho-ERK, anti-STAT3, anti-phospho-STAT3, anti-PTEN, and anti-beta actin antibodies were purchased from Cell Signaling Technology . The membrane was then incubated with diluted primary antibodys for overnight at 4°C, rinsed and washed, and incubated with the appropriate HRP-conjugated secondary antibody  for 2h at room temperature. The membrane was washed and rinsed as before, and the expressed proteins were detected with the Pierce ECL Plus Western Blotting Substrate . Each experiment was performed in triplicate.Cells were centrifuged at 300 g for 3 min, washed with PBS, and fixed in 70% ethanol at −20°C. After incubation with fluorescently labeled antibodies for 30 min at 4°C, cells were washed with PBS buffer and analyzed on a FACSCalibur flow cytometer  with CellQuest Pro software (10000 events were recorded for each sample).Total mRNA was purified from cells using TRIzol reagent  according to the manufacturer's protocol for cultured epithelial cells. The High Capacity RNA-to-cDNA Kit  was used to generate cDNA from 1 μg of total mRNA. qPCR analysis was performed on the ABI qPCR system (Applied Biosystems) using SYBR Green PCR Master Mix (Applied Biosystems). The RT-PCR amplification program used was 5 min at 94°C followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The PCR products were analyzed on 1% agarose gels  in 1% Tris-borate-EDTA buffer stained with DYNE Loading STAR , and they were visualized by a UV transilluminator and recorded using a gel photo imaging system.ΔΔCT.The primers used in the PCR reaction were as follows: PD-L1 forward primer 5′-ttgggaaatggaggataaga-3′ and reverse primer 5′-ggatgtgccagaggtagttct-3′, CCL2 forward primer 5′- ccccagtcacctgctgttat-3′ and reverse primer 5′-tggaatcctgaacccacttc-3′, CCL21 forward primer 5′-gccttgccacactctttctc-3′ and reverse primer 5′-caaggaagaggtggggtgta-3′, CXCL1 forward primer 5′-gggaattcaccccaagaac-3′ and reverse primer 5′-caccagtgagcttcctcctc-3′, VEGF forward primer 5′-aaggaggagggcagaatcat-3′ and reverse primer 5′-atctgcatggtgatgttgga-3′, and GAPDH forward primer 5′-gcctcaagatcatcagcaatgcct-3′ and reverse primer 5′-tgtggtcatgagtccttccacgat-3′. The relative quantity of mRNA  was calculated as RQ = 2The changes in release of CCL2, CCL21, VEGF and CXCL1 were evaluated using ELISA. Supernatants were collected after 24h treatment with lapatinib. The experiment was conducted using individual Human ELISA Kits  according to the manufacturer's protocols. The absorbance was read at 450 nm using a SPECTRA max PLUS 384 and data were analyzed with SoftMax Pro software .Tissue microarray construction was performed for 251 patients with gastric cancer who underwent gastric resection between 2003 and 2004 at Seoul National University Bundang Hospital, as previously described . Paired All the experiments were repeated three times. The values shown in the tables and figures represent the mean ± standard deviation of three assays. Associations among groups were evaluated using chi-square test, linear-by-linear association, and one-way analysis of variance (ANOVA). This study for patients’ information was approved by the Institutional Review Board of SNUBH (B-1701/378-305). All analyses were performed using Microsoft Excel, SigmaPlot software, and SPSS .


N-glycosylation. We show that glycogen synthase kinase 3β (GSK3β) interacts with PD-L1 and induces phosphorylation-dependent proteasome degradation of PD-L1 by β-TrCP. In-depth analysis of PD-L1 N192, N200 and N219 glycosylation suggests that glycosylation antagonizes GSK3β binding. In this regard, only non-glycosylated PD-L1 forms a complex with GSK3β and β-TrCP. We also demonstrate that epidermal growth factor (EGF) stabilizes PD-L1 via GSK3β inactivation in basal-like breast cancer. Inhibition of EGF signalling by gefitinib destabilizes PD-L1, enhances antitumour T-cell immunity and therapeutic efficacy of PD-1 blockade in syngeneic mouse models. Together, our results link ubiquitination and glycosylation pathways to the stringent regulation of PD-L1, which could lead to potential therapeutic strategies to enhance cancer immune therapy efficacy.Extracellular interaction between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) leads to tumour-associated immune escape. Here we show that the immunosuppression activity of PD-L1 is stringently modulated by ubiquitination and  Programmed Death ligand-1 (PD-L1) protein mediates immune suppression in cancer. Here, the authors show that in breast cancer, PD-L1 expression can be up regulated post-translationally by glycosylation, which in turn acts through inhibiting GSK3β-mediated PD-L1 degradation. Programmed death ligand-1 (PD-L1), also known as B7 homologue 1 or B7-H1, is a 33 kDa type 1 transmembrane protein that binds to its receptor, programmed cell death protein-1 (PD-1), which leads to the inhibition of T lymphocyte proliferation, cytokine production, and cytolytic activity and suppression of the body's immune response1N-glycosylation plays a critical role in determining protein structure and function. In particular, glycosylation of membrane receptor proteins is important for protein–protein interactions, such as those between ligands and receptors, and has been shown to affect protein activitiesN-glycosylation continues in the Golgi apparatus10N-glycosylation affects a number of biological processes, including the modulation of protein–protein interactions13N-glycan structure of vascular endothelial growth factor (VEGF) receptor was shown to sensitize anti-VEGF refractory tumours to VEGF inhibitionGlycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase that was originally identified as a regulator of glycogen metabolism. It was later shown to be a key component of the Wnt signalling pathway, which