Graphene has become a bridge across optoelectronics, mechanics, and bio-chemical sensing due to its unique photoelectric characteristics. Moreover, benefiting from its two-dimensional nature, this atomically thick film with full flexibility has been widely incorporated with optical waveguides such as fibers, realizing novel photonic devices including polarizers, lasers, and sensors. Among the graphene-based optical devices, sensor is one of the most important branch, especially for gas sensing, as rapid progress has been made in both sensing structures and devices in recent years. This article presents a comprehensive and systematic overview of graphene-based microfiber gas sensors regarding many aspects including sensing principles, properties, fabrication, interrogating and implementations. Microfibers with sub-wavelength geometry and large index contrast between the fiber core and surroundings ,2 has beGraphene is a unique two-dimensional material composed of carbon in a honeycomb lattice with atomic thickness ,8, and hAmong them, graphene-based gas detection is one of the most creative and successful applications, showing the potential to achieve ultimate sensitivity: single molecule gas detection . MoreoveWhen deposited on microfibers, graphene interacts with the evanescent field, enabling different kinds of graphene-based microfiber gas sensors. Here we conclude the recent progress in this fiend regarding their sensing principles, fabrication and performances. Typical graphene-based microfiber gas sensors include biconical tapers, gratings, interferometers, coils, ring cavities, and so on. These fiber-optic graphene gas sensors are categorized in both passive (such as interferometric) and active (such as pumping laser-based) techniques, which shows the potential applications in highly-sensitive gas sensing in combining with microfiber and new materials. Although some review articles on graphene-based electrical gas sensors have been published ,10, ther±E(κ) F|κ|= ±ħν, where κ is the wave vector, Fν ≈ 106 m/s is the Fermi velocity, and ħ is Planck’s constant  + 1}−1 is the Fermi–Dirac distribution, ħ = 1.05 × 10−34 J·s, Bk = 1.3806505 × 10−23 J/K is Boltzmann’s constant, and e = −1.6 × 10−19 C is the unit charge. External electric tuning majorly influences the g,interσ, which is also directly related to the saturable absorption, which is driven by the photoexcited electron kinetics [Re(gσ) and Im(gσ) are the real and imaginary parts of gσ, respectively. By regarding the graphene monolayer thickness Δ = 0.4 nm, the refractive index of the graphene layer can be derived from [The theoretically calculated results are shown in kinetics . Consideved from :(5){Re [effn vs. |FE|” correlation of a graphene-based microfiber with core diameter ≈ 8 µm. It can be seen that by changing the Fermi level of graphene, both the transmission phase and the transmission loss of a graphene-based microfiber could be modified remarkably. Such a mechanism can not only be applied to sensing, but has also been widely used for fast optical modulation [Once incorporated with a microfiber, the graphene performs as a part of the cladding, and can influence the optical transmission via mode effective refractive index modulation. According to different graphene–microfiber hybrid geometries, the mode field distributions and complex effective index od (FEM) ,44. Figuod (FEM) ,45, loweod (FEM) . The moddulation ,48.A is the optical amplitude, Re(effn) and Im(effn) are the real and imaginary parts of the graphene-based microfiber, z is the transmission distance, t is the time delay, c is the light velocity in vacuum, and ω is the optical frequency. As a conclusion, the Fermi-level modification of graphene would modulate both the output phase and output power of the microfiber. In optical nonlinear processes, such an effect also alters the phase-matching and nonlinear threshold [It is known that the electromagnetic field distributed along microfibers obeys the Bessel equations in cylindrical coordinates , which hhreshold .3, NO2, and H2O. It is worth pointing out that for graphene oxide (GO) or other functionalized graphene, non-polar gas such as H2 is also detectable [When gas molecules are adsorbed on the surface of graphene, the Fermi level of graphene is tuned ,55. Figutectable . Figure tectable . As expoSince the first graphene-based microfiber optical gas sensor reported in 2012 , a varieTypically, microfibers are fabricated from commercial glass fibers or bulks, by using the fusing & drawing method , which cSometimes one not only wants a microfiber-guiding evanescent field, but also hopes to keep the inner microstructures of the original fiber ; then, chemical etching becomes a choice ,68. For 4 gas in a dilute H2 environment over the surface at 1000 °C. With the exposure of Cu foil in a CH4/H2 environment, the nucleation of graphene islands starts taking place randomly, eventually aggregating into a continuous graphene film. The chemical vapor deposition (CVD) and wet transfer techniques provide inch-level large-scale monolayer graphene for incorporation with microfibers ,70,71,723 solution; covering the PMMA/graphene flexible film on a substrate or wrapping it on a microfiber; removing the PMMA by using acetone, leaving only graphene remaining.Graphene can be either attached on or wrapped around a microfiber, as schematically shown in To characterize the quality of graphene on the microfibers, Raman spectroscopy ,78,79, sIn recent years, graphene gas sensors with microfibers develop rapidly, some of them are also reviewed in Refs. ,82,83,84Based on the graphene-attached microfiber scheme, we reported gas sensors based on both optical intensity detection and interferometric demodulation by using mode field analysis or Mach-Zehnder Interferometer (MZI) ,84. Figu3 gas detection [3 trace detection, 0.3 ppm resolution and 0.5 s response delay was achieved.The regeneration time for the acetone gas detection was in the rage of several minutes. The interferometer scheme was a big step forward to achieve a much higher sensitivity. etection . It illu2 gas. A sensitivity of 0.5 ppm was achieved in this work. As the reduced GO film was thicker and fluffier than the CVD graphene, its response time was limited to minute-level. In this type of sensor, it is also important to enhance the mode distribution ratio out of the core and optimize the microfiber diameter (or the thickness of the remained cladding), as discussed by Zhang et al. in Ref. [H ~ ln(S)ln(D)/A, the best diameter could be around 10 µm for graphene-wrapped silica fiber-Bragg-gratings (FBGs) with standard 8 µm core.Similarly,  in Ref. . Figure 3 gas sensing and NO2 gas sensing, respectively. As shown in pλ = 2effΛn, where Λ is the MFBG period. The gas adsorption based on effn modulation was measured by detecting the spectral shift of the MFBG. The maximum sensitivity of the CVD graphene-coated MFBG reached 0.2 ppm for NH3 gas and 0.5 ppm for xylene gas, respectively.Since the scheme of graphene-microfiber attachment spatially limits the light–graphene interaction, since 2014, graphene-wrapped microfiber structures have become a trend. For instance, based on graphene-wrapped micro fiber Bragg gratings (MFBGs), the footprint of the sensors can be dramatically miniaturized. Wu et al.  and Srid21 mode is more sensitive to local refractive index alteration than the in-core HE11 [3 gas detection and ~0.2 ppm for H2O vapor detection were achieved. By using GO-ZnO film, Hu et al. also recently realized a NH3 gas sensor, demonstrating sub-ppm sensitivity, as shown in 3 gas. Additionally, in 2014, Mishra et al. also reported a graphene Surface Plasma Resonance (SPR) sensor based on microfibers for sensing NH3 gas [Another method to enhance light–graphene interaction in graphene-based microfiber structures is to excite high-order mode propagation with larger mode-field area or to excite plasmons. In 2014, Yao et al. demonstrated a graphene-based microfiber multimode interferometer, as shown in ore HE11 , hence i NH3 gas . Mishra Q resonance, the interferometric resolution can be effectively improved. Yu et al. made such an attempt by building a GO-deposited microfiber knot resonator [Q factor. In future investigations, by optimizing the graphene coverage region [Limited by the spectral resolution of the optical interference and the linear loss of the passive devices, sub-ppm seemed to be the limitation for a graphene-based microfiber gas sensor. Taking advantage of high esonator . Figure e region , the res3 gas detection, which is a globally leading number comparable to other advanced techniques [Av is the acoustic velocity, c is the light velocity in vacuum, pn and sn are the effective indexes of the pump mode and the generated Stokes mode, pf and sf are the pump frequency and the Stokes frequency, eγ is the electro-strictive coefficient, and BΓ is the lifespan of the phonons. Gas adsorption on the reduced GO film enables an RF spectral shift 200 kHz/ppm, while the uncertainty of the Brillouin optomechanical generation is only 200 Hz, due to the extremely high Q factor (106). Moreover, such an optomechanical micro-resonator kept an exceptional dynamic range from 1 ppb to 400 ppm, crossing over five orders.Moreover, in 2017, based on graphene-enhanced Brillouin scattering, a microfiber interrogated Whispering Gallery Mode ptomechanical gas sensor was realized. chniques ,96. SuchThe above review summarizes the progress in optical graphene gas sensors based on microfibers. By optimizing the optical sensing structures, this type of sensor has made significant advancements in sensitivity, which is summarized in 3, H2O, or NO2, and these sensors lack selectivity. Towards the realization of graphene-based microfiber sensors for other gas detection, the graphene material itself would be further functionalized, such as by using element-doped films [In this article, we review the principles, fabrications, implementations, and performances of optical graphene gas sensors with microfibers, which have attracted intense interest in research and development and play an important role in industry. The mode of “graphene and microfiber” has become a widely used platform for—but not limited to—gas detections. In this way, higher sensitivity and better selectivity are constant pursuits, while still remaining challenges. On one hand, to increase the sensitivity, more and more new optical mechanisms and techniques are being reported, such as graphene-based laser sensing , high-ored films , graphened films , or fluoed films . With th


