The search for new optical materials capable of absorbing light in the frequency range from visible to near infrared is of great importance for applications in optoelectronic devices. In this paper, we report a theoretical study of the electronic and optical properties of hybrid structures composed of fullerenes adsorbed on graphene and on graphene nanoribbons. The calculations are performed in the framework of the density functional theory including the van der Waals dispersive interactions. We found that the adsorption of the C Nanostructures based on carbon allotropes have been intensively studied in the last two decades due to the possibility of modulating and controlling their optical and transport properties for novel optoelectronic applications  and deveSeveral hybrid nanostructures with particular geometric configurations are formed with different carbon allotropes through covalent or non-covalent interactions, with complexes such as graphene–nanotubes, graphene–fullerenes, nanoribbon–fullerenes, nanotube–fullerenes and nanobuds . These hacitors,  and it hacitors, . Very rebricated .It is known by experimental and theoretical studies that the adsorption of fullerenes on pristine graphene is governed by van der Waals interactions ,11. CompIn this paper, we study theoretically the electronic structure and optical and electro-optical properties of graphene–fullerene nanohybrids. In the framework of the density functional theory (DFT), we have calculated the electronic band structure, the density of states (DOS) and the optical spectrum of two different types of hybridized configurations: COur results show that the energy spectrum and DOS for all hybrid structures are an ensemble of the overall features of its components, which is an expected result due to the type of interactions between the components. We found that the adsorption of the CIt is well known that graphene nanoribbons hold unique geometrical-dependent electronical properties that can be tuned by varying the width of the nanoribbon and the structure of its edges . Our calIn addition, we included an external electric field applied across the ribbon edges and we investigated the electro-absorption spectrum of two representative hybrids GR–CIn The adsorption energy is also influenced by the finite width of the nGRs, and in ulations , that ingraphite . In contFinally, in The main effects of the adsorption of fullerenes on graphene are manifested in its optical response, which is displayed in z direction. Each figure shows results for the (a) pristine nGR, and the hybrids: (b) nGR–CNext, in π bands coming from the pristine nanoribbon and localized states generated by the adsorption of the fullerene molecules. As a general observation, we can infer that the modification of the electronic structure of each nGR due to the fullerene adsorption is strongly dependent on the size of the fullerene molecule. The DOS for the studied complexes shows the emergence of many new resonances apart from those proper of the geometrical characteristics of the nanoribbons.Similarly, as in the case of the SLG–fullerenes systems, the energy spectrum of nGRs with adsorbed fullerenes displays a superposition of π bands of the graphene nanoribbons are clearly modified as a consequence of the re-hybridization with the molecule orbitals. In particular, this is more evident in Our results show that the main alterations in the electronic band structure of the pristine nanoribbons occur for the GRs of smaller band gap, 8GR  and 12GRz) and the right panels for perpendicular light polarization (across the ribbon-x). It can be observed that the principal signature of the adsorption of molecules on the nanoribbons is the strong light absorption in the range between 2 and 3 eV. This is true for all nanoribbons considered, independent of their width and of the light polarization considered.The optical response of the hybrid structures shows many rich features, as noted in In the range of lower energies . The fullerene physisorption on SLG and GR is assessed by the van der Waals density functional as proposed by Dion et al. [x and y directions for a SLG and the z direction for GRs was employed. For the SLG, we used an A represents a SLG or a nGR and the subindex B represents a fullerene.Our DFT calculations were carried out by using the SIESTA ab initio package, reference  which emn et al.  (equivalgraphene . This fugraphene . The supThe optical absorption spectrum was obtained through the dipolar approximation implemented in the SIESTA code. For each one of the systems, we have employed a Gaussian broadening of In this work, we have presented theoretical calculations of the electronic and optical properties of graphene–fullerene nanohybrids in the framework of the density functional theory using the SIESTA package including the van der Waals dispersive interactions. We have studied two different hybridized complexes: C


In sarcoidosis, the proinflammatory cytokines interferon gamma, tumour necrosis factor and interleukin-6 are released by monocyte-derived macrophages and lymphocytes in the lungs and other affected tissues. Regulatory receptors expressed on monocytes and macrophages act to suppress cytokine production, and reduced expression of regulatory receptors may thus promote tissue inflammation. The aim of this study was to characterise the role of regulatory receptors on blood monocytes in patients with sarcoidosis. Cytokine release in response to stimulation of whole blood was measured in healthy controls and Caucasian non-smoking patients with sarcoidosis who were not taking disease modifying therapy. Expression of the regulatory molecules IL-10R, SIRP-α/β, CD47, CD200R, and CD200L was measured by flow cytometry, and functional activity was assessed using blocking antibodies. Stimulated whole blood and monocytes from patients with sarcoidosis produced more TNF and IL-6 compared with healthy controls. 52.9% of sarcoidosis patients had monocytes characterised by low expression of CD200R, compared with 11.7% of controls (p < 0.0001). Patients with low monocyte CD200R expression produced higher levels of proinflammatory cytokines. In functional studies, blocking the CD200 axis increased production of TNF and IL-6. Reduced expression of CD200R on monocytes may be a mechanism contributing to monocyte and macrophage hyper-activation in sarcoidosis. Sarcoidosis is characterised by increased inflammatory activity within tissue granulomata, with accumulation of activated lymphocytes and monocyte-derived macrophages (epithelioid macrophages) and local release of proinflammatory cytokines1256et al. reported that sarcoidosis blood monocytes produced smaller amounts of IL-10 than controls and were less able to suppress T cell proliferation14Regulation of inflammatory responses is vital to initiate resolution and prevent excessive tissue damageex vivo perturbation of monocytes. Monocytes are shown to be a source of TNF and IL-6 within stimulated whole blood assays, and monocytes from sarcoidosis patients produced these cytokines to a greater extent than healthy volunteers. Patients with sarcoidosis more commonly had monocytes expressing low levels of CD200R, whereas other regulatory receptors  were expressed at normal levels. Finally, blockade of CD200R or CD200L led to increased production of TNF and IL-6. Collectively, the data argue that reduced expression of CD200R is an important mechanism underlying monocyte/macrophage hyper-responsiveness in sarcoidosis.The aim of the present study was to assess the role of regulatory receptors in modulating monocyte cytokine production in sarcoidosis. A whole blood assay was chosen for monocyte stimulation to minimise The demographics and clinical details of study participants are shown in ex vivo stimulation studies. Significantly higher concentrations of secreted TNF and IL-6 were found in stimulated whole blood from patients with sarcoidosis compared with healthy controls  compared with 9.3% ± 0.82, p < 0.05) and reduction in classical CD14++/CD16− monocytes  (+/CD16++ monocytes between the cohorts (7.9% ± 1.51 compared with 7.1% ± 0.76).Monocytes appeared to be responsible for elevated TNF and IL-6 responses in sarcoidosis patients, so monocyte subsets were characterised based on relative expression of CD14 and CD16 < 0.01) . There wMonocyte- and macrophage-derived TNF can be regulated by a variety of cytokines and cell surface receptor-ligand interactionslow compared with 11.7% of healthy donors (P < 0.0001)  . Using a 0.0001) . Monocytcontrols .+ T lymphocytes was similar in sarcoidosis patients and healthy subjects (data not shown). However, because of the T cell lymphocytopenia there were fewer T cells expressing CD200L in sarcoidosis blood .Expression of the ligand CD200L was absent on monocytes, and the percentage of CD200LTo investigate how CD200L/CD200R interactions regulate proinflammatory cytokine release, monocyte CD200R expression was correlated with stimulated cytokine release in whole blood assays and PBMC cultures. In sarcoidosis, IL-6 responses were significantly higher in subjects with low monocyte CD200R expression when compared with high CD200R expressers , and a sTo determine whether the CD200L/CD200R axis was important for regulating cytokine production in whole blood, blocking studies were performed using antibodies. CD200R blockade increased cytokine release from isolated PBMCs from healthy subjects . TNF inclow monocyte phenotype. Low monocyte CD200R expression was associated with heightened TNF and IL-6 production in response to PHA stimulation, and blockade of CD200R or CD200L in healthy controls led to increased TNF and IL-6 secretion by monocytes, recapitulating the hyper-activated monocyte state seen in sarcoidosis.This study is the first to characterise the expression of the regulatory receptor CD200R and its ligand CD200L on blood mononuclear cells in sarcoidosis. We have demonstrated that the distribution of monocyte CD200R expression in a cohort of patients with sarcoidosis is distinctly different from healthy control subjects, with a significantly higher proportion of sarcoidosis patients exhibiting a CD200Rlow monocyte phenotype is not a manifestation of systemic inflammation. Thus monocyte CD200R appears to reflect a unique aspect of disease activity in sarcoidosis. Further studies are required to assess its value in predicting disease progression and response to anti-inflammatory therapy. Whether a similar hyper-responsive phenotype is exhibited by local tissue macrophages in sarcoidosis has yet to be determined; responses in peripheral blood monocytes may not translate directly to the macrophages within granulomata. Murine models suggest that in lung inflammation, monocytes actively contribute to resident macrophage populations5Monocyte CD200R expression was not associated with serum ACE activity, suggesting that CD200R is not simply a marker of total granuloma burdenDespite the evidence for active cellular immunity within tissue granulomata, previous research on peripheral blood in sarcoidosis has been subject to contradictory reports suggesting immune responses may be enhanced2628et al. reported that sarcoidosis monocytes had reduced IL-10 productionet al. studied monocytes that had been isolated by magnetic bead negative selection and stimulated with lipopolysaccharide+ CD14++ blood monocyte population was