plays important roles in embryonic development and tumorigenesis17171819While examining PD-L1 protein expression in human tumour tissues and cancer cell lines, we noticed that the majority of PD-L1 was detected at ∼45 kDa (black circle), but a smaller fraction at 33 kDa (arrowhead) also appeared . KnockinN-glycosidase F; PNGase F) to remove the entire N-glycan structure and then subjected the cell lysates to western blot analysis. A significant portion of the 45-kDa PD-L1 was reduced to 33 kDa upon PNGase F treatment . Thus, to determine whether this pattern corresponds to PD-L1 glycosylation, we treated MDA-MB-231 and BT549 cells with recombinant glycosidase - and green fluorescent protein (GFP)-tagged PD-L1 had an ∼15-kDa molecular weight shift from their actual size when treated with PNGase F . Interes-L1 ref. . Glycosyycin TM;  in a doscosidase . TogetheN-glycans, including the complex type, were identified for each of the 4 N-glycosylation sites - or intracellular domain-specific antibodies indicated that PD-L1 glycosylation occurred on its ECD . To pinpon sites , supporton sites . SubstitBecause the levels of glycosylated PD-L1 were significantly higher than the levels of its non-glycosylated form , we nextNext, we asked how N192, N200 and N219 glycosylation might regulate PD-L1 protein stability. We noted that PD-L1 contains a consensus GSK3β phosphorylation motif  adjacent to N192, N200 and N219 glycosylation sites ). Protei19To determine whether PD-L1 is a potential substrate of GSK3β, we conducted an immune-complex kinase assay. GST-PD-L1 was strongly phosphorylated by GSK3β CA  but not GSK3β kinase dead , and site-directed mutation of the consensus motif on PD-L1  completely abolished PD-L1 phosphorylation . Among tTo determine whether GSK3β-mediated phosphorylation involves PD-L1 degradation, we assessed the E3 ligase and its physiological effects on PD-L1. We noted that PD-L1 contains a putative β-TrCP destruction box, D/LSGXXS, that is present next to GSK3β phosphorylation sites 2425. Non242426in vitro and in vivo. Cells with PD-L1 3SA exhibited more PD-1 protein binding to the cell surface than did cells with PD-L1 WT  and p-GSK3β  but negatively with granzyme B , insulin-like growth factor-1, hepatocyte growth factor, fibroblast growth factor and transforming growth factor (TGF)-β. Among those examined, only EGF strongly induced PD-L1 expression in BT549 and MB-468 cells . Similar2in vitro, gefitinib enhanced anti-PD-1 antibody efficacy in a 4T1-luciferase (4T1-Luc) syngeneic BALB/c model. Tumour size was reduced, and mouse survival improved in both gefitinib- and anti-PD-1 antibody-treated mice . Both EGF-induced PD-L1 expression  and ecto28ted mice , with noted mice  and minited mice . The tumted mice . In addited mice . In factUnlike CTLA-4 or PD-1, which are primarily expressed in immune cells1431High AKT activation in cells with loss of PTEN has been shown to upregulate PD-L1 expressionIn summary, we demonstrated a novel interchange between glycosylation and phosphorylation regulating ubiquitination and degradation of PD-L1. This regulatory event is critical for BLBC cells that escape immune surveillance via PD-L1/PD-1 interaction. Importantly, inhibition of EGF-mediated PD-L1 stabilization enhances a therapeutic efficacy of PD-1 blockade to promote tumour-infiltrating cytotoxic T-cell immune response . Thus, tAll cell lines were obtained from the American Type Culture Collection  and have been independently validated using STR DNA fingerprinting at MD Anderson, and tests for mycoplasma contamination were negative. These cells were grown in DMEM/F12 or RPMI 1640 medium supplemented with 10% fetal bovine serum. PD-L1-stable transfectants in MDA-MB-231, MDA-MB-468, BT549 and HEK 293T cells were selected using puromycin . For transient transfection, cells were transiently transfected with DNA using SN liposomes4 cells in 50 μl medium mixed with 50 μl Matrixgel Basement Membrane Matrix  were injected into the mammary fat fad. For treatment with antibody, 100 μg anti-PD-1 antibody  or rat IgG (Bio X Cell) as control was injected intraperitoneally on days 3, 6, 9 and 12 after 4T1 cell inoculation. For drug treatment, mice were treated with daily oral doses of 10 mg kg−1 gefitinib for 2 weeks (5 days per week). Tumour was measured weekly with a caliper, and tumour volume was calculated using the formula: π/6 × length × width2.All BALB/C mouse  procedures were conducted under the guidelines approved by the Institutional Animal Care and Use Committee at MD Anderson Cancer Center. Mice were divided according to the mean value of tumour volume in each group. 4T1-Luc cells  and DNase (Sigma), and lymphocytes were enriched on a Ficoll gradient (Sigma) and then T cells were isolated using Dynabeads untouched mouse T-cell kit (Invitrogen). T cells were stained using CD3-PerCP , CD4-FITC , CD8-APC/Cy7 (BioLegend), CD45.1-PE (BioLegend) and IFNγ-Pacific Blue antibodies. Stained samples were analysed using BD FACSCanto II (BD Biosciences) cytometer.Mice receiving a 5 × 10−1 puromycin (InvivoGen).The lentiviral-based shRNA (pGIPZ plasmids) used to knock down expression of human or mouse PD-L1 ref.  was purcThe human PD-L1 clone was obtained from the shRNA/ORF Core Facility  and cloned into pCDH lentiviral expression vectors to establish PD-L1-Flag or PD-L1-Myc expression cell lines. In addition, it also cloned into pEGFP-N1 and pCMV-HA mammalian cell expression vectors for transient transfection. Using the pCDH/PD-L1-Flag expression vector as a template, we made PD-L1-Flag NQ mutants , and 4NQ (N35Q/N192Q/N200Q/N219Q) were developed by performing a site-directed mutagenesis . To creaQuantitative reverse transcriptase PCR (qRT–PCR) assays were