TBX5 3′ untranslated region (UTR) variations with congenital cardiac malformations. This study aimed to investigate the relationship between TBX5 3′UTR variants and risk for congenital heart disease (CHD) susceptibility in two Han Chinese populations, and to reveal its molecular mechanism. The relationship between TBX5 3′UTR variants and CHD susceptibility was examined in 1 177 CHD patients and 990 healthy controls in two independent case–control studies. Variant rs6489956 C>T was found to be associated with increased CHD susceptibility in both cohorts. The combined CHD risk for the CT and TT genotype carriers was 1.83 times higher than that of CC genotype, while the risk for CT or TT genotype was 1.94 times and 2.31 times higher than that of CC carriers, respectively. Quantitative real-time PCR and western blot analysis showed that T allele carriers exhibited reduced TBX5 mRNA and protein levels in CHDs tissues. Compared with C allele, T allele showed increased binding affinity to miR-9 and miR-30a in both luciferase assays and surface plasmon resonance analysis. Functional analysis confirmed that miR-9 and miR-30a downregulated TBX5 expression at the transcriptional and translational levels, respectively. The assays in zebrafish model were in support of the interaction of miR-9/30a and TBX5 3′UTR . We concluded that TBX5 3′UTR variant rs6489956 increased susceptibility of CHD in the Han Chinese population because it changes the binding affinity of two target miRNAs that specifically mediate TBX5 expression.TBX5 is a vital transcription factor involved in cardiac development in a dosage-dependent manner. But little is known about the potential association of  Congenital heart diseases (CHDs) are the most prevalent type of human structural birth defects worldwide, affecting between 19 and 75 per thousand live births ,3,4,5. HTBX5 invariably result in Holt-Oram syndrome (HOS), an inherited disease characterized by upper limb and cardiac deformities [TBX5 might be associated with increased CHD risk through altered gene expression or dosage effects.Genome-wide association studies (GWASs) suggest that a large number of non-coding variants contribute to the risk of various diseases, including cardiovascular disease ,8,9,10.ormities , 17. ThuTBX5 is a transcription factor with well-defined roles in heart and forelimb development. Multiple studies in animal models have confirmed that cardiac development is sensitive to Tbx5 dosage , 18, 19.TBX5 in the promoter [TBX5 3′UTR to CHDs remains unknown. Our previous study demonstrated that miR-10a and miR-10b significantly repressed TBX5 protein levels by targeting its 3′UTR [TBX5 3′UTR that significantly increases CHD risk in a Han Chinese cohort composed of 1 177 CHD patients and 990 healthy controls. We further investigated the molecular mechanism underlying that TBX5 3′UTR variants influence risk for CHD susceptibility.Recently, several non-coding variants of promoter , intron promoter  and enhapromoter  were repts 3′UTR . In presTBX5 3′UTR were identified. Among them, four variants had minor allele frequency (MAF) >0.05 and were chosen for genotyping in 288 cases and controls of the Shandong group. Among them, the prevalence of variant rs6489956 (c.* 1101C>T) was significantly different between the CHDs and the control subjects. This variant was then selected for further validation in the other two independent case–control cohorts , as well as 272 CHDs and 384 controls in the Shanghai cohort . The combination of these two independent studies showed a 1.83-fold increased CHD risk when the dominant model is used for TBX5 rs6489956 (P=3.62×10−8) in a total number of 1 177 CHDs patients and 990 controls (P=8.21×10−8) and 2.31  for the CT and TT genotypes, respectively. Therefore, The variant rs6489956 was significantly associated with an increased risk of CHD in two cohorts separately and together. Similarly, the allele analysis showed that carriers of the susceptible T allele exhibited a higher risk of CHD than carriers of the C allele in both cohorts, resulting in a 1.86-fold combined higher susceptibility of CHD . All genotype frequencies were in accordance with the Hardy-Weinberg expectation among the control subjects (P>0.05).As the homozygous TT genotype was very rare, we applied the dominant model of inheritance to enhance the statistical power of the study, which combined homozygous TT alleles with heterozygous CT alleles to compare with the wild-type CC alleles in the association studies. Logistic regression analyses demonstrated heterozygote CT and homozygote TT subjects had a significantly increased risk of CHD compared with those with the wild-type CC allele in a study of 905 CHDs and 606 controls in the Shandong group (odds ratio (OR)=2.33, 95% confidence interval (CI)=1.70–3.19, controls , with anTBX5 c.