expanded. Since this monocyte subset has been shown to express the highest levels of the TNF receptor TNFR1The present study implicates blood monocytes as potentially important regulatory cells in sarcoidosis. Cranshaw + lymphocytes will be present in sarcoidosis blood for potential ligation of monocyte CD200R.Although the whole blood assay used in the present study has an advantage in minimally perturbing cells such as monocytes that are highly pleiotropic and respond rapidly to changing environments on isolation and in tissue culture, the assay does have some limitations. For example, due to T cell lymphocytopenia, fewer CD200L18F-fluorodeoxyglucose uptake on positron emission tomography imaging in an open label studyde novo development of sarcoidosis-like granulomatain vitro and in mice in vivo383940Anti-inflammatory drug treatment in sarcoidosis is generally considered to be clinically indicated if, following an initial observation period, there is evidence of progressive disease. Corticosteroid treatment reduces macrophage TNF release and leads to improvement in disease markers, but the effect of steroids on long term outcomes is uncertain and the benefits have to be balanced against the potential for serious side effects32The present study provides evidence that the CD200R/CD200L axis is important in modulating proinflammatory cytokine release from monocytes in sarcoidosis, and raises the possibility that disease progression could be propagated by reduced monocyte CD200R expression. There is a need for improved treatment options for patients with sarcoidosis, and the CD200L/CD200R axis is potentially tractable for future therapyEthical approval for the use of blood samples from healthy volunteers was obtained from the Ethics Committee, Hull York Medical School. Regional ethical approval was obtained to obtain blood and tissue samples from sarcoidosis patients from the Hull and East Riding local research ethics committee (LREC 08/H1304/54). Written informed consent was obtained from all subjects, and all methods were performed in accordance with the relevant guidelines and regulations of Good Clinical Practice. Patients with sarcoidosis were diagnosed by multi-disciplinary assessment and displayed non-caseating granulomata on histopathological analysis. Patients with other granulomatous diseases were excluded. Exclusion criteria for sarcoidosis patients and healthy volunteers were concomitant inflammatory or immunological diseases, treatment with corticosteroids or other immune-modulatory therapies, and current tobacco smoking. Blood was collected in sodium heparin tubes (BD) and processed within 1 hour. The results of each set of experiments informed subsequent experimental protocols so not all patients and controls contributed to all of the experiments. To avoid bias, subjects included in each set of experiments were not selected in any way. The numbers of subjects contributing to each experiment are shown in the figure legends.See 2 atmosphere.Heparinised whole blood was diluted 1:1 with sterile phosphate-buffered saline (PBS) and layered over Histopaque 1077 in a 1:1 ratio. The gradient was centrifuged at 600 × g for 30 minutes without braking. The PBMC layer was removed with a sterile pipette and washed twice before suspending in RPMI medium. Whole blood or isolated PBMCs were incubated at 37 °C in a 5% COStaphylococcus aureus enterotoxin A (SEA) were added to 1 ml aliquots of fresh whole blood in microfuge tubes and incubated at 37 °C for 16 hrs (1–72 hrs for time course experiments). Following centrifugation at 3000 × g for 8 minutes, plasma was collected and stored at −20 °C. Isolated PBMCs were resuspended in the same volume of RPMI media as the original blood volume and incubated under the same conditions as whole blood. ELISA kits  were used to measure IFN-γ, IL-10, TNF, IL-6, IL-4 and IL-12(p70) according to the manufacturer’s instructions. Plates were analysed using a Multiskan FC Microplate Photometer . Results below the minimum detectable limits with 1:2 dilutions of plasma were recorded as zero.Phytohaemagglutinin (PHA) or 5 cells) were suspended in 50 μl of FACS blocking solution containing antibody (10 μg/ml) and incubated at 4 °C in the dark for 1 hour. Cells were washed and resuspended and analysed using a FACSCalibur flow cytometer and Cellquest Pro software . Additional analyses were performed using Flowing Software 2.5.0 (Perttu Terho). Gates were defined using isotype control antibodies.PBMCs  or PBS for 1 hour at 37 °C, prior to addition of 100 μg/ml PHA. Supernatants were collected after 16 hours and stored at −20 °C prior to ELISA.5 cells) in 96 well round bottom plates were treated with Brefeldin A to ensure cytokine accumulation for 1–16 hrs. Cells were blocked for 1 hour, incubated with fluorophore-conjugated CD3 or CD14 antibodies for 1 hour on ice, fixed with 4% paraformaldehyde, permeabilised with ice cold 90% methanol for 30 minutes, and blocked. Intracellular staining with directly conjugated IL-6, IL-10, TNF, IFNγ antibodies or isotype control IgG (10 μg/ml) was performed at 4 °C for 1 hour followed by flow cytometric analysis.PBMCs . Statistical significance was determined using Mann-Whitney U, Kolmogorov–Smirnov, or Kruskal-Wallis tests for non-parametric distributions and Student’s T-test, one-way ANOVA or two-way ANOVA for parametric distributions. Multiple comparisons underwent Sidak, Tukey’s or Bonferroni post-hoc analyses as appropriate.How to cite this article: Fraser, S. D. et al. Reduced expression of monocyte CD200R is associated with enhanced proinflammatory cytokine production in sarcoidosis. Sci. Rep.6, 38689; doi: 10.1038/srep38689 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We discuss how our methods can be used for the rapid determination of graphene quality and can complement Raman Spectroscopy techniques. Lastly, we explore a PN junction embodiment which further validates that our fabrication techniques can rapidly adapt to alternative device architectures and greatly broaden the research applicability.Historically, graphene-based transistor fabrication has been time-consuming due to the high demand for carefully controlled Raman spectroscopy, physical vapor deposition, and lift-off processes. For the first time in a three-terminal graphene field-effect transistor embodiment, we introduce a rapid fabrication technique that implements non-toxic eutectic liquid-metal Galinstan interconnects and an electrolytic gate dielectric comprised of honey. The goal is to minimize cost and turnaround time between fabrication runs; thereby, allowing researchers to focus on the characterization of graphene phenomena that drives innovation rather than a lengthy device fabrication process that hinders it. We demonstrate characteristic Dirac peaks for a single-gate graphene field-effect transistor embodiment that exhibits hole and electron mobilities of 213 ± 15 and 166 ± 5  However, to explore such scientific phenomena one must be trained and certified in sophisticated optical characterization and microfabrication techniques to design and fabricate graphene devices such as graphene field-effect transistors (GFETs). Furthermore, the complexity of the required fabrication steps must be conducted in a controlled environment to increase device yield and minimize exposure to harmful chemicals. This process can be quite time-consuming and demanding depending on the scope of the device architecture and the allotted time given for expenditure of project funds. Such drawbacks reduce the accessibility of graphene research across academic and private institutions.The lowering cost of graphene synthesis has created opportunities that include lightweight electronics such as wearable, flexible, and electromagnetic sensors7. Unfortunately, numerous articles have reported degradation in graphene transistor performance due to mechanical and electrical strain put on graphene that is a direct result of photolithography and physical vapor deposition10. In addition, graphene has been shown to exhibit high contact resistance with standard electrode materials relative to its size that create non-ideal performance across the board11. This performance limitation will potentially force a researcher back into the cleanroom to identify drawbacks in his or her fabrication process.In a typical GFET fabrication process, graphene is synthesized via chemical vapor deposition and transferred onto a target substrate via delamination and the graphene quality is measured via Raman Spectroscopy. Lift-off processes are then used to construct contact electrodes, gate-dielectrics, and gate-contactsLM-GFET). We demonstrate comparable GFET performance with the proposed inexpensive, non-traditional materials to much more common ionic gel materials13. Galinstan is a non-toxic liquid-metal alloy consisting of 68.5% gallium, 21.5% indium, and 10% tin14 that possesses desirable conformal properties. In a previous study, Galinstan device interconnects exhibited less than a 5.5% change in resistance for a graphene two-terminal device when subjected to repeated deformations as small as 4.5 mm radius of curvature15. Moreover, graphene transistors can greatly benefit from the conductivity of Galinstan (2.30 × 106 S/m), a desirable vapor pressure (<1 × 10−6 Pa at 500 °C) compared with mercury (0.1713 Pa at 20 °C), and a stable liquid state across a broad temperature range (−19 °C to 1300 °C)16. Honey is typically produced via sugary secretions from bees, harvested, and packaged under various brand names for commercial food consumption. Honey contains various concentrations of water, vitamins, minerals, amino acids, and sugars: fructose, glucose, sucrose that can be controlled via bee production and honey extraction techniques17. To our benefit, honey formulates an ionic gel-like solution analogous to ion gels. Ion gels consist of room-temperature ionic liquids and gelating triblock copolymers13. Recently, ion gels have demonstrated ideal performance as electrolytic gate dielectrics for flexible GFET devices due to an ability to produce extremely high capacitance and high dielectric constants required for high on-current and low-voltage operation12. The introduction of honey as an electrolytic gate dielectric is advantageous for rapid fabrication of GFET devices due to the commercial availability, non-toxicity, control of ionic content that can be used to alter dielectric properties, and quick mixing that reduces preparation time of honey. Currently, ion gels require special preparation in an atmospheric controlled environment to mitigate outgassing and combustion, which only adds to its complexity.In this paper, we demonstrate the rapid fabrication of a three-terminal graphene-field effect transistor embodiment with the use of commercially available eutectic liquid-metal Galinstan interconnects and electrolytic gate dielectric comprised of honey imide ([EMIM][TFSI]) ionic liquid in Polystyrene-Poly (Ethylene Oxide) (PS-PEO) diblock copolymer. A detailed description of the ion gel synthesis is given in the Methods section. Figure ON/OFF, ratio as a function of top-gate voltage (ION/IOFF − VTG). The transport characteristics are presented as a ratio of ION and IOFF due to the significant difference in drain-to-source current between the sample measurements. The maximum operating current of the device with ion gel was 124 ± 10 μA with an ION/IOFF = 1.7, where Vds = 1.2 V. The maximum operating current of the device with honey was 620 ± 40 μA with an ION/IOFF = 3.1. A model fit determined the hole and electron mobilities for the ion gel device was 61 ± 3 and 189 ± 3 cm2/V · s and 100 ± 4 and 126 ± 5 cm2/V · s respectively for the Honey LM-GFET.Transport characteristics were compared with an ion gel ON/OFF ratios. The high capacitance of honey (~2 μF/cm2) enabled a significant electric field-effect, hence enabled a large drain current. The opposite was seen for the lower capacitance of the ion gel (0.1 μF/cm2). The reduced field-effect of the ion gel device may be limited by a relatively high gate leakage current that is typical of electrolytic gate dielectrics23. The gate leakage current of the ion gel was measured to be 20 μA, which was 4x greater than the honey. Although, the gate leakage was high for the ion gel device, the formation of the electric double layer at the graphene/ion gel interface allowed for ambipolar operation. In addition, the dirac peaks measured for ion gel devices were not as sharp as the dirac peaks measured by the honey devices. This phenomenon may be indicative of charge inhomogeneity at the electric double layer surface and can be caused by improper/incomplete mixing of the ionic liquid and copolymer solutions.The importance of a high capacitance electrolytic gate dielectric became clear in our comparison of the honey and ion gel Due to the liquid properties of honey and liquid-metal, these materials may be rinsed in boiling deionized water after initial measurements have been completed. The authors include an investigation of the electrical performance of graphene before and after rinsing of the liquid-metal electrodes and electrolytic gate dielectric made of honey. It was determined there is a slight change in the electrical performance of the graphene devices after rinsing, Fig. cm2/V · s respectively at a drain bias of 100 mV. After rinsing, hole and electron mobilities are 88 ± 3 and 165 ± 5 cm2/V · s respectively, at a drain bias of 100 mV. Notably, the extracted hole mobility increased, while the electron mobility slightly decreased. While this can be due to the nominal variance of each of the extracted values, it is believed that the DI water with subsequent heating removed several charge impurities that would have otherwise contributed to cross-sections of electron/hole scattering24. Therefore, the experimental results suggest that the proposed rinse process minimally impacts device performance, and yet allows designers to rapidly and prudently explore new device architectures with the same graphene material. The reuse of graphene is an incentive to reduce carbon waste.Despite a DI water rinse, there is no significant shift of the charge neutrality point (Dirac peak). Additionally, the mobilities extracted from the model fit show negligible, if any, degradation. The hole and electron mobilities before rinsing the LM-GFET are 46 ± 6 and 189 ± 1 25. However, the equipment required to perform these measurements is costly, and the measurements can be time-consuming. Due to the optical magnification necessary for spatially dependent graphene Raman measurements, investigations are limited to a few grain boundaries. Moreover, inhomogeneity across graphene due to topographical imperfections and varying concentration of dopants can change quite drastically in large scale devices26. In GFET devices, charge carriers encounter numerous grain boundaries from source to drain. Transport characteristics such as Current-Voltage (I–V) measurements average many grain boundaries and enable a way to analyze the electrical performance of large graphene channels .Raman spectroscopy is the industry standard for graphene characterization and provides a researcher with the number of graphene layers, as well as the impurities or dopants present within a graphene materialG band of graphene, Fig. G Band is not easily identifiable and one must take great care when analyzing Raman data. To identify the G band, static measurements are required and consist of several prolonged exposure acquisitions that increase the signal-to-noise ratio27, Fig. ID2/IG) ratios can be computed to extract the number of graphene layers. The authors’ best attempt for Raman Spectroscopy characterization of graphene on PET with post-processing to remove the PET signature took approximately 1 hour per sample for a single spot. To conduct Raman measurements of three separate samples (which is the industry standard) will take up to almost 3 hours with post-processing included. Moreover, a Raman Spectroscopy map of a graphene channel will take much longer.Raman spectroscopy of graphene transferred onto polymer substrates is quite challenging for unspecialized laboratories. The reason being, there exist strong polymeric vibrational modes thousands of times more sensitive to Raman scattering near the LM-GFET rapid fabrication methods to compare graphene on PET samples with both high and low quality that were previously determined via 514.5 nm Raman spectroscopy. The intention for this experiment was to validate the use of our methods as a useful tool to complement Raman Spectroscopy data for large scale graphene devices. As previously discussed, Fig. ID2/IG) ratios for Sites A–C are 3.10, 1.92, and 2.13 respectively, thus indicated high quality monolayer graphene. As was determined from I–V measurements, the hole and electron mobilities for the high quality graphene was on the order of 213 ± 15 and 166 ± 5 cm2/V · s respectively for a drain bias of 100 mV. On the other hand, the extracted (ID2/IG) ratios for a sample of low quality graphene was determined to be 2.44, 1.52, and 0.70 respectively. Despite being labeled low quality via the Raman spectroscopy measurements, our rapid fabrication methods determined the hole and electron mobilities were 128 ± 4 and 101 ± 4 cm2/V · s. Although, the computed mobilities are lower than what was measured in the high quality samples, the low quality samples were respectively still quite comparable.The authors utilized their proposed 25, and the location of the Dirac peak can provide crucial information on the impurities present.It has become common practice by commercial graphene manufacturers to provide graphene quality via Raman spectroscopy data of a few (1–3) spots with purchased graphene samples. Due to comparable results for the high and low quality graphene samples, there is reason to believe that our methods can complement Raman Spectroscopy measurements of large samples. One may consider graphene to be of low quality immediately after undesirable Raman spectroscopy measurements, and therefore may not utilize and dispose of the graphene sample. Our proposed methods provide an additional metric to explore graphene quality beyond spatially dependent Raman spectroscopy and in a transistor embodiment. The devices in this paper were relatively large. Users simply need to perform any I–V measurements of their fabricated devices, then use an automated code to extract electrical performance. The extracted mobility can be correlated to graphene quality as previously demonstratedFETs operate by the formation of junctions through an inherent electric field within a bulk semiconductor. The electric field can be created via chemical doping, yet will often be fixed upon fabrication. One can create a similar adaptive effect in a GFET device by manufacturing a lateral electric field with collinear wires suspended above graphene either in a dielectric, electrolyte, or slightly conductive liquid. Moreover, the electric field does not need to be referenced to graphene or any gate electrodes.Rds − VTG demonstrates typical GFET transport characteristics with a single Dirac peak. However, as the E-lateral is turned on, two Dirac peaks occur and the charge carrier distribution can be clearly identified.An assessment of such a transistor comprised of graphene and honey, shows that the newly formed lateral electric field  creates an effective PN junction because any free electrons within the graphene channel are swept to one side of the channel and any holes are swept to the other end. A representation of this idea is illustrated in Fig. VL of Fig. VR is set to a positive voltage. This creates a forward bias scenario when the source is set to ground. For example, when VL = −15 V and VR = +15 V, E-lateral = 30 V between the two terminals. Case B  as E-lateral was biased more negatively. This effect can be attributed to an excess of charge carrier diffusion to the electric double layer that increases with E-lateral30 and was similarly seen for a single-gated LM-GFET device operation. The opposite is true when the VL and VR were biased both positive and n-type doping occurred (right shift). An additional effect of biasing is the change in the drain-to-source resistance Rds maximum as the lateral electric field strength is altered, Inset of Fig. There was a noticeable shift in the overall transport characteristics of Case A and Case C. To explore this phenomena, Case B and Case D demonstrate cases in which Our transistors have demonstrated a method to rapidly characterize graphene materials with the use of non-toxic eutectic liquid-metal Galinstan interconnects and honey gate dielectrics in a three-terminal graphene field-effect transistor embodiment. The devices characterized in the paper were fabricated within less than 30 minutes and in a general laboratory setting. Our methods are repeatable, therefore one can adopt our methods into an automated quality assurance process at a per chip level for end-to-end fabrication increasing yield and eliminating tedious testing. Despite not being fabricated in a conventional cleanroom, our devices provided comparable performance to the current state-of-the-art. We demonstrated transport characteristics for a single-gate graphene field-effect transistor and introduced adaptive control over PN junction properties with only an applied lateral electric field bias. We anticipate an adaptive PN junction capability can be adopted into diodes. Furthermore, the manipulation of the physical characteristics of Galinstan is a precursor to flexible devices. Liquid-metal Galinstan can be embedded in microfluidic enclosures and exhibit shape deformability. There are many devices that can result from reconfigurability such as wearable diagnostics and conformal RF devices. Moreover, the liquid state of honey provides the potential for uniform and flexible gate dielectrics that are currently an issue for PVD-based gate dielectrics. In this paper, the authors only demonstrated a rigid architecture with inexpensive materials. The authors encourage the readers to explore alternate embodiments utilizing the liquid materials described and further explore the potential for flexible applications. The authors admit the use of liquid-metal Galinstan and honey for graphene devices in this paper was discovered by accident. We predict our transistors will lead towards the exploration of alternative materials that are slightly unconventional in the hopes these innovative discoveries provide a new class of materials that are non-toxic, biodegradable, and require minimal preparation time.nm (Green) Micro-Raman Spectroscopy System for three different graphene sites. The absence of a defect band D and analysis of the peak intensity ratio of the 2D and G Bands (ID2/IG ≈ 2) indicated high quality graphene in all three sites, Fig. mm3 each were then dispensed with a blunt-tip syringe to act as source and drain electrodes. Honey was commercially acquired and dispensed from a plastic dropper at a volume of 1.0 mm3 between the two liquid-metal droplets to act as the electrolytic gate dielectric. For the PN junction LM-GFET embodiment, two wires were suspended above graphene and inside the honey gate dielectric. Both wires were biased with an Agilent E3648A Dual Power Supply. Current, voltage, and capacitance measurements were performed with an Agilent 4155C Semiconductor Parameter Analyzer, probe station, and Hioki IM3570 Impedance Analyzer in air and in a standard laboratory environment.In this work, graphene was commercially acquired and a quality measure was conducted with a Renishaw InVia 514.5 LM-GFET devices with a plastic dropper and measurements were all conducted in a fume hood to reduce exposure to the ion gel.Due to the toxicity and degradation of the ion gel components in contact with atmospheric oxygen, synthesis was conducted in a nitrogen-purged atmospheric controlled glove box (Vacuum Atmospheres Company OMNI-LAB). Only when the glove box oxygen content was reduced to below 2 ppm, 1-Ethyl-3 Methylimidizalium Bis(Trifluoromethylsufonyl)imide ([EMIM][TFSI]) ionic liquid  was mixed with Polystyrene-Poly (ethylene oxide) (PS-PEO) diblock copolymer (weight 0.036 g) using a magnetic stirrer. 