performed to measure the expression of mRNA37The following antibodies were used: Flag , Myc , HA , PD-L1 , PD-L1 , PD-L1 , PD-L1 , PD-1 , Granzyme B , EGFR , GSK3β , phospho-GSK3β Ser 9 , β-TrCP , α-Tubulin , β-Actin . EGF, cycloheximide and TM were purchased from Sigma-Aldrich. Gefitinib, erlotinib, lapatinib, cetuximab and AG1478 were obtained from Calbiochem Corp .The mouse anti-phospho-PD-L1 T180 antibody was raised against a phosphorylated synthetic peptide (C-QVLSGKTT(p)TTNSKREE-NH2), and the mouse anti-phospho-PD-L1 S184 antibody was generated from a phosphorylated synthetic peptide (C-GKTTTTNS(p)KREEKLF-NH2). Antibodies were generated as previously describedg for 30 min to remove debris. Cleared lysates were subjected to immunoprecipitation with antibodies. To measure 2-DG incorporation on PD-L1 protein, cells were incubated with IRDye 800CW 2-DG Optical probe (LI-COR Biosciences) for overnight, and then we performed immunoprecipitation. Uncropped scans of the most important western blots are shown in Western blot analysis was performed as described previously39For immunocytochemistry, cells were fixed in 4% paraformaldehyde at room temperature for 15 min, permeabilized in 5% Triton X-100 for 5 min and then stained using primary antibodies. The secondary antibodies used were anti-mouse Alexa Fluor 488 or 594 dye conjugate and/or anti-rabbit Alexa Fluor 488 or 594 dye conjugate (Life Technologies). Nuclei were stained with 4′,6-diamidino-2-phenylindole . After mounting, the cells were visualized using a multiphoton confocal laser-scanning microscope .To measure PD-1 and PD-L1 protein interaction, cells were fixed in 4% paraformaldehyde at room temperature for 15 min and then incubated with recombinant human PD-1 Fc protein (R&D Systems) for 1 h. The secondary antibodies used were anti-human Alexa Fluor 488 dye conjugate (Life Technologies). Nuclei were stained with DAPI . And then we measured the fluorescence intensity of Alexa Fluor 488 dye using a microplate reader Synergy Neo  and normalized to the intensity by total protein quantity. To take an image, after mounting, the cells were visualized using a confocal laser-scanning microscope (Carl Zeiss). To monitor a dynamic PD-1 protein binding on live cell surface, PD-L1 WT or 3SA-expressing BT549 cells were incubated with Alexa Fluor 488 dye conjugate PD-1 Fc protein and taken a time-lapse image at every hour using the IncuCyte Zoom microscope (Essen Bioscience).−1) and IL-2 (10 ng ml−1). After 96 h, RFP and green fluorescent (NucView 488 Caspase 3/7 substrate) signals were measured. Green-fluorescent cells were counted as dead cells.T-cell-mediated tumour-cell-killing assay was performed according to the manufacturer's protocol (Essen Bioscience). To analyse the killing of tumour cells by T-cell inactivation, nuclear-restricted red fluorescent protein (RFP)-expressing tumour cells were co-cultured with activated primary human T cells (Stemcell Technologies) in the presence of caspase 3/7 substrate (Essen Bioscience). T cells were activated by incubation with anti-CD3 antibody  as described by the manufacturer. To stain glycosylated PD-L1 protein, we stained purified PD-L1 protein using the Glycoprotein Staining Kit (Pierce/Thermo Scientific) as described by the manufacturer.To confirm glycosylation of PD-L1 protein, we treated the cell lysates with PNGase F, Endo H and χ2-test were used and a P value less than 0.05 is considered statistically significant. The intensity of staining was ranked into four groups: high (+++), medium (++), low (+) and negative (−) according to histological scores.Human breast tumour tissue specimens were obtained following the guidelines approved by the Institutional Review Board at MD Anderson Cancer Center, and written informed consent was obtained from patients in all cases at the time of enrolment. IHC staining was performed as described previously39−1. MS and MS/MS data were acquired under the higher-energy collisional induced dissociation (HCD) product ion trigger collision-induced dissociation (CID) mode, using a 3-s top-speed-mode cycle time. Collision energy used for HCD (fourier transform mass spectrometry (FTMSn)) was 28% stepped at 5% and the product ions m/z 204.0867 or 366.1396 were used to trigger CID (ion trap mass spectrometery (ITMSn)) at 30% collision energy. The raw data obtained were then converted to the Mascot generic format using Proteome Discoverer 1.4, and the resulting HCD MS2 data were searched for tentative glycopeptide matches using Byonic v. 2.2.9 (Protein Metrics) with the following parameters: peptide tolerance, 2 p.p.m.; fragment tolerance, 6 p.p.m.; missed cleavages, 1; modifications, carbamidomethyl cysteine (fixed), methionine oxidation (common2) and deamidation at N (rare 1). The returned positive glycopeptide hits were then further validated manually by considering both HCD and CID MS2 results.Purified Flag-tagged PD-L1 protein was reduced by 10 mM dithiothreitol for 1 h at 37 °C, and then alkylated by 50 mM iodoacetamide for 1 h in the dark at room temperature, both in 25 mM ammonium bicarbonate buffer. Reduced and alkylated PD-L1 was then treated with sequencing grade trypsin  at 37 °C overnight, after which the digested products were diluted with formic acid to a final concentration of 0.1% and desalted with ZipTip C18 (Millipore) before nano LC-MS/MS analysis on an Orbitrap Fusion Tribrid fitted with an UltiMate 3000 RSLCnano System (Thermo Scientific). Briefly, the peptide mixture was loaded on an Acclaim PepMap 100  trap column and then separated on an Acclaim PepMap RSLC 25 cm × 75 μm i.d. column (Dionex) by a gradient of 5–35% acetonitrile/0.1% formic acid in 60 min at a flow rate of 500 nl minχ2-test and Mann–Whitney test. Student's t-test was performed for experimental data. A P value<0.05 was considered statistically significant.Data in bar graphs represent the mean fold change relative to untreated or control groups with s.d. of three independent experiments. Statistical analyses were performed using SPSS . The correlation between protein expression was analysed using Pearson All data supporting the findings of this study are available with the article, or from the corresponding author upon reasonable request.How to cite this article: Li, C.-W. et al. Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity. Nat. Commun. 7:12632 doi: 10.1038/ncomms12632 (2016).Supplementary Figures 1-10 and Supplementary Table 1


