I–V characteristics of the graphene/SiC-based sensor has been proposed as an indicator of presence of the heavy metals. Since the calculations suggested the strongest charge transfer between Pb and graphene, the proposed sensing platform was characterized by good selectivity towards lead atoms and slight interferences from cadmium and mercury. The dependence of the sensitivity parameters on the concentration of Cd, Hg and Pb is studied and discussed.A vertical diode structure comprising homogeneous monolayer epitaxial graphene on silicon carbide is fabricated by thermal decomposition of a Si-face 4H-SiC wafer in argon atmosphere. Current–voltage characteristics of the graphene/SiC Schottky junction were analyzed by applying the thermionic-emission theory. Extracted values of the Schottky barrier height and the ideality factor are found to be 0.4879 ± 0.013 eV and 1.01803 ± 0.0049, respectively. Deviations of these parameters from average values are smaller than those of previously observed literature data, thereby implying uniformity of the Schottky barrier height over the whole diode area, a stable rectifying behaviour and a good quality of ohmic palladium–graphene contacts. Keeping in mind the strong sensitivity of graphene to analytes we propose the possibility to use the graphene/SiC Schottky diode as a sensing platform for the recognition of toxic heavy metals. Using density functional theory (DFT) calculations we gain insight into the nature of the interaction of cadmium, mercury and lead with graphene as well as estimate the work function and the Schottky barrier height of the graphene/SiC structure before and after applying heavy metals to the sensing material. A shift of the  As a result of reckless and uncontrollable human activity many dangerous non-biodegradable substances are released into the atmosphere and water sources . Among tTo get a full picture of the state of the art describing all meaningful existing approaches to detect heavy metals, we also need to mention anode stripping voltammetry (ASV). ASV uses carbon, mercury and bismuth electrodes (and others) and is a well-known method for the identification of heavy metals –13. Neve2/g) [Taking the above discussion into account, one can conclude that the need to create accurate and real-time analytical instruments for detection of heavy metals still exists