5.8 mL of acetonitrile solvent (weight 4.56 g) was added with a standard syringe needle to the ionic liquid/copolymer solution to thoroughly dissolve the copolymer. The final ion gel solution consisted of 9.21% [EMIM][TFSI] ionic liquid, 0.71% PS-PEO copolymer, and 90.07% acetonitrile solvent that was evaporated before I-V measurements. The ion gel was dispensed on the


Immune checkpoint blockade targeting PD-1/PD-L1 has shown efficacy in several types of cancers. However, the correlation between PD-L1/PD-1 expression and the specific clinicopathological features in papillary thyroid carcinoma (PTC) has not been investigated..We examined the immunohistochemical expression of PD-L1, PD-1, and BRAF V600E on whole-tissue sections from 126 cases of primary PTC more than 1 cm in size. The correlation between the PD-L1/PD-1 expression and the clinicopathological features was evaluatedPD-L1 was positively expressed in 53.2% PTCs, and its expression was positively correlated with rich tumor-infiltrating lymphocytes (TILs), background chronic lymphocytic thyroiditis (CLT), female gender, absence of psammoma bodies, and PD-1 expression. Among these parameters, rich TILs, female gender, and absence of psammoma bodies were independent factors affecting PD-L1 expression on the multivariate logistic regression analysis. PD-1 expression was detected in the TILs and was positively correlated with rich TILs, background CLT, and absence of stromal calcification. Lack of stromal calcification was an independent factor affecting PD-1 expression. Neither PD-L1 nor PD-1 expression showed significant correlation with BRAF V600E expression.Our results show that the distinctive pathological features of PTCs, including TILs, background CLT, female gender, psammoma bodies, and stromal calcification, are useful parameters for predicting PD-L1 or PD-1 expression. PTC is the most common malignancy of the endocrine organs, and its incidence has increased in the past decade. Although PTC overall has a good prognosis, more than 10% of patients with PTC develop recurrence or distant metastasis after surgery, and some of these patients with succumb to PTC –4. The BAs the largest endocrine organ in humans, the thyroid is frequently affected by organ-specific and systemic autoimmune diseases, including chronic lymphocytic thyroiditis (CLT), Graves’ disease, and Riedel’s thyroiditis. Among them, the most frequent condition associated with PTC is chronic lymphocytic thyroiditis , 9. ManyThe purpose of the present study is to answer the following questions: (1) What is the correlation between the PD-L1/PD-1 expression and the clinicopathological parameters, including a background of CLT in PTCs? (2) What is the relationship between the PD-L1/PD-1 expression and the BRAF V600E expression? (3) What clinicopathological factors affect PD-L1/PD-1 expression in PTCs?One hundred twenty-six (126) consecutive patients with a primary PTC measuring more than 10 mm in diameter who underwent surgical treatment in 2013 at Peking University Cancer Hospital, Beijing, China, were enrolled, including 96 females and 30 males. The patients’ age at surgery was 43.94 ± 13.15 (mean ± s.d.). All patients had no evidence of distant metastasis at the time of surgery. All histological subtypes of PTCs were included except microcarcinoma .Sections stained with hematoxylin and eosin from the 126 cases of PTC were histologically evaluated by two pathologists, BY and ND, to confirm the histological subtypes and to examine TILs, psammoma bodies, stromal calcification, bone formation within the tumor mass, and extrathyroid invasion, multifocality as well as background disease. Of these 126 PTCs, 114 cases were conventional type, 8 were follicular variants, 2 were solid variants, 1 was tall cell variant, and 1 case was a Warthin-like tumor variant.The evaluation of TILs in PTC was according to the evaluation of TILs in breast cancer recommended by an International TILs Working Group in 2014 . Most PTThe background disease was classified as CLT and non-CLT in the present study. According to Mizukami’s definition, CLT was divided into four groups: chronic thyroiditis, oxyphilic; chronic thyroiditis, mixed; chronic thyroiditis, hyperplastic; and chronic thyroiditis, focal . The firThe pT, pN, and stage groupings were categorized according to the cancer staging of the seventh edition of American Joint Committee on Cancer.Formalin-fixed, paraffin-embedded (FFPE) whole-tissue sections were cut to generate 4-μm unstained slides in 126 PTCs, including the tumor center and tumor invasive front, for PD-L1, PD-1, and BRAF V600E staining. All stained sections were examined microscopically and scored independently by three pathologists .PD-L1 staining was performed with an anti-human PD-L1 rabbit monoclonal antibody at working solution  on an automated staining platform  using a Bond Polymer Refine Detection kit  according to the manufacturer’s instructions. To verify the specificity of the immunostaining, primary antibody was replaced by phosphate-buffered saline from the staining procedure in a set of control sections. Patterns with 5% or greater positive tumor cells were considered positive, and those with less than 5% positive cells were considered negative .2O2 solution at room temperature for 10 min. Then the sections were incubated with mouse anti-PD-1 monoclonal antibody at working solution  at 37 °C for 15 min, followed by incubation with anti-mouse IgG-HRP conjugate  at room temperature for 25 min. Antibody binding was visualized using an EnVision FLEX DAB CHROMOGEN kit  according to the manufacturer’s instructions. For general negative controls, the primary antibodies were replaced by phosphate-buffered saline. Positive immunostaining of PD-1 was regarded as staining on ≥3 TILs per high power field  in the hot spot inside the tumor or in the tumor invasive front.For PD-1, the FFPE sections were deparaffinized and rehydrated, and then antigen retrieval was carried out in a PTLink machine . Endogenous peroxidase activity was blocked by incubation in 3% HBRAF V600E staining was performed using the anti-BRAF V600E mouse monoclonal primary antibody at working solution  on the Benchmark XT platform . The specific visualization of V600E-mutated BRAF protein was accomplished using the ultraView Universal DAB Detection Kit . Pre-validated BRAF V600E positive and negative PTC specimens were used as positive and negative controls, respectively. According to previous studies together with our experience, the immunoreactivity of V600E-mutated BRAF protein was evaluated as positive if unequivocal diffuse cytoplasmic staining of tumor cells was identified , 6, 22. P-value) was less than 0.05. Data analysis was performed using SPSS 17.0 statistical software.Time-independent and categorical data were evaluated using the chi-squared test, continuity correction, or Fisher’s exact probability test as appropriate. The multivariate logistic regression model was used to evaluate the multivariate analyses affecting PD-L1 and PD-1 expression. The differences were considered significant when the probability (PD-L1 expression was located in the membrane with or without cytoplasmic staining. The staining pattern was diffuse or focal Fig. . Two of PD-1 expression was identified in the lymphocytes both within the cancer and in the non-neoplastic thyroid tissue including CLT. Only PD-1 expression on the TILs both inside of and in the invasive front of PTC was counted Fig. . PD-1 waP = 0.004, Table P = 0.030, Table P = 0.020, 0.032, and 0.001, respectively, Table The presence of CLT usually indicates autoimmune disease of the thyroid, although it may overlap with non-specific lymphocytic thyroiditis. The presence of CLT was significantly correlated with both PD-L1 and PD-1 expression. PD-L1 expression was in 77.8 and 46.5% cases of PTCs with and without CLT in the background thyroid tissue, respectively (P = 0.011, Fig. Positive BRAF V600E staining Fig.  was seenClinicopathological features that are correlated with PD-L1 expression including rich TILs, a background disease of CLT, female gender, and the absence of psammoma bodies, together with PD-1 expression, were subsequently analyzed using the multivariate logistic regression analysis model. Among them, rich TILs, female gender, and the absence of psammoma bodies, were independent factors to predict PD-L1 expression Table . Rich TIIn this study, we examined the expression of PD-L1 and PD-1 in 126 cases of PTCs and analyzed their association with clinicopathological parameters. Both PD-L1 and PD-1 expression were correlated with CLT in the background thyroid tissue. Many studies have indicated that up to one-third of PTCs arose in a background of chronic thyroiditis. However, these studies did not have serologic evidence of preexisting thyroiditis . In our Expression of PD-L1 and that of PD-1 were significantly correlated each other in the present study, and PD-1 expression may influence the clinical significance of PD-L1 expression, and vice versa. A previous study has classified the tumor microenvironments into four groups based on PD-L1 expression and TILs . DiffereRecently, high expression of PD-L1 was reported by Chowdhury et al. to be correlated with invasiveness of PTC and poor disease-free survival . Our casThe constitutively active BRAF V600E protein has been associated with worse clinical outcomes in thyroid cancer . Angell We examined the expression of PD-L1, PD-1, and BRAF V600E in 126 cases of PTC, and found that distinctive pathological features in PTC, including TILs, a background of CLT, psammoma bodies, and stromal calcification, were useful parameters for predicting PD-L1 or PD-1 expression, which was a novel finding and should be highlighted in the forthcoming era of immunotherapy. Further studies are necessary to determine the prognostic and therapeutic value of these findings in PTCs.