Because of recent research advances in nanoscience and nanotechnology, there has been a growing interest in functional nanomaterials for biomedical applications, such as tissue engineering scaffolds, biosensors, bioimaging agents and drug delivery carriers. Among a great number of promising candidates, graphene and its derivatives—including graphene oxide and reduced graphene oxide—have particularly attracted plenty of attention from researchers as novel nanobiomaterials. Graphene and its derivatives, two-dimensional nanomaterials, have been found to have outstanding biocompatibility and biofunctionality as well as exceptional mechanical strength, electrical conductivity and thermal stability. Therefore, tremendous studies have been devoted to employ functional graphene nanomaterials in biomedical applications. Herein, we focus on the biological potentials of functional graphene nanomaterials and summarize some of major literature concerning the multifaceted biomedical applications of functional graphene nanomaterials to coated substrates, patterned arrays and hybrid scaffolds that have been reported in recent years. Over the last several decades, the development of nanoscience and nanotechnology has made dramatic progress in research towards the understanding of nanomaterials. In addition, numerous attempts have been devoted at that time to employ nanomaterials in various research and industrial fields. It is commonly acknowledged that nanomaterials are defined as materials having at least one dimension, such as length, thickness, width, or diameter, of smaller than 100 nm in size and the plenty of attentions has been paid to various nanomaterials, including carbon nanotube, nanoparticle, polymeric nanofiber, quantum dot, graphene and nanocomposite ,3,4. Nan2-bonded carbon atoms arranged in a honeycombed lattice structure. It was first described by Boehm and coworkers in 1986 . Th. Th32]. −1 for the HOPG sample, the multi-layer graphene sample and the sample with a single-layer to few-layer graphene, respectively. On the other hand, there is also a significant increase in the carrier concentration, especially in the samples containing a mixture of single-layer to several layers of graphene: 3.58 × 1018, 14.9 × 1018 and 46.3 × 1018 cm−3 for HOPG sample, multi-layer graphene sample and samples containing a mixture of single-layer to several layers of graphene, respectively. In addition, it was revealed that the superior electrical properties of graphene nanomaterials result in highly sensitive detection of the micromolar concentration of dopamine—a neurotransmitter—on graphene-coated surfaces by Raman spectroscopy and microscopy.In other studies, the graphene nanomaterial-coated substrates hold the potentials for neural cells ,46,48,572+ oscillations, which represents neural signal transmission . Th. Th83]. On the other hand, recently, the natural and synthetic polymers have been extensively used to fabricate biological scaffolds for tissue engineering applications. Polymeric biomaterials have superior biocompatibility and biodegradability but their intrinsic poor thermal and mechanical properties are often quoted as disadvantages. Therefore, much research has been suggested to compensate the poor thermal and mechanical properties of polymeric biomaterials by functionalization of graphene possessing exceptional thermomechanical properties. Shin et al. demonstrated that the impregnation of GO can not only reinforced the poor mechanical properties of polymer nanofiber scaffolds but can also promote myoblast growth and differentiation . DespiteMore recently, studies concerning the development of three-dimensional (3D) scaffolds using graphene nanomaterials have been increasingly reported ,99,101. Splendid progress in nanoscience and nanotechnology has invigorated interest in application of nanomaterials to biomedical fields. Thanks to this interest, there has also been tremendous advances in research on graphene nanomaterials and their biocompatibility and biofunctionality having been gradually established. In this review, some of recent literature concerning the multifaceted biomedical applications of functional graphene nanomaterials was summarized and discussed. According to the recent studies, it is obvious that the functional graphene nanomaterials can be employed in a variety of ways to biomedical applications. In addition, the conventional approaches can be further developed by unique properties of graphene nanomaterials themselves, such as exceptional thermomechanical, excellent physicochemical, outstanding electrical and specific biological properties. In particular, many studies support the fact that graphene nanomaterial-coated substrates, -patterned arrays and hybrid scaffolds are typical approaches for biomedical applications of graphene nanomaterials, which allows us to more easily and intuitively understand the potential of graphene nanomaterials.Although we have focused on three ways to apply graphene nanomaterials in biomedical applications, there are many other ways to employ them in biomedical fields and the potentials for application of graphene nanomaterials to biomedical fields will continue to evolve. Even if more research remains a significant challenge that should be addressed through comprehensive and systematic studies to fundamentally understand the functional graphene nanomaterials, we envision that the functional graphene nanomaterials will become promising novel candidates, which can open the way to handle unsolved problems in the current biomedical field.