*1101C>T (rs6489956) is related to multiple types of CHDs (P=9.70×10−6). In isolated phenotypes, similar statistical significance was noted in atrial septal defect (ASD) cases (P=3.67×10−3) and ventricular septal defect (VSD) cases (P=2.69×10−5).A stratified analysis was performed according to standard CHDs classifications as previously described , 24. It  of CHDs . NotablyTBX5 c.*1101C>T (rs648995) variant is located in the TBX5 3′UTR, we assumed that this variant might be involved in either TBX5 transcription or translation. Thus, we examined TBX5 mRNA expression using quantitative RT-PCR on the cardiac tissue samples. Of the 30 cardiac samples tested, 25 had the rs6489956 CC genotype, four had the CT genotype, and only one had the TT genotype. The relative TBX5 mRNA expression level in the CT/TT genotypes was only 49.3% of that of the CC genotype (P<0.05) (P=0.0037)  . Consist=0.0037) . AlthougTBX5 mRNA expression associated with the variant rs6489956 was further confirmed by in vitro luciferase assays in different cell lines. Consistent with the tissue sample results, the plasmid containing the mutant-T allele showed a 26%, 28% and 31% reduction in luciferase activity when compared with the wild-type C allele in HEK 293T, HCM and H9C2 cells, respectively (P<0.01)  . TherefoTBX5 3′UTR variant rs6489956, there are 16 software-predicted miRNAs including five upstream miRNAs, seven downstream miRNAs and four on-site miRNAs   . For the×10−6m) , it mighescribed .TBX5 caused by the 3′ UTR variant c.*1101C>T was demonstrated by a psiCHECK luciferase reporter assay as well as the rescue experiments from miRNA inhibitors. The luciferase gene expression of psiCHECK-C and psiCHECK-T were both inhibited in a linear manner with increased miRNAs concentration, and the miRNAs displayed a more pronounced inhibition effect on psiCHECK-T : wild-type (WT) control, standard MO control (5 ng), tbx5 MO (5 ng), tbx5 MO (2.5 ng), tbx5 MO (0.5 ng), MO (5 ng)+C allele mRNA (100 pg), MO (5 ng)+T allele mRNA (100 pg), MO (5 ng)+C allele mRNA (100 pg)+hsa-mi9 (100 pg), MO (5 ng)+T allele mRNA (100 pg)+hsa-miR-9 (100 pg), MO (5 ng)+C allele mRNA (100 pg)+hsa-miR-30a (100 pg) and MO (5 ng)+T allele mRNA (100 pg)+hsa-miR-30a (100 pg). As previously reported [tbx5 loss-of-function in zebrafish caused heart developmental defects, including different degrees of heart looping arrested, heart edematous, string-like heart, and so on, which were confirmed in our results  was found to significantly increase CHD risk in Han Chinese by interacting with miR-30a and miR-9 . Although we first postulated that a functional variant in the TBX5 3′UTR may increase the risk of CHD, a recent study indirectly supported our findings by reporting that a TBX5 enhancer variant causes isolated congenital heart disease [TBX5 3′UTR and the risk of CHD need further exploration in other ethnic groups. However, our study elucidated that the sporadic CHDs could be attributed to the combined contribution of the common regulatory variations in the TBX5 gene, as well as the epigenetic factors such as miR-30a or miR-9.It is well known that the mutations in syndrome ,34,35,365 dosage , 37, 38. disease . BecauseTBX5 non-coding variants with increased CHD risk may be explained by the sensitive nature of the dosage effect of TBX5 on heart development. While severe loss-of-function mutations in TBX5 coding regions led to both heart and forelimb phenotypes, non-coding variants were associated with compartmentalized phenotypes, which might reflect the differing sensitivities of the impacted tissues to the altered TBX5 expression level. This contention is supported by the fact that the TBX5 intronic variant rs7312625 was reported to be associated with altered PR intervals . In African Americans, and intronic variant rs3825214 was shown to reduce lone atrial fibrillation risk in Han Chinese [The possible mechanism of underlying the association of  Chinese , 40. TheTBX5 expression can be observed throughout the epicardium and myocardium, including the free walls and septa of all four cardiac chambers, while its expression in the endocardium is limited to the left ventricle [Tbx5 mRNA expression is restricted to the atria and left ventricle. Consistent with these expression patterns, HOS patients with TBX5 mutations may have a number of significant heart defects, such as septal defects and certain complex malformations , APVR and hypoplastic left heart) [n=830, P=9.70×10−6). Additionally, we speculated that the failure to detect any correlation between variant rs6489956 and APVR and outflow tract malformation susceptibility