Escherichia coli compared to bagasse-derived graphene, with both surfaces showing antifouling efficacy comparable to copper. Attachment of Gram-positive coccal Staphylococcus aureus was lower on the surfaces of both types of graphene compared to that on copper, with bagasse-derived graphene being particularly effective. Toxicity to planktonic bacteria estimated as a reduction in colony-forming units as a result of sample exposure showed that both graphenes effectively retarded cell replication.Antifouling efficacy of graphene nanowalls, i.e., substrate-bound vertically-oriented graphene nanosheets, has been demonstrated against biofilm-forming Gram-positive and Gram-negative bacteria. Where graphene nanowalls are typically prepared using costly high-temperature synthesis from high-purity carbon precursors, large-scale applications demand efficient, low-cost processes. The advancement of plasma enabled synthesis techniques in the production of nanomaterials has opened a novel and effective method for converting low-cost natural waste resources to produce nanomaterials with a wide range of applications. Through this work, we report the rapid reforming of sugarcane bagasse, a low-value by-product from sugarcane industry, into high-quality vertically-oriented graphene nanosheets at a relatively low temperature of 400 °C. Electron microscopy showed that graphene nanowalls fabricated from methane were significantly more effective at preventing surface attachment of Gram-negative rod-shaped  Recent times have witnessed a significant increase in the use of nanomaterials, especially graphene, for a wide range of applications, ranging from electronics to agriculture and manufacturing ,2,3. HowThe use of natural resources for the production of graphene and graphene-based products has been studied widely ,8. RecenEscherichia coli and Gram-positive Staphylococcus aureus. A fast and reactive plasma-enabled process is used for reforming sugarcane bagasse and methane gas into graphene. This particular graphene growth process does not involve toxic or hazardous gases and does not require any catalyst or external substrate heating to produce thin vertical graphene sheets on the same substrate. This method is also environmentally friendly, does not produce any chemical residue or waste, and is energy and material-efficient.This work investigates the antibacterial activity of graphene fabricated from raw, multicomponent, low-cost resources, compared to that of graphene derived from high-purity carbon precursor, against pathogenic multi-drug resistant bacterial species, namely Gram-negative −1 (G band), 1320 cm−1 (D band) and 2600 cm−1 (2D), which confirms the formation of graphene from different precursors.Successful formations of graphene from both precursors were confirmed by Raman spectroscopy. The Raman spectra were collected using a Renishaw inVia spectrometer  with laser excitations of 633 nm. The ratio of intensity of 2D and G bands indicates the number of layers of graphene. The increased ratio indicates the formation of thinner graphene flakes, whereas the ratio is smaller for thicker ones. Here, the thinnest flakes were obtained on plasma-treated copper substrate, where methane was converted to graphene.The morphology and elemental distribution of the graphene are characterized by Field Emission Scanning Electron Microscopy  employing an electron gun voltage of 10 kV with an energy dispersion spectrum .The SEM images in The energy dispersion spectra (EDS) for the samples are shown in Further characterization of the graphene nanosheets was performed using Transmission Electron Microscopy (TEM). The crystal images of graphene nanosheets were collected using JOEL 2100F HR-TEM . The electron beam energy used for this analysis was 200 keV.TEM samples were prepared by placing a drop of graphene dispersed in isopropanol on a carbon-coated copper grid and subsequently evaporating the isopropanol. E.coli and Gram-positive coccal S.aureus bacteria were investigated in terms of cell attachment and toxicity to planktonic cells. For this purpose, cell cultures were incubated in the presence of different graphene samples and copper in 30 µL of Luria broth at room temperature. After 4 h of incubation, the surfaces of the samples were visualised using SEM. As evident from E. coli compared to bagasse-derived graphene, with both surfaces showing antifouling efficacy comparable to copper. Attachment of S. aureus was lower on the surfaces of both types of graphene compared to that on copper, with bagasse-derived graphene being particularly effective.The antibacterial efficacy of two different graphene samples against Gram-negative rod-shaped E. coli bacteria, graphene samples fabricated from bagasse (GNW_B) showed considerable toxicity against planktonic bacteria. Although the cell numbers gradually increased over the period of incubation, the cell numbers were below (at 1 h) or similar to (at 2 h) those on copper. On the other hand, graphene derived from methane (GNW_M) effectively retarded cell replication, with cell numbers increasing only slightly during the first hour of exposure, and then deceasing to below the initial seed values. The efficacy of methane-derived graphene was significantly better than that of copper, a known broad-spectrum antibacterial agent. Cells incubated in the presence of copper surfaces first experienced limited antibacterial action from copper, with cell numbers reaching 9.3 × 107 CFU/mL. However, after 2 h of exposure, there was a significant reduction in the number of viable cells, attributed to the diffusion of copper ions from the surface of the substrate.Toxicity of the surfaces to planktonic bacteria was investigated by estimating the number of colony-forming units  at different times during the incubation period. The results of this study are summarized in E. coli bacteria, the growth of S. aureus was effectively retarded by graphene nanowalls from bagasse (GNW_B) and methane (GNW_M), with the latter being characterized by slightly lower numbers of surviving organisms at 2 h. The growth of cells incubated in the presence of copper was limited in the first hour of incubation. However, the cell numbers increased significantly after 2 h of incubation, and were substantially higher than those observed for cells incubated in the presence of graphene samples, reaching approximately 9.6 × 108 CFU/mL.In contrast to E. coli and S. aureus.As a broad-spectrum antifouling and antibacterial agent, GNW_M is more efficient than GNW_B for the pathogens tested, i.e., −1, D band at 1320 cm−1 and 2D band at 2600 cm−1 (2D). This confirms the formation of a graphitic structure [In this experiment, we successfully synthesised graphene using methane gas and sugarcane bagasse as the precursors. The Raman spectra of the obtained graphene  shows thtructure . Moreovetructure . TherefoThe high-resolution SEM images in The morphology of graphene layers grown by means of plasma-enhanced synthesis has been previously shown to depend on the chemistry and state of the precursor, as well as on the properties of the catalyst. The growth of vertical graphene is considered to be a step-flow process on the basis of nucleation at the bottom, and the thickness of graphene will depend on the number of layers that nucleates from the bottom .TransitiE. coli and S. aureus, shown in E.coli, resulting in contact bacterial inactivation on the surface [The antifouling efficacy of methane- and bagasse-derived graphene surfaces was comparable to or better than that of copper. There are several possible mechanisms that may be responsible for the bactericidal activity of graphene . The obs surface .S. aureus was lower on the surfaces of both types of graphene compared to that on copper, with bagasse-derived graphene being particularly effective. The electron transfer mechanism from a microbial membrane to graphene is another mechanism for the destruction of bacteria [Attachment of bacteria , which mbacteria .In addition to mechanisms associated with direct cell-surface contact, oxidative stress induced by graphene nanowalls may inhibit the bacterial metabolism and disrupt essential cellular functions, eventually leading to cellular inactivation. Oxidative stress can include reactive oxygen species (ROS)-dependent or ROS-independent pathways. In the former, the stress is induced by excess accumulation of extracellular ROS species, such as hydrogen peroxide, superoxide anions, hydroxyl radicals, and singlet molecular oxygen. These ROS species induce lipid peroxidation, intercellular protein inactivation and gradual disintegration of cell membrane, followed by the eventual cell death. ROS-independent oxidative stress oxidizes the vital cellular structure without ROS production, which can be induced by the charge transfer from cellular membrane to graphene, where graphene acts as an electron pump ,28.S. aureus is more efficient that E. coli in terms of surface attachment and production of extracellular polymeric substances on the surface of copper, which may mask the surface and prevent the process of contact-mediated membrane peroxidation. This is supported by the SEM images  and argon (Ar). Growth is carried out over a range of plasma exposure times, with best results obtained at 10 min. A gas mixture of H2/Ar/CH4 at a flow rate of 85/10/5 sccm, respectively, was fed into the chamber for deposition.In 2/Ar was fed into the chamber at a flow rate of 50 and 15 sccm, respectively. In both the experiments, the chamber pressure was maintained at 2.0 Pa and plasma was generated using RF power of 760 W and the deposition was carried out for 10 min.E.coli and S.aureus, bacterial cultures were refreshed on nutrient agar plates from a stock culture and grown overnight at 37 °C in a 5 mL of nutrient broth [600 = 0.1 bacterial suspension was placed on the surface of graphene nanowalls on fabricated on copper and nickel substrates and was allowed to incubate for 4 h at room temperature (22 °C) in a cell culture plate. Untreated copper and nickel substrate was used as control. After every 1 h, a 10 μL aliquot was taken and a 10× dilution series (10−1 to 10−8) was made, and from the resulting 100 μL solution, 30 μL was plated on nutrient agar media, in triplicate, for each solution. Plates were incubated overnight at 37 °C, and colonies for each aliquot between 3 and 30 were counted and recorded with their respective dilution factor. This experiment was conducted in triplicate.For the qualitative analysis of bactericidal activity of graphene against nt broth . The culProduction of high-quality, large-area graphene sheets in a cost-effective way has always been a challenge ,32. Low-