Functional walking requires the ability to modify one’s gait pattern to environmental demands and task goals—gait adaptability. Following incomplete spinal cord injury (ISCI), gait rehabilitation such as locomotor training (Basic-LT) emphasizes intense, repetitive stepping practice. Rehabilitation approaches focusing on practice of gait adaptability tasks have not been established for individuals with ISCIs but may promote recovery of higher level walking skills. The primary purpose of this case series was to describe and determine the feasibility of administering a gait adaptability retraining approach—Adapt-LT—by comparing the dose and intensity of Adapt-LT to Basic-LT.Three individuals with ISCIs  completed three weeks each (15 sessions) of Basic-LT and Adapt-LT. Interventions included practice on a treadmill with body weight support and practice overground . Adapt-LT focused on speed changes, obstacle negotiation, and backward walking. Training parameters  were compared and outcomes assessed pre and post interventions. Based on completion of the protocol and similarities in training parameters in the two interventions, it was feasible to administer Adapt-LT with a similar dosage and intensity as Basic-LT. Additionally, the participants demonstrated gains in walking function and balance following each training type.Rehabilitation that includes stepping practice with adaptability tasks is feasible for individuals with ISCIs. Further investigation is needed to determine the efficacy of Adapt-LT. Recovery of walking function is a primary goal and focus of rehabilitation for individuals with incomplete spinal cord injuries (ISCIs) . To walkIn our search of the literature, only two reports have focused on strategies for retraining gait adaptability skills following ISCI , 8. In aboth spinal and cortical neural networks [all of these features—repetitive stepping practice, increased speeds, as well as practice of adaptability tasks.Spinal cord injury (SCI) gait rehabilitation principles that emphasize repetitive stepping practice and speed  are basenetworks , it may Although SCI gait rehabilitation that incorporates all of these features has not been established, different types of adaptability training have been successfully applied to adults post-stroke, and in other populations –22. For Overall, these prior studies suggest that gait rehabilitation with adaptability tasks may be beneficial for individuals with ISCIs, but there may be challenges to achieving a sufficiently high dosage (number of steps) and intensity during adaptability training. Furthermore, adaptability training could be particularly difficult to implement for individuals with ISCIs due to common impairments such as severe bilateral weakness and spasticity. To address these challenges, a gait rehabilitation approach, referred to as Adapt-LT was developed. Adapt-LT includes basic stepping practice (Basic-LT), but also implements repetitive practice of gait adaptability tasks—obstacle negotiation, backward walking, and speed changes. Therefore, the goals of this case series were to describe the Adapt-LT approach and determine the feasibility of delivering Adapt-LT at a dosage and intensity similar to training with Basic-LT. We specifically focused on whether a similar number of steps, stepping speeds, and self-reported exertion levels could be achieved for both Adapt-LT and Basic-LT. Walking function and balance also were assessed at the end of each intervention.This case series was conducted at the Malcom Randall Veterans Affairs Medical Center Brain Rehabilitation Research Center in Gainesville, Florida. Institutional and federal regulations concerning ethical use of human volunteers were followed; all protocols were approved by the University of Florida and Veterans Affairs Medical Center . Participants provided informed consent prior to enrollment. Eligibility criteria included ≥ 18 years old with a singular, motor ISCI (≥6 months post-injury), medically stable, discharged from physical therapy, and able to ambulate at speeds of ≥0.3 m/s at the time of enrollment.Three adult males (26–77 years) with ISCIs (durations > 18 months) were enrolled. Descriptive information was obtained through review of medical records, participant self-report, and assessment at time of enrollment. A licensed physical therapist completed all clinical assessments. The International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) American Spinal Injury Association Impairment Scale (AIS) was used to classify each participant’s neurologic level of injury  common tasks representing different adaptability domains; , 28 (b) Obstacle negotiation -Obstacles were delivered bilaterally and included foam blocks and boxes of variable sizes . Progression of the task included increasing obstacle frequency, varying rates of obstacle delivery, and increasing obstacle size. 2. Speed changes-Speed changes consisted of abruptly and unexpectedly changing from faster speeds to and from slower speeds. 