might be due to the limited and insufficient sample size.In both embryonic and adult human heart tissues, entricle . Howeverentricle  and chicentricle , Tbx5 mRt heart) , 44. In in vivo and in vitro assays on the function of variant rs6489956 indicated that the T allele led to reduction in TBX5 gene expression. We further identified two miRNAs, miR-9 and miR-30a, that interacted with this variant. This is not surprising, because miRNAs are predicted to function in the post-transcriptional regulation of nearly one third of all human genes [TBX5 mRNA, but reduced its protein concentration, while miR-9 downregulated both transcription and translation of TBX5. It has been extensively reported that miRNAs help to regulate cardiac growth and remodeling, and contribute to specific myocyte properties. miRNAs are implicated in numerous cardiovascular diseases, such as heart hypertrophy, heart failure, atherosclerosis, acute myocardial infarction, VSD, TOF and arrhythmias [TBX5 was disturbed during a sensitive time window of development, it increases the likelihood of inducing CHD. As a new model trigger, the interactions between miRNAs and TBX5 regulatory variant increase the CHD risk through the downregulated TBX5, either at the post-transcriptional or translational level.Both an genes . It was hythmias . Thus, wHand2-deficient mice [Because miRNAs are vital factors that mainly target 3′UTR sequences of mRNA, we suspected that miRNAs might have important roles in CHDs. Both bioinformatic predictions and functional analyses showed that both miR-30a and miR-9 have a higher affinity to the minor T allele. We further confirmed these results using luciferase assays, quantitative RT-PCR, western blot analysis and whole-embryo microRNA sensor assay in zebrafish. Recently, many studies have implicated miRNAs in various cardiovascular diseases. For example, miRNA-1–2 knockout mice have decreased levels of the cardiac transcription factor Hand2, which results in heart malformations (VSD) similar to those observed in ent mice , 54. ComTBX5 3′UTR variant rs6489956 on the increased susceptibility of CHD in two independent Han Chinese populations. It was well known that both Tbx5 overexpression and insufficiency result in heart defects in developing embryos. In view of the decreased TBX5 mRNA levels detected in the heart tissues of CHD patients with the CT and TT genotypes, the individuals with the T allele are susceptible to develop CHD due to the TXB5 deficiency. Functional studies clearly showed that the consequent repression of TBX5 expression on the post-transcriptional level resulted from an enhanced binding affinity of the T allele to miRNAs. Our data highlighted an important function of the underlying genetic variations in the 3′UTR of TBX5 in CHDs, providing new insight into risk assessment for these common birth defects.In summary, our study demonstrated a significant effect of We analyzed samples from two independent case–control groups. The Shandong group consisted of 905 CHD patients and 606 matched controls recruited from the Cardiovascular Disease Institute, General Hospital of Jinan Military Command  between August 2009 and September 2011. The Shanghai group was composed of 272 CHD cases and 384 matched controls enrolled in the Children’s Hospital of Fudan University  between August 2010 and November 2011. All subjects were unrelated ethnic Han Chinese. All of the controls were non-CHD outpatients from the same geographical area who were matched to the affected individuals in terms of age and sex over the same period. CHDs patients who were considered to be syndromic and not isolated CHDs, or who had a positive family history of CHD in a first-degree relative , were excluded from the study.There were no newborn samples in our study. We classified the 1 177 CHDs cases into seven broad categories according to the commonly accepted criteria . SpecifiTBX5 gene, we selected a subset of 32 subjects for resequencing. Half of these subjects were CHD patients, and half were controls. Fifty-three cardiovascular tissue samples were obtained with informed consent from CHD patients who had undergone cardiac operations from January 2011 to September 2011 in the Cardiovascular Disease Institute, General Hospital of Jinan Military Command  and Children’s Hospital of Fudan University , among which 30 were used for quantitative real-time PCR (RT-PCR) assay and 23 for western blot analysis. The 53 collected cardiac tissue samples comprised 10 atrium samples, 4 ventricle samples, 4 appendage sample, 8 atrial septum samples, 4 ventricular septum samples, 15 outlet samples, and 8 aorta samples. Normal heart tissue samples were collected by the Department of Forensic Medicine, Fudan University from adults with no history of heart disease who died of accidents.To screen for genetic variants in the 3′UTR of All study protocols were reviewed and approved by the medical ethics committee of Children’s Hospital of Fudan University, and written consent was obtained