P < 0.05) and progression-free survival (PFS)  than those in chemotherapy groups, especially PD-L1 positive patients. Anti-PD-1/PD-L1 antibodies improved the objective response rate (ORR) compared with docetaxel . In addition, the anti-PD-1/PD-L1 antibody therapy had fewer treatment-related adverse events (AEs)  than docetaxel, especially the grade ≥3 AEs . In conclusion, our study revealed that, compared with docetaxel, anti-PD-1/PD-L1 antibody therapy improved clinical efficacy and safety in previously treated advanced NSCLC patients. This therapy may be a promising treatment for advanced NSCLC patients.Antibodies against the immune checkpoint proteins PD-1 and PD-L1 are novel therapeutic drugs for the treatment of advanced non-small cell lung cancer (NSCLC). Many clinical trials involving these drugs achieved breakthroughs in patients previously treated for advanced NSCLC. However, the results of these clinical studies are not consistent. In this report, we performed a meta-analysis to assess the efficacy and safety of anti-PD-1/PD-L1 antibodies compared with docetaxel treatment for advanced NSCLC patients from 5 randomized clinical trials. We demonstrated that the patients in anti-PD-1/PD-L1 antibody therapy groups had significantly longer overall survival (OS) (HR = 0.69, 95% CI 0.63–0.75,  Lung cancer is one of the most common malignancies and is the leading cause of cancer-related deaths worldwide . Each yeImmunotherapy is emerging as a promising therapeutic strategy for the treatment of NSCLC. Cancer immunotherapy aims to restore the immune responses of CD4+ and CD8+ T cells, enabling them to function in an anti-tumor manner . ImmunotReg cells), which is engaged by the tumor-expressed ligands PD-L1/L2 to inhibit T-cell activation and promote tumor immune escape [Programmed death protein-1 (PD-1) receptor is expressed on activated T cells  seem to be controversial. Several previously published meta-analyses have analyzed the efficacy and toxicity of anti-PD-1/PD-L1 antibodies , 10, 11,Two investigators independently identified the articles eligible for further review by screening titles and abstracts. As a result, a total of 3228 records were identified according to the primary search strategy; 2800 records remained after removing the duplicates; 2698 records were removed after screening; 53 were excluded after screening the titles and abstracts; and 44 studies were excluded after reviewing each publication. Finally, we enrolled 5 published clinical trials involving a total of 3025 patients. The flow chart of our study is shown in Figure 2, the KEYNOTE-010 analysis included two studies with different doses of treatment agents compared with the docetaxel group [The characteristics of the 5 included studies are listed in Table el group . We asseP < 0.001).All trials reported the overall survival (OS) data. The median overall survival (OS) and the 95% confidence interval (95% CI), hazard ratio (HR) and the 95% CI for the treatment group versus control group were retrieved from the published edition as well as the PD-L1 is a potential biomarker that is expressed on tumor cells and tumor-infiltrating immune cells. The PD-L1 expression level plays a crucial role in the prognosis of cancer patients , 17. TheTo better analyze the importance of PD-L1 expression, we redefined the positive PD-L1 as more than 1% or TC1/2/3 or IC1/2/3 based on the included 5 RCTs and analyzed the OS/PFS in the subgroups according to PD-L1 expression. We also defined the PD-L1 negative as less than 1% or TC0 and IC0.P < 0.001). In addition, for the PD-L1 negative subgroup, anti-PD-1/PD-L1 antibody therapy also significantly improved the OS compared with docetaxel .The subgroup analysis according to PD-L1 expression level showed that in the PD-L1 positive subgroup, anti-PD-1/PD-L1 antibody therapy significantly improved the OS compared with docetaxel  remains controversial in several randomized clinical trials Table . In the P < 0.05).The pooled HRs showed a significant improvement in PFS for anti-PD-1/PD-L1 therapy compared with docetaxel , but not in the PD-L1 negative group .The subgroup analysis based on the PD-L1 expression status showed that anti-PD1/PD-L1 antibody treatment improved PFS in the PD-L1 positive group , which suggested a higher clinical response rate for anti-PD-1/PD-L1 antibodies than for docetaxel in advanced NSCLC patients.All the studies included in this meta-analysis reported the partial or complete overall response rate according to RECIST (version 1.1). We compared the overall response rate of anti-PD-1/PD-L1 antibodies  with docetaxel for advanced NSCLC patients. The polled odds ratio (OR) for overall response rate (ORR) was 1.64 . Moreover, we compared the severe adverse events  of anti-PD-1/PD-L1 antibody therapy with chemotherapy for advanced NSCLC patients. In this analysis, the anti-PD-1/PD-L1 antibody therapy also showed significantly lower toxicity than the chemotherapy groups .Anti-PD1/PD-L1 antibody therapy showed significantly lower toxicity than chemotherapy for treatment-related adverse events  is a co-inhibitory molecule expressed by activated T cells. When it binds its ligands, PD-L1 or PD-L2, T-cell activation is inhibited and antitumor immune response is dampened , 19. PD-In recent years, antibodies targeting the PD-1/PD-L1 pathway have been widely explored in clinical trials and have exhibited satisfactory results . Nivolumet al. [et al. [These PD-1/PD-L1 inhibitors are breakthroughs in the treatment of NSCLC . Some clet al.  wrote a  [et al.  conducteSome phase II/III clinical trials published recently provided more information about the safety and efficacy of anti-PD-1/PD-L1 antibody therapy , 16. In 2. The expression of PD-L1 in tumor specimens was detected by immunohistochemistry (IHC). All clinical trials were conducted under the guidance of previously designed protocols, and all participants were followed up regularly during the clinical trials.In these clinical trials, all patients with stage IIIB or IV NSCLC had previous treatment, such as surgical resection, radiation therapy or platinum-based chemotherapy, and these patients had tumor recurrence or progression during or after the regular treatment. All patients enrolled in the experimental groups received the anti-PD-1/PD-L1 antibodies intravenously at an appropriate dose identified by the previously conducted phase I clinical trials. In the control groups, the participants received docetaxel intravenously at a dose of 75 mg/mOur meta-analysis demonstrated that immune checkpoint inhibitors significantly improved efficacy in previously treated advanced NSCLC patients using OS/PFS/ORR as the primary or secondary endpoints.P < 0.001) and significantly prolonged PFS (P < 0.001). However, the improvement of OS between the two treatments in the PD-L1 negative subgroup (P = 0.02) was not as much as that in the PD-L1 positive subgroup (P < 0.001), and there was no significant difference in PFS between the two groups (P > 0.05). We found that PD-L1 expression might be an important prognostic factor for the efficacy of PD-1/PD-L1 inhibitors in advanced NSCLC. However, we did not compare objective response rate (ORR) or adverse events (AEs) in the subgroup according to PD-L1 expression due to the lack of data.PD-L1 is a potential biomarker for anti-PD1/PD-L1 antibodies; a positive status is defined differently in these clinical trials. In CheckMate-017 and CheckMate-057, more than 1% of positive IHC staining cancer cells were defined as PD-L1 positive. The KEYNOTE-010 clinical trial only enrolled patients with PD-L1 expression ≥1%. In POPLAR and OAK, IHC staining of PD-L1 expression was detected on both tumor cells (TC) and tumor-infiltrating immune cells (IC). TC1/2/3 or IC1/2/3 was defined as PD-L1 positive, and TC0 and IC0 were defined as PD-L1 negative. To better reflect the role of PD-L1 expression in PD1/PD-L1 inhibitors treatment, we redefined the positive PD-L1 as more than 1% or TC1/2/3 or IC1/2/3 based on the included 5 RCTs and analyzed the OS/PFS in the subgroups according to PD-L1 expression. In the PD-L1 positive subgroup, anti-PD-1/PD-L1 antibody therapy showed significantly improved OS compared with chemotherapy  including fatigue, decreased appetite, nausea, diarrhea, and anemia were identified in all trials. The side effects of anti-PD1/PD-L1 antibody therapy were less than the docetaxel groups. This finding might be related to the damage of epithelium-derived cells and renewing cell populations caused by docetaxel. Although anti-PD1/PD-L1 antibodies caused few chemotherapy-related adverse events, the immune-mediated adverse events, including inflammatory pneumonitis, interstitial nephritis, hyperthyroidism, and hypothyroidism, occurred more frequently in pulmonary, endocrine, mucocutaneous and renal sites and even immunologically privileged sites such as the eye. Most of these immune-mediated adverse events were moderate and could be controlled by following guidelines. Occasionally, the side effects were life threatening, such as severe inflammatory pneumonitis, and required cessation of therapy and treatment with immunosuppressants such as corticosteroids . It was However, our study has some limitations. First, we extracted data from published articles without individual patient data, which might result in the bias of data analysis. Second, the definition of PD-L1 expression on the tumor and tumor-infiltrating cells remains inconsistent in different clinical trials. For this reason, we formulated a uniform definition of PD-L1 expression in patients within all these clinical trials. Third, we only included RCTs using docetaxel because it is the most common drug used as the second line of chemotherapy in advanced NSCLC. Therefore, the number of studies included in this meta-analysis is small. Because of the above limitations in our study, further studies based on the information from ongoing trials are needed to verify the efficacy and safety of anti-PD1/PD-L1 therapy versus docetaxel in patients with advanced NSCLC.In conclusion, our study indicates that anti-PD-1/PD-L1 antibody therapy improves PFS, OS and ORR and shows less toxicity in patients with advanced or metastatic NSCLC. Despite some limitations, our study suggests that immune checkpoint inhibitors may provide a promising therapeutic strategy for patients with advanced NSCLC.We searched relevant databases to select corresponding clinical trials, such as Pubmed (Medline), EMBASE, the Corane library, clinicaltrial.gov, and ASCO meeting abstracts . The following terms were used to select trial publications or presentations: non-small cell lung cancer, NSCLC, nivolumab, pembrolizumab, atezolizumab, PD-1, PD-L1, immunotherapy, and randomized clinical trial. We searched for publications and unpublished trials written in the English language.The eligible literature was confined to randomized clinical trials written in English. The studies included met the following criteria: (1) Published studies comparing anti-PD-1/PD-L1 antibodies with docetaxel for patients with pretreated advanced non-small cell lung cancer; (2) The outcomes of the trials were available: overall survival (OS), progression-free-survival (PFS), objective response rate (ORR), adverse events (AE), and hazard ratio (HR). The exclusion criteria were (1) The phase I trials and (2) studies with no available outcomes.Two investigators conducted the literature research and reviewed the studies independently to avoid bias. Disagreements were resolved by discussion and adjudicated by a third investigator. For the included studies, we extracted the following data: authors, year of publication, abbreviations of the trials, registered number, trial phase, dose of drugs, number of enrolled patients, tumor histology, PD-L1 expression level, as well as the outcomes mentioned above.et al [The quality of the studies included was assessed using the method reported by Jadad et al . We scorP < 0.05 was considered to be statistically significant. The I2 statistic and Q statistics were used to test statistical heterogeneity of included studies, with a predefined significant threshold of I2 < 50% or p > 0.1. If the I2 was ≤ 50%, then the trials were considered to be homogeneous, and a fixed-effect model was used. Otherwise, a random-effect model was used.We used the Review Manager 5.3.5 to perform the statistical analysis under the guidance of the Cochrane library. The pooled HRs (hazard ratio) with 95% CIs for OS and PFS, and the ORs (odds ratio) with 95% CIs for ORR and AEs were calculated using the Review Manager 5.35. HRs > 1 favored the docetaxel arm while HRs < 1 favored the anti-PD-1/PD-L1 antibodies arm. ORs > 1 for ORR and AEs meant a higher response rate and toxicity, whereas ORs < 1 reflected lower response rate and safety.