3. Backward walking -For progression, backward walking speed was increased and body weight support was decreased. During overground training, participants performed the same tasks and the goal was to spend a similar amount of time on each task.Thus, based on these factors, Adapt-LT emphasized the same general principles as Basic-LT and also included practice of adaptability tasks  Figs.  and 2a–dFeasibility was determined based on the successful completion of the Adapt-LT protocol and focused on comparisons of dosage (amount of steps) and intensity  parameters across Basic-LT and Adapt-LT. Additionally, these parameters were assessed because of their importance in motor relearning  and association with improved outcomes in studies of walking function after SCI –11.During training on the treadmill, steps were counted for 15–30 s at each speed and the total number of steps was estimated for each session. During training overground, the total number of steps was counted by an assistant.The average maximal treadmill training speed was determined for sessions 11–15 based on the highest speed sustained for at least 30 s or, as in the case of speed changes during Adapt-LT, a speed achieved at least twice during the session.For each training bout, participants were asked to report their exertion level using the 20-point Borg Rating of Perceived Exertion Scale . ReporteClinical assessments of walking function and balance were administered before and after 15 sessions of Basic-LT and 15 sessions of Adapt-LT  and Borg ratings of exertion were similar for both training types . SCI01’s walking pattern was characterized as stiff and he had particular difficulty in flexing his lower extremity joints. Therefore, the treadmill environment and hands-on assistance may have been helpful in achieving sufficient stepping practice. Additionally, the adaptability tasks may have encouraged less stiff movements  and enabled training at higher speeds.Basic and Adapt-LT Outcomes. Following 3 weeks of Basic-LT, no gains in walking function or balance were seen for SCI01. In contrast, after Adapt-LT, SCI01 showed a reduced TUG time (∆ = ↓10.22 s), increased MBT score (∆↑ = 2), and increased ABC score (∆ = ↑12.81%).Overall Outcomes. At the conclusion of the study, SCI01 demonstrated increased walking function (∆TUG = ↓2.44 s) and increased balance  compared to baseline. The changes, however, did not exceed MCID values for individuals with SCIs. Prior to his enrollment, SCI01 could not stand without support for >5 s. It is, therefore, note-worthly that following the completion of both interventions he stood without support for >1 min. SCI01 reported that he perceived improvement in his walking function and general mobility. He stated that he now believed getting better was possible, whereas he did not believe this was possible before training.Dosage and Training Intensity. SCI02 achieved similar average treadmill step counts during both types of training , but performed nearly double the number of steps overground during Basic-LT . For this individual who walked at a faster speed, the adaptability tasks likely took a relatively greater time to complete. His average maximal training speeds and Borg ratings were similar for both training types .Basic and Adapt-LT Outcomes. SCI02 demonstrated greater improvements following Basic-LT. His gains in gait speed (∆ = ↑0.38 m/s) exceeded the MCID value for individuals with SCIs [ith SCIs . He alsoOverall Outcomes. At the conclusion of the study, SCI02 demonstrated an increase in walking function  and increased balance  compared to baseline. SCI02 did not complete the ABC prior to Basic-LT; therefore, this overall change was obtained from comparing post Basic-LT scores to post Adapt-LT scores. The increase in gait speed exceeded the MCID value (0.13 m/s) for individuals with SCIs [ith SCIs . SCI02 aDosage and Training Intensity. SCI03 performed an average of 2161 steps during each session of Basic-LT on the treadmill and 2006 steps during Adapt-LT. During each session of overground training an average of 147 steps were practiced during Basic-LT and 203 steps for Adapt-LT. Average maximal treadmill speeds were similar  and Borg ratings of exertion also were similar for both training types .Basic and Adapt-LT Outcomes. SCI03 showed greater positive changes in almost all clinical outcomes following Adapt-LT compared to BLT. He demonstrated increased gait speed (∆ = ↑0.11 m/s), reduced TUG time (∆ = ↓3.27 s), a higher MBT score (∆ = ↑2) and a higher ABC score (∆ = ↑4.38%). In contrast, only the SCI-FAP score (∆ = ↓261.2) showed a greater positive change following Basic-LT. The improvements following Adapt-LT exceeded the MCID value (0.13 m/s) for gait speed in individuals with SCIs [e ∆ = ↑4.%. In conOverall Outcomes. At the conclusion of the study, SCI03 demonstrated increased walking function  and balance . The increase in gait speed and decrease in TUG time both exceeded the MCID values for individuals with SCIs  [ctively) . InteresThe focus of this case series was to describe Adapt-LT and determine the feasibility of administering Adapt-LT at a dosage and intensity similar to Basic-LT. Three adults with chronic ISCIs completed 15-sessions of Adapt-LT and the amount of stepping practice, treadmill speeds, and perceived exertion were similar to Basic-LT. Moreover, outcomes at the conclusion of training indicated that participants improved in walking and balance function, suggesting that ongoing improvements are achievable in individuals with chronic ISCIs. Overall, our goal in developing Adapt-LT was to build off of previous SCI rehabilitation strategies and address the challenges of implementing a gait adaptability intervention with a similar dosage and intensity of Basic-LT .Gait rehabilitation principles applied in the development and administration of Adapt-LT are well-established , 13 and A recent study of retraining gait adaptability post-ISCI