from the parents of subjects and/or the subjects themselves before study enrollment.TBX5 3′UTR fragments were amplified using PCR to amplify a 1757-bp fragment from 114793401 to 114791645 (NM_000192) in 32 unrelated individuals randomly selected for variant screening by sequencing. Direct dye terminator sequencing of the PCR products was carried out using the ABI Prism BigDye system according to the manufacturer’s instructions . Selected single-nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) >0.05 were genotyped using SNaPshot analysis (ABI). In the first stage of the present study, a total of 288 CHD cases and the same number of controls of the Shandong group were collected and genotyped in October 2010. The variants associated with CHD risk were chosen for further case–control study in the following Shandong and the Shanghai samples. The samples were sequenced and genotyped on an ABI 3730 automated sequencer and analyzed using Lasergene 7.0 and Peakscanner 1.0, respectively. All of the PCR and sequencing primers were listed in Genomic DNA was isolated from the peripheral venous blood using conventional regents. Total RNA was extracted from human cardiovascular tissue samples preserved in RNAlater  and converted to cDNA using random hexamers, oligo(dT) primers and PrimeScript RT reagent Kit . Quantitative Real-time PCR was performed with SYBR Premix Ex Taq (TaKaRa) using a StepOne Real-Time PCR system (ABI) with β-actin as an internal reference gene. Each reaction was performed in triplicate. The primers were described in Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide electrophoresis gels and blotted on to polyvinylidene fluoride membranes . Non-specific binding was blocked with 5% skimmed milk in TBS. Blots were probed with either mouse horseradish peroxidase (HRP)-conjugated anti-β-actin monoclonal antibody , or rabbit anti-TBX5 polyclonal antibody  followed by HRP-conjugated goat anti-rabbit secondary antibody . Proteins were detected using ECL reagents (Millipore).TBX5 3′UTR containing either the C or T allele was amplified from genomic DNA and subcloned into the downstream region of Renilla luciferase using XhoI and NotI restriction sites in psiCHECK-2 vector . Primers were listed in To construct the reporter plasmids, TBX5 ORF fused to PCMV6-XL4 was purchased from Origene Technologies . The mutant TBX5 was generated by the the Quick Change Site-Directed Mutagenesis Kit (Stratagene). Constructs were linearized using PvuII and the capped and polyA tailed mRNA was synthesized with mMessage mMACHINE T7 Ultra Kit  for the zebrafish microinjection.Human wild-type Human embryonic kidney 293T (HEK 293T), human cardiac myocytes (HCM) and rat cardiac myocyte (H9C2) cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum.5, 2.0×105 and 1.0×105 per liter, respectively. Twenty-four hours later, the cells were transfected with 400 ng of each TBX5 reporter plasmid using Lipofectamine 2000 . To screen the predicted candidate miRNAs, HEK 293T cells were co-transfected with 25 ng of psiCHECK-C/T plasmid and 75 ng of miRNA expression vector in 96-well culture plates. To further explore the interaction between TBX5 3′UTR and selected candidate miRNAs, HEK 293T, HCM or H9C2 cells were co-transfected with 50 ng psiCHECK-C/T plasmid as well as 0 ng, 50 ng, 100 ng, 150 ng or 200 ng of miRNA expression vector. For small interfering RNA assays, HEK 293T cells were co-transfected with 100 ng psiCHECK-C/T plasmid and 100 nm miRNA inhibitors  in 24-well culture plates.HEK 293T, HCM or H9C2 cells were seeded in 24-well culture plates at the density of 4.0×10Cells were lysed 24 h after transfection for luciferase assays that employed the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Three independent transfection experiments were performed, and each luciferase assay was conducted in triplicate.m of miRNA inhibitors in 6-well culture plates. Cells were harvested and lysed 48 h after transfection in RIPA buffer and assayed for the relative protein concentrations of intracellular TBX5  and β-actin (Kangchen Bio-tech) using western blot analysis. RNA was also purified from these cells for quantitative RT-PCR analysis of TBX5 mRNA levels.HEK 293T cells were transfected with 2 μg of miRNA expression vector or 100 nm. Single-stranded RNA  harboring 32 bp of 3′UTR wild-type-C allele RNA or 3′UTR mutant-T-allele was diluted into a variety of concentrations in PBST buffer  and flowed over the biotinylated miRNAs. At the end of each cycle, 5 mm NaOH was applied to regenerate the sensor surface. Data were converted by BIA evaluation software.SPR analysis was conducted using the ProteOn XPR36 protein interaction array system . Biotinylated miRNAs were immobilized to an individual channel of the streptavidin-modified sensor chip at a fixed concentration of 400 nTBX5 mRNA and 100 pg miRNAs using a microinjector . Twelve hours post-injection, the dead embryos were removed, leaving only viable embryos that were used for further analysis. Consistent with the previously published studies [−1 tricaine/1% 1- phenyl-2-thiourea  and embedded in 0.6% low melting agarose. Confocal imaging analysis was performed using Imaris software. Two transgenic zebrafish lines: Tg(vmhc:eGFP) and Tg(vmhc:mCherry-NTR) were used as described in previous work [The morpholino (MO) antisense oligonucleotide MO-Tbx5a (5′-GAAAGGTGTCTTCACTGTCCGCCAT-3′) was purchased from Gene Tools . Human miR-30a, mir-9 and a negative control were purchased from Thermo Fisher Scientific . Zebrafish were raised under standard conditions at 28.5 °C. Each 1-2-cell stage embryo was injected with a constant injection of 5 ng MO, 100 pg  studies , 55, allous work .