Of the 36 Xp11.2 RCC patients, 9 (25.0%) had tumors with positive PD-L1 expression and 27 (75.0%) had tumors with negative PD-L1 expression. Positive PD-L1 expression correlated with advanced tumor stage (P = 0.001), regional lymph node metastasis (P < 0.001), and distant metastasis (P < 0.001). A multivariate analysis identified positive PD-L1 expression was an independent adverse prognostic factor for both progression free survival  and overall survival . The median PFS and OS for the whole cohort were 13.0 months  and 36.0 months , respectively. Our findings suggest that positive PD-L1 expression is indicative of worse clinical outcome in Xp11.2 RCC. Further studies are needed to explore the potential efficacy of targeting PD-L1 in Xp11.2 RCC.Programmed death ligand-1 (PD-L1), a promising antitumor target, has proven clinical value against many malignancies. However, the PD-L1 content of Xp11.2 translocation renal cell carcinoma (Xp11.2 RCC) and its correlation with clinical outcomes remain unclear. This study aimed to investigate PD-L1 expression in Xp11.2 RCC and to assess its prognostic value. Formalin-fixed paraffin-embedded specimens from 36 adult patients that were histologically confirmed (by fluorescence  Xp11.2 translocation RCC (Xp11.2 RCC) is a rare subtype that was recognized as a distinctive pathological entity in the 2004 World Health Organization renal tumor classification3. Xp11.2 RCC is characterized by several translocations on chromosome Xp11.2, resulting in gene fusion between TFE3 and at least six possible partners6.Renal cell carcinoma (RCC) is widely recognized as a heterogeneous disease with various histological subtypes. The most common histopathological subtypes are clear cell (60%–75%), papillary (10%–15%), chromophobe (5%), and collecting duct carcinomas7. However, previous studies indicate that Xp11.2 RCC presents at an advanced stage with a rapid clinical course9. As a result, systematic treatment may be indispensable for most patients. Anti-VEGF drugs are reported to have activity against metastatic Xp11.2 RCC11. However, Xp11.2 RCC has poor prognosis regardless of treatment13. Therefore, new, effective treatments are desperately needed for patients with this tumor type.As it is a rare RCC subtype, the best treatment for Xp11.2 RCC has not been defined. Surgery is the optimal treatment for localized Xp11.2 RCC patients, including those with positive regional lymph nodes16. However, its prognostic and clinical value in patients with Xp11.2 RCC subtypes is unknown. In this study, we sought to investigate the levels of PD-L1 expression and its correlation with clinical outcome in a series of patients with Xp11.2 RCC that was histologically confirmed using TFE3 break-apart fluorescence in situ hybridization (FISH).Monoclonal antibodies (mAbs) targeting the programmed death 1 (PD1)/programmed death ligand-1 (PD-L1) pathway have achieved impressive response rates in patients with melanoma, non-small cell lung cancer, and bladder cancer, and PD-L1 has been validated as a predictive biomarker for the outcome of mAb therapy in many studies). The clinicopathological features of the patient cohort are summarized in Table Representative images of the TFE3 break-apart FISH assay show the classical TFE3 rearrangement associated with Xp11.2 translocation  had positive PD-L1 expression and 27 (75%) had negative PD-L1 expression , sex (P = 0.161), clinical manifestation (P = 0.845), tumor laterality (P = 0.700), surgical option (P = 0.121), tumor size (P = 0.783), and ISUP grade (P = 0.355). There was significant correlation between positive PD-L1 expression and a higher tumor stage (P = 0.001). Approximately 80% of TNM T3–4 stage tumors, but only 22% of T1–2 stage tumors had positive PD-L1 expression. Positive PD-L1 expression was more common in tumors with regional lymph node metastasis (89%) than in N0 stage tumors  and in the tumors of patients presenting with distant metastasis than in those with localized disease (P < 0.001).PD-L1 expression levels and clinicopathological parameters are listed in Table P = 0.001): 78% (7/9) of patients with positive tumor PD-L1 expression had a median OS of 17 months , whereas only 15% (4/27) of those with negative tumor PD-L1 expression died of the disease within the study period .The PFS was shorter for patients who had tumors with positive PD-L1 expression  than for those with for tumors with negative PD-L1 expression , N stage , M stage , and PD-L1 expression status  were associated with PFS. Further, tumor N stage , M stage , and PD-L1 expression status  were identified as independent prognostic factors of PFS by multivariate analysis. Advanced tumor T stage , regional lymph node metastasis , distant metastasis , ISUP grade  and positive PD-L1 expression  were associated with shorter OS. After adjusting for all the clinical and pathological parameters tested in this study, only distant metastasis  and positive PD-L1 expression  were independently associated with OS.The prognostic value of each clinicopathological factor, including PD-L1 expression status, was evaluated for PFS Table  and OS . Univari17.PD-L1  is highly expressed in a number of tumor types and is recognized as a strong prognostic factor for affected patients. PD-L1 binding to its receptor, PD-1, on activated T lymphocytes and other immune cells negatively regulates T-cell proliferation and activity. This interaction allows tumor cells to evade immune surveillance and eradication. Many malignancies with positive PD-L1 expression was associated with poor patient prognosis. This is especially so for cancers originating in the epithelium, such as esophageal cancer, gastric cancer, and oropharyngeal squamous cell carcinomaet al. were among the first to identify the clinical significance of PD-L1 expression in clear cell RCC (ccRCC) patients using immunohistochemical methods20. These authors found that PD-L1-positive tumors were more likely to have adverse pathological features including a late tumor stage (III or IV), high International Society of Urological Pathology (ISUP) grade (III or IV), larger tumor size, and a higher risk of cancer-specific mortality for patients. Krambeck et al. reported that survivin expression status combined with PD-L1 expression status gave a more accurate prognosis for ccRCC patients. High survivin expression combined with PD-L1 positivity was significantly associated with a high risk of death for ccRCC patients in both univariate  and multivariate  analyses. In addition to immunohistochemical analyses, circulating PD-L1 expression has also been examined in patient peripheral blood samples. In Frigola’s study, preoperative circulating PD-L1 levels in ccRCC patients were quantified by ELISA21. Higher preoperative PD-L1 levels in blood samples were associated with larger tumors and those with an advanced stage, higher grade, and necrosis. A doubling of soluble PD-L1 levels was associated with a 41% increased risk of death. For the metastatic ccRCC patients treated with VEGF-targeted therapies, Toni K et al.’s study also suggested that increased tumor cell PD-L1, or PD-L1 plus tumor CD8-positive T-cell counts, were associated with shorter survival22. As a result, accumulating evidence indicates that elevated PD-L1 is a negative predictor for survival in ccRCC patients.Thompson et al. reported that out of 10 Xp11.2 RCC patients, 3 had positive PD-L1 expression in tumor cells and 9 harbored PD-L1-positive tumor infiltrating immune cells23. However, the prognostic value of these findings and their correlation with clinical variables were not analyzed due to limited patient numbers. In our study, from 2246 consecutive patients, 36 Xp11.2 RCC patients were enrolled which were histologically confirmed by TFE3 break-apart FISH analysis. We demonstrated that positive PD-L1 expression was considerablely associated with advanced T stage (T3–4), regional lymph nodes or distant metastasis. Additionally, positive tumor PD-L1 expression indicated a worse prognosis for Xp11.2 RCC patients.So far, only one study has attempted to measure PD-L1 expression in Xp11.2 RCCs. Choueiri 24. Of 46 patients with melanoma, NSCLC, or RCC, 25 tumors were positive and 21 were negative for PD-L1. Tumor PD-L1 expression was evaluated immunohistochemically. A treatment response was seen in nine PD-L1-positive patients (36%) but in none of the PD-L1-negative patients. The authors also noted that multiple tumor sections should be analyzed and that a patient was considered PD-L1 positive if at least one biopsy sample was positive. Powles et al. investigated the atezolizumab responsive of bladder cancer patients stratified by PD-L1 expression status25. The response rate was 43% for patients with PD-L1-positive tumors, but only 11% for patients with PD-L1-negative tumors. Recently, McDermott et al. reported that atezolizumab, a humanized anti-PD-L1 antibody, had promising antitumor activity in patients with metastatic RCC26. The objective response rate reached 18% in those with PD-L1-positive staining of >1% of the tumor area, but dropped to 9% in those with PD-L1-positive staining of <1% of the tumor area. The objective response rate for patients with ISUP grade IV and/or sarcomatoid histology was 22%. For the patients who previously received antiangiogenic therapy, nivolumab also performed better than traditional second-line treatment of everolimus. In R.J. Motzer’s study, overall survival was longer and fewer grade 3 or 4 adverse events occurred with nivolumab than with everolimus. The objective response rate of the nivolumab reached to 25% which was greater than everolimus group which was only 5% 27. In our study, positive PD-L1 expression is indicative of a worse clinical outcome in Xp11.2 RCC patients. Prospective clinical trials are warranted to evaluate the treatment efficacy for Xp11.2 RCC patients, especially for those with advanced disease stage and a poor response to various therapeutic agents.Recently, PD-1/PD-L1-targeted immunotherapy has been shown to be clinically effective and thus a promising therapeutic option for several malignancies. Clinical trials have also suggested that clinical response rates strongly correlate with PD-L1 expression status. This was first demonstrated in a phase I study of nivolumab in solid tumors (CA209-003)The current study had several limitations. First, its retrospective nature in a single institution may introduce selection bias. Prospective multicenter clinical trials are needed to validate the results. Second, although PD-L1 its expression status was closely associated with patient prognosis, the therapeutic effect of anti-PD-L1 mAbs in Xp11.2 RCC patients was not evaluated because these drugs are not yet available in China. Lastly, the dual color, break-apart FISH assay used in this study cannot detect each partner of the specific translocation, although it is an accurate and convenient diagnostic tool in formalin-fixed paraffin-embedded tissues.In conclusion, positive PD-L1 expression is independently associated with tumor progression and predicts an adverse prognosis for Xp11.2 RCC patients. Importantly, our findings suggest that immunotherapy targeting the PD-1/PD-L1 pathway may represent a potential novel treatment for Xp11.2 RCC patients.28. Patient medical records were reviewed to collect demographic data, clinical manifestation, surgical techniques, pathological findings, clinical outcomes, and follow-up information. Tumor sizes were defined as the largest diameter of the surgically removed tumor mass. The 2010 American Joint Committee on Cancer TNM staging system was used to classify tumors. The study was carried out in accordance with the ethical standards of the Helsinki Declaration II and approved by the Institution Review Board of Fudan University Shanghai Cancer Center. Informed consent was obtained from each patient and the study was approved by our Institutional Ethics Committee.Tumor specimens from 2246 consecutive patients who underwent radical or partial nephrectomy to treat RCC from January 2008 to January 2015 at Fudan University Shanghai Cancer Center were analyzed. A total of 36 patients were confirmed to have Xp11.2 RCC using TFE3 break-apart FISH, as previously described and the Envision detection kit . Tissue sections (4 μm thick) obtained from archived formalin-fixed, paraffin-embedded tissue blocks were deparaffinized in a xylene series and rehydrated with a graded ethanol series. Thereafter, endogenous peroxidase was quenched by incubation in 0.3% H2O2 for 15 min at 37 °C, and nonspecific binding was blocked by incubation in 10% normal goat serum for 60 min at room temperature. For antigen retrieval, sections were autoclaved in 0.01 M sodium citrate buffer, pH 6.0 (at 20 psi) for 10 min. Tissues sections were then incubated overnight with primary antibody at 4 °C. Chromogenic antigen detection was carried out using a peroxidase-conjugated secondary antibody (60 min incubation) and DAB reagents . Tissue sections were counterstained with Meyer’s hematoxylin .PD-L1 immunostaining of 36 Xp11.2 RCC tissue samples was performed using a rabbit monoclonal anti-PD-L1 antibody 29.All sections were examined and scored by two experienced pathologists blinded to all clinical data in an open discussion. The percentages of tumor cells with positive PD-L1 staining were quantified. In accordance with previous studies, PD-L1 tumor positivity was defined as membrane staining of ≥5% tumor cellsp values of <0.05 were considered statistically significant.To evaluate the relationship between the PD-L1 expression and clinicopathological parameters, the Student’s t-test and chi-square test were used for continuous and categorical variables, respectively. Univariate and multivariate Cox regression models were performed to evaluate the prognostic value of all parameters. Progression free survival (PFS) and overall survival (OS) for patients groups classified according to tumor PD-L1 expression level were calculated by the Kaplan–Meier method and compared using the log-rank test. All statistical tests were done using SPSS version 20 . Two-tailed