reported potential limitations of their approach were reduced amounts of practice and slower training speeds . To overOverall, the general training parameters we applied across Basic- and Adapt-LT were consistent with previously-reported studies describing intense SCI gait rehabilitation. Specifically, the duration of training and training speeds used during both interventions were consistent with prior reports . In addiA potential limitation in the design of this case series was the consistent order of the two types of training. Specifically, the Adapt-LT training parameters and outcomes may have been different had Basic-LT not been administered first. However, based on the goals of this case series, Basic-LT was provided prior to Adapt-LT for a variety of reasons. First, since our focus was to establish feasibility of Adapt-LT, we thought it was important to provide each participant with an established intervention to determine their ability to safely participate and identify baseline training responses. The use of only three adaptability tasks—obstacle negotiation, speed changes, and backward walking—also poses another potential limitation. While these tasks are important elements of community mobility, other features such as uneven terrains, doorways, and stairways were not practiced . FinallyAdapt-LT, a gait rehabilitation intervention focused on repetitive practice of walking and tasks requiring gait adaptability was feasible for the three individuals with chronic ISCIs. In most instances, the training parameters of amount of practice, walking speed, and perceived exertion were consistent across Basic- and Adapt-LT. Although responses to training were varied, gains in walking and balance function were achieved. Overall, the outcomes provide preliminary insight into how individuals with ISCIs may respond to varied forms of training. Future studies are necessary to assess the efficacy of Adapt-LT and to further develop this approach to maximize its potential effectiveness for promoting walking recovery.


Idiopathic pulmonary fibrosis (IPF), a fibrosing interstitial pneumonia of unknown etiology, primarily affects older adults and leads to a progressive decline in lung function and quality of life. With a median survival of 3–5 years, IPF is the most common and deadly of the idiopathic interstitial pneumonias. Despite the poor survivorship, there exists substantial variation in disease progression, making accurate prognostication difficult. Lung transplantation remains the sole curative intervention in IPF, but two anti-fibrotic therapies were recently shown to slow pulmonary function decline and are now approved for the treatment of IPF in many countries around the world. While the approval of these therapies represents an important first step in combatting of this devastating disease, a comprehensive approach to diagnosing and treating patients with IPF remains critically important. Included in this comprehensive assessment is the recognition and appropriate management of comorbid conditions. Though IPF is characterized by single organ involvement, many comorbid conditions occur within other organ systems. Common cardiovascular processes include coronary artery disease and pulmonary hypertension (PH), while gastroesophageal reflux and hiatal hernia are the most commonly encountered gastrointestinal disorders. Hematologic abnormalities appear to place patients with IPF at increased risk of venous thromboembolism, while diabetes mellitus (DM) and hypothyroidism are prevalent metabolic disorders. Several pulmonary comorbidities have also been linked to IPF, and include emphysema, lung cancer, and obstructive sleep apnea. While the treatment of some comorbid conditions, such as CAD, DM, and hypothyroidism is recommended irrespective of IPF, the benefit of treating others, such as gastroesophageal reflux and PH, remains unclear. In this review, we highlight common comorbid conditions encountered in IPF, discuss disease-specific diagnostic modalities, and review the current state of treatment data for several key comorbidities. Post hoc analyses of clinical trial datasets also suggest that anti-fibrotic therapy may reduce the risk of acute exacerbations and improve overall survival in those with IPF (Idiopathic Pulmonary Fibrosis (IPF), a fibrosing interstitial lung disease (ILD) of unknown etiology, primarily affects older adults and leads to a progressive decline in lung function and quality of life –4. With with IPF –14.While the identification of therapies that effectively slow IPF progression represents a monumental step forward in the care of patients with IPF, pharmacotherapy is but one component of the multi-pronged approach necessary to optimally manage patients with IPF. Other equally important pieces include evidence-based prognostication  improvemRoughly 70–80% of individuals with IPF endorse a history of cigarette smoking, which has long been an established IPF risk factor –20. Not High-resolution computed tomography (HRCT) is part of the routine diagnostic evaluation of all patients with suspected IPF , and rouCompared to those in the general population, individuals with IPF have a nearly 5-fold increased risk of developing lung cancer, with 3–22% of patients affected and an estimated incidence of 11 cases per 100,000 person-years –32. The Survival among those with IPF and comorbid lung cancer is poor , 39 and As for emphysema, HRCT serves as a reasonable modality for lung cancer screening, but many HRCT protocols still perform non-contiguous imaging, which can miss early nodules and other local changes; contiguous imaging is required . Most luPreliminary studies suggest that OSA may be an underappreciated comorbid condition in those