χ2-test. Differences in demographic features and allelic or genotypic frequencies between the CHD cases and controls were evaluated using Pearson’s χ2-test. To evaluate associations between the genotypes and CHD risk, ORs and 95% CIs were calculated by unconditional logistic regression analysis with adjustments for age and sex. In multiple comparison testing, we employed the Bonferroni correction. The significance level was adjusted to P=0.0025 given the four SNPs being tested for 5 genetic models in 288 cases and the same number of controls.The Hardy-Weinberg equilibrium test was performed on controls using the t-test with Bonferroni correction. The P<0.05 was defined as statistical significance. Statistical analyses were conducted with SPSS 16.0 software .Data were calculated as the mean±s.d. Significant differences between the test and control groups were analyzed by means of an independent


It is reported recently Tropomyosin-related receptor Kinase B (TrkB) plays key roles in the anoikis resistance during the processes of tumorigenesis and metastasis. However, its prognostic significance for clinical patients remains inconclusive. In order to establish a correct and practicable link between increased TrkB and prognostication of human solid tumors, a meta-analysis was performed in this article. A systematic literature research in the electronic databases PubMed, Embase and Web of Science was performed to identify eligible studies. A fixed-effects meta-analytical model was employed to correlate TrkB expression with OS, DFS and clinicopathological features. A total of 11 studies covering 1516 patients with various solid tumors were recruited in this meta-analysis. TrkB over-expression was associated with poorer OS and poorer DFS in multivariate analysis. Additionally, the pooled odds ratios (ORs) indicated that TrkB over-expression was associated with large tumor size, lymph node metastasis, distant metastasis and a higher clinical stage. Overall, these results indicated that TrkB over-expression in patients with solid tumors might be related to poor prognosis and serve as a potential predictive marker of poor clinicopathological prognosis factor. Anoikis is a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding extracellular matrix –3. AnoikTropomyosin-related receptor Kinase B (TrkB) is a tyrosine kinase receptor for brain-derived neutrophic factor (BDNF), which triggers several intracellular signals –10. TrkBIn this study, we conducted a meta-analysis to investigate the significance of over-expressed TrkB in the prediction of prognosis of corresponding patients. The results showed that increased TrkB expression in patients with solid tumors related to poor prognosis and served as a potential prediction of poor clinicopathological prognosis factor.The initial search identified 315 publications, of which, 18 studies were of acceptable relevance. However, four of these studies were excluded because the survival curve was based on NTRK2 expression, and three were excluded because of the absence of information about clinicopathological characteristics. Ultimately, 11 studies met the eligibility criteria and were included in the current meta-analysis  was observed when using a fixed-effects model to analyze the pooled HR of the OSs.The pooled hazard ratio (HR) revealed that over-expressed TrkB was significantly associated with poor overall survival (OS) for cancer victims in multivariate analysis .A significant correlation between over-expressed TrkB and disease-free survival (DFS) was also observed in the patients with solid tumors in multivariate analysis , which triggers several intracellular signals. TrkB is over-expressed in various human malignancies and growing evidence demonstrated its association with tumour cell proliferation, invasion and metastasis –31. TrkBIn this meta-analysis, we evaluated survival data from 1516 solid tumor patients from 11 eligible studies, which met the inclusion criteria, were organized according to OS and DFS. For all studies, TrkB expression was detected by IHC. By meta-analysis of the 11 studies, we identified the pool HRs which indicated that TrkB was a factor in poor prognosis in various cancers. Because there is no significant heterogeneity among our included studies, so we did not perform further subgroup analyses. Additionally, no