Conductance quantization is the quintessential feature of electronic transport in non-interacting mesoscopic systems. This phenomenon is observed in quasi one-dimensional conductors at zero magnetic field B, and the formation of edge states at finite magnetic fields results in wider conductance plateaus within the quantum Hall regime. Electrostatic interactions can change this picture qualitatively. At finite B, screening mechanisms in narrow, gated ballistic conductors are predicted to give rise to an increase in conductance and a suppression of quantization due to the appearance of additional conduction channels. Despite being a universal effect, this regime has proven experimentally elusive because of difficulties in realizing one-dimensional systems with sufficiently hard-walled, disorder-free confinement. Here, we experimentally demonstrate the suppression of conductance quantization within the quantum Hall regime for graphene nanoconstrictions with low edge roughness. Our findings may have profound impact on fundamental studies of quantum transport in finite-size, two-dimensional crystals with low disorder. Conductance quantization is the hallmark of non-interacting confined systems. The authors show that the quantization in graphene nanoconstrictions with low edge disorder is suppressed in the quantum Hall regime. This is explained by the addition of new conductance channels due to electrostatic screening. B, conductance quantization arises due to the formation of transverse subbands in confined, quasi one-dimensional (1D) systems such as quantum point contacts (QPC) or quantum wires2. As B increases, the system gradually enters the quantum Hall (QH) regime, where propagating modes evolve from magnetoelectric subbands interacting with both edges, to chiral edge states surrounding an incompressible, gapped bulk2. Within a one-electron picture, both propagation states lead to a quantized two-terminal conductance given by G = Ne2/h 2. The situation changes when taking into account Coulomb interactions13 between injected carriers and/or their coupling to an external gate. For example, the conductance of a 1D channel with repulsive electron–electron interactions vanishes in the presence of any scattering potential at |B| = 0 T ref3. Furthermore, the observation of the so-called 0.7 anomaly12 or the 0.25 feature13 in the conductance quantization of QPCs at |B| = 0 T are also signatures of electron–electron interactions. In a perpendicular B, the interplay between screening mechanisms and the Hall potential causes a reconstruction of the edge states into alternating conductive (compressible) and insulating (incompressible) regions no longer strictly linked to the topology of the conductor8. Compressible zones are characterized by partially filled Landau levels (LLs) pinned at the Fermi energy with a variable electron concentration. Conversely, incompressible regions (strips) consist of fully occupied LLs and display the typical insulating behavior of a QH state8.At zero magnetic field 9. This universal transport regime should occur in narrow, ballistic systems confined by hard-wall potentials11, where a large accumulation of charge carriers near sharp edges and a pronounced inner depletion inhibits the formation of stable incompressible strips9. Although both interactions between carriers and their coupling to the external gate can affect conductance9, it is the electrostatic screening of the gate potential which is the main contributing mechanism10 to the CQS effect.We focus on the ballistic conductance of gated quasi-1D systems, where screening theories predict conductance quantization suppression (CQS) in the QH regime24. Commonly studied QPCs in two-dimensional (2D) electron gases have soft-confining potentials because the gates and dopant layers are far away from the actual carrier layer2. Graphene, on the other hand, provides extraordinary opportunities to examine the physics of the QH effect16. First, it exhibits a natural hard-wall confinement at its borders. Furthermore, the distance to the gate can be arbitrarily selected since electrons in strict 2D materials reside right at the surface. Both features enable the possibility of designing specific device geometries which are (heavily) dominated by screening effects. An example of such a geometry is a narrow graphene strip with a width comparable or smaller than the thickness of the dielectric spacer10. Indeed, CQS in a perpendicular magnetic field has been predicted to occur in gated graphene nanoribbons11. In these systems, the suppression of conductance quantization is related solely to the simultaneous existence of compressible strips in the center of the ribbon and the appearance of additional counter-propagating states11. Nevertheless, experiments conducted with different types of narrow, high-quality graphene devices have so far not confirmed these predictions24.To date, the experimental realization of such narrow, disorder-free, sharp-edged devices has been inherently difficultB18, similar to gate-defined, soft-potential, narrow ballistic graphene channels19. These discrepancies between experiments and theoretical predictions motivate us to investigate devices which have been designed to meet the required theoretically predicted conditions for CQS;11 specifically, a device geometry able to produce a large charge density gradient across the nanostructure, a narrow ballistic channel, and low edge disorder.In more detail, the magnetoconductance of ballistic graphene constrictions remains quantized when increasing By addressing these factors, we experimentally demonstrate the suppression of conductance quantization within the QH regime for graphene nanoconstrictions with low edge roughness. Our findings are a strong experimental confirmation that the single-electron picture is inadequate for describing the transport behavior of finite-size, two-dimensional crystals with low disorder.8, incompressible strips must be wider than the magnetic length lB to be stable. For a given LL with level index k, the minimum charge carrier density gradient across a graphene nanostructure, which prevents the formation of a stable incompressible strip, is (Methods)ε is the permittivity of the dielectric and vF ~ 106 ms−1 the Fermi velocity in graphene.According to QH theoriesB| and k, normalized by the average density navg = 1016 m−22 as dielectric material. Values of |B| of 0–10 T, k of 0, 1, 2, and navg are experimentally accessible in our study. For |B| ≤ 10 T and k ≤ 2, an estimated threshold of 7 m−1 prevents incompressible strips from forming in graphene devices. Figure Figure W and dielectric thicknesses b to examine the stability condition  used for the theoretical prediction of the CQS9 (green curve) shows x > 0.13W across the device. This length is comparable to lB = 0.16W at |B| = 10 T, preventing the appearance of stable incompressible strips. A similar situation occurs (blue curve) in slightly wider constrictions (W = 100 nm) on a dielectric with b = 100 nm. Notably, wider geometries have the added advantage of reducing the significance of edge disorder in experimental devices. Much wider constrictions with sizes close to samples reported in literature18  show lB at |B| = 10 T. This condition remains satisfied for smaller |B| and k, and so this geometry enables the formation of incompressible strips8 and results in a quantized magnetoconductance18.Here, we consider two distinct geometries (ribbons and constrictions) with different widths ion (Eq. ). The riL = W ~ 100 nm on b = 100 nm SiO2 substrates (Methods). Our graphene flakes were exfoliated on hydrophobic SiO225, resulting in mean free paths larger than L,W  of the two types of studied sample. Their geometry and fabrication steps are similar with the exception of the last etching step, which defines the edge disorder of the nanoconstriction26. While all our devices have a certain degree of edge disorder, Sample type 1 , a clear indication of size quantization18. These modulations are significant in Sample type 1, with step heights ΔG up to ~ 2e2/h. Further analysis at |B| = 0 T can be found in the Supplementary Information , dramatically differing from the single-electron picture. Particularly, when increasing the gate voltage Vg, G shows a peak whose value is larger than the expected quantization plateau and cannot be explained by accounting for geometrical corrections in spatially uniform and homogeneous conductors28 , in agreement with the well-known, geometrical effects for homogeneous devices with L > W [k = 1, 2 in Sample type 2 showing G values lower than the expected quantization values. These trends are confirmed in further devices and analysis , which exhibits a quantized = 0 Fig. . Furtherth L > W  (Supplemmentclasspt{minima10 (Methods). This potential corresponds closely to those generated by more complex models, such as self-consistent solutions within the Hartree approximation29. Although such approaches can account for both Coulomb interactions between injected carriers and their coupling to the external gate, the electrostatic screening of the gate potential is the primary factor determining the charge density distribution in these systems30. The interpretation given above is supported by tight-binding calculations, where the inhomogeneous electrostatic potential across the device is introduced using the analytical model proposed by Silvestrov and EfetovFigure 9. These new dispersive states support propagation in both directions and unlike QH states, are susceptible to backscattering due to the overlap between forward and backward propagating states9 and disorder11. Therefore the channels lose their exact quantization as edge disorder is increased, forming peak-like conductance features at low edge disorder levels, before being completely suppressed by stronger scattering  is the electron density across the device at |B| = 0 T, ε is the dielectric constant of the insulating material, kth incompressible strip, and Ek is the Landau spectrum. In the case of spin-degenerate graphene, we get 16. Thus, for the graphene nanodevice to be free of incompressible strips, the charge carrier density gradient The competition between the Hall and screened potentials determines the stability of incompressible strips in a perpendicular magnetic fieldmentclass2pt{minimnel(x) can be calculated33 using the expression Ez(x) is the perpendicular electric field component in the corresponding gated devices at y = 0 at a distance z = 0.5 nm above the flake and ε = 3.9ε0 is the permittivity of the SiO2. Ez(x) can be obtained for any geometry by solving the Poisson equation in the device using a finite-element method33 solver , achieved by the mechanical exfoliation of graphene on hydrophobic25 Si/SiO2 substrates (SiO2 thickness b = 100 nm) and contact resistance Rc below 600 Ω. To test these initial device parameters , we first shape, contact and measure the magnetotransport properties of rectangular two-terminal devices with a width of ~1 µm  and Au (30 nm) at low pressure (<5 × 10−7 mbar). The subsequent definition of the nanoconstrictions is done via electron beam lithography using polymethyl-methacrylate developed at −5 °C in a 1:3 IPA:H2O solution.We fabricate devices with field-effect mobility 26. Devices with a higher amount of edge disorder (Sample type 2) are defined by plasma ashing, which, despite being known to introduce instabilities and localized states in graphene nanodevices, is widely used to shape graphene nanostructures24. In contrast, devices with a much lower amount of edge disorder (Sample type 1) were produced by RIE26 . We achieve an edge roughness ≤1 nm with the RIE etching procedure, as demonstrated in the transmission electron micrograph shown in the Supplementary Fig. The edge quality in our constrictions is defined with two complementary etching processes: oxygen plasma ashing and RIE34. After these 18 h, the devices are dipped for 5 s in acetone, 5 s in IPA, and then dried with nitrogen.Prior to measuring their electrical properties, we dip our devices for 18 h in a pure hexamethyldisilazane solution to reduce the effect of environmental contaminants that may have been adsorbed on the basal plane of graphene or at the edges during the processing stepsOur measurements were done in an Oxford Instrument Teslatron PT cryostat. Measurements of differential conductance were performed using a Stanford SR830 lock-in amplifier with an excitation voltage of 80 μV at a frequency of 17.77 Hz.L = W = 100 nm) and different degrees of edge disorder (Supplementary Note In our simulations, we consider zigzag nanoribbons with similar dimensions to the experimentally measured constrictions (i. The hopping parameters tij take the values t1 = −2.7 eV, t2 = −0.2 eV, and t3 = −0.18 eV, respectively35.The electronic structure is described by a single π-orbital third-nearest-neighbor tight-binding HamiltonianThe effect of a magnetic field is included using the Peierls’ phase approach. This involves introducing a field-dependent phase factor in the tight-binding hopping parametersy-direction.We choose the Landau gauge GR and GA are the retarded and advanced Green’s functions respectively.The conductance through the ribbon is evaluated in terms of the transmissionThe effect of a gate voltage is introduced by fixing the Fermi energy and instead changing the onsite energy potentials according to25n010A uniform carrier density can be included using an infinite plane capacitor26, which gives rise to the stronger CQS observed for LL0 in our experiments (Supplementary Fig. Furthermore, in Fig. The data that support the findings of this study are available from the corresponding authors on request.Supplementary InformationPeer Review File