with IPF. Three investigations have shown OSA to be present in 58–88% of patients with IPF, with up to 68% having moderate-to-severe OSA based on an apnea–hypopnea index ≥15 events/hour –54. In aAs with the general population, moderate-to-severe OSA is generally treated with continuous positive airway pressure (CPAP). CPAP initiation has been shown to improve quality of life measures and sleep instruments in those with IPF and comorbid moderate-to-severe OSA, though CPAP non-adherence was common . The optIdiopathic pulmonary fibrosis and CAD share several risk factors, including increasing age, male gender, and smoking history. Among the largest retrospective studies conducted to date, the estimated prevalence of CAD in those with IPF ranges from 4 to 25% –63. A prThe U.S. Preventative Services Task Force concluded that there was insufficient evidence to recommend for or against routine CAD screening in asymptomatic, high-risk individuals in the general population . Among sPulmonary hypertension defined as mean pulmonary artery pressure (mPAP) ≥25 mm Hg , commonlPulmonary hypertension should be suspected in patients with dyspnea or oxygen desaturation out of proportion to pulmonary function, disproportionately low DLCO, evidence of right heart failure on physical exam, or evidence of pulmonary artery enlargement and/or right ventricular hypertrophy on imaging studies. TTE, which estimates RVSP as a surrogate for mPAP, is perhaps the most commonly utilized modality to screen for PH. Although TTE-estimated RVSP has been shown to correlate poorly with mPAP determined by right heart catheterization , 76, an There are currently no approved therapies for the treatment of PH in patients with IPF and the last decade has seen a disappointing number of negative clinical trials using vasodilator therapies. Several studies have investigated the use of PH therapies for IPF in general (regardless of the presence of PH) and failed to demonstrate efficacy in slowing IPF progression –79, and post hoc subgroup analysis of patients with evidence of PH by TTE showed that sildenafil therapy did improve walk distance as well (The phosphodiesterase-5 inhibitor sildenafil was studied in patients with advanced IPF (defined by a baseline DLCO of less than 35%). While it did not significantly alter the primary functional endpoint of walk distance, it did show improvements in dyspnea score, oxygenation, and quality of life . A post  as well . Based o as well .Relatively few studies have assessed the epidemiology and clinical consequences of PE and more broadly VTE in patients with IPF. A study utilizing U.S. insurance claims data suggested that 2.7% of individuals with a diagnosis code for IPF also carried a diagnosis of PE . AnotherLongitudinal analyses of patients with IPF suggest that the risk of incident VTE is 3–6 times higher among patients with IPF compared to control subjects, with an estimated 6–9 new events per 1,000 person-years , 86. As post hoc analysis of IPF clinical trial datasets (The treatment of PE/VTE requires prolonged blockade of the coagulation cascade, which facilitates clot resolution. The American College of Chest Physicians recommend 3 months of anticoagulant therapy in those with VTE provoked by surgery or other known VTE risk factor. These guidelines also recommend at least 3 months of anticoagulant therapy in patients with a first-time unprovoked VTE, after which time the risk–benefit ratio for extended therapy should be considered . This asdatasets , 91. TheGastro-esophageal reflux is another common comorbid condition in patients with IPF , but theThe ideal modality for diagnosing GER remains unclear. While all patients should be screened for GER-associated symptoms, including heartburn, choking, and regurgitation, symptom-based screening has a low sensitivity for detecting pathologic GER , 98. Flupost hoc analysis of three clinical trial datasets (post hoc analysis of a separate clinical trial dataset (It has been hypothesized that GER may contribute to the progression of IPF in some patients. Several studies, with mixed results, have explored the influence of anti-acid therapy on IPF disease course. GER therapy was associated with improved survival in a retrospective, multi-center cohort analysis  and lessdatasets . These f dataset . AdditioA likely contributor to the high prevalence of GER in patients with IPF is HH, which has been described in 40–53% of patients with IPF , 108. WhWhile 1–2% of men and 5–9% of women in the general population carry a diagnosis of hypothyroidism –112, a rCase–control analyses performed in Japan, Mexico, and the U.K. estimated the prevalence of DM type 2 to be 10–33%, among individuals with IPF, which was significantly higher than that of matched control populations , 93, 113Idiopathic pulmonary fibrosis progression commonly manifests as worsening dyspnea, declining pulmonary function, and hypoxemia. While these manifestations undoubtedly impact quality of life, they have been shown to correlate with depression and anxiety –116. DesA small IPF cohort study based in Europe suggested that increasing duration of disease correlates with standardized depression scores , while aIdiopathic pulmonary fibrosis remains a devastating diagnosis for patients and their families, and its management requires a multi-pronged approach. Comorbidity assessment and management is a cornerstone of comprehensive management of IPF and we have reviewed the most commonly associated comorbidities that clinicians should consider Table . AggressJO and HC contributed to the conception and writing of this review. Both authors have reviewed and approved the submitted work.JO and HC have no relevant conflicts to disclose related to the submitted work.