publication bias was observed. These results indicated over-expressed TrkB was positive related to poor OS and DFS in solid tumor patients. In addition, pooled results of the correlations were identified between over-expressed TrkB and clinicopathological features of patients with solid tumors. The results showed the expression of TrkB was positively associated with large tumor size, lymph node metastasis, distant metastasis and a higher clinical stage. We can explain this result by TrkB’s ability to enhance anoikis resistance, promote tumour cell proliferation, invasion and metastasis. Because of its involvement in these processes, TrkB is likely to be causally involved in tumor progression and, consequently, increased levels of TrkB would be expected to indicate a poor prognosis.This meta-analysis was properly conducted, however, further analysis with several limitations would be considered in the future. first, need more trials to analysis; second, some of the survival data were extracted from Kaplan-Meier curves and might be less reliable than a direct analysis of variance; third, we need to search more non-English publications. In addition, the possible existence of unpublished studies could also result in potential publication bias. In general, concerning these limitations mentioned above, we must interpret these results with adequate caution.In conclusion, our meta-analysis indicated that over-expressed TrkB was positive related to poor OS and DFS in solid tumor patients. Over-expressed TrkB could be served as a potential marker for poor clinicopathological prognostic factors in patients with various solid tumors. However, additional studies related to specific tumor types are necessary to illuminate the clinical utility of increased TrkB in solid tumors.This systematic review and meta-analysis is reported in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) Statement. The literatures relevant to TrkB expression and survival in solid tumors were searched in the PubMed, Embase and Web of Science databases through March 31, 2017. The search terms included the following key words in various combinations: TrkB, NTRK2, prognosis, prognostic, survival, and overall survival. The hits were limited to human studies and those published in English. The references list of review and bibliographies were further sifted to identify additional potentially relevant studies to avoid omission due to the electronic search approach.The collected studies included in this meta-analysis had to meet the following criteria: (1) a pathological diagnosis of cancer was made; (2) TrkB expression in patients with any type of tumor was measured via immunohistochemistry; (3) associations of TrkB expression with OS, DFS or clinicopathological features were described; (4) HRs and 95% confidence intervals (CIs) were reported or could be calculated (based on the information in the paper); and (5) when the same author reported repeated results from the same population, the most complete report was included. The exclusion criteria for this meta-analysis were as follows: (1) unpublished papers; (2) laboratory articles, reviews and letters; (3) non-English language articles; (4) overlapping articles or ones with duplicate data; (5) articles with only animal experiments; (6) studies without information about survival curves; and (7) TrkB expression in patients with any type of tumor was analyzed only using RT-PCR method.All data were extracted independently by two investigators (Chunze Zhang and Dan Gao). For each eligible study, the following characteristics were extracted: first author’s name, publication year, region, type of cancer, number of patients, patients’ ages, follow-up times, detection methods, staining position, cut-off values, survival data (including OS and DFS) and clinicopathological parameters, such as tumor differentiation, tumor size, lymph node metastasis and TNM stage. For studies that presented only Kaplan-Meier curves was used to extract the survival data. The Newcastle-Ottawa-Scale (NOS) was adopted to assess the study quality of each individual study. The NOS score ranged from 0 to 9, and studies with NOS score ≥ 7 were defined as high-quality studies.Q test) and I-squared statistical test were used to analyze the heterogeneity between studies. When the result of a Q-test (I2 > 50% or P < 0.05) indicated heterogeneity, the random-effects model was used for the meta-analysis. Otherwise, a fixed-effects model was used. HR with its value over 1.0 indicated poor prognosis patients with increased TrkB expression. Funnel plots were used to graphically represent the publication bias. Begg’s (rank correlation) test was adopted to confirm the publication bias.This meta-analysis was performed using Stata 12.0  software. Pooled estimates of HRs and their 95% CIs were used to estimate the association between TrkB expression and patients’ survival. The chisquared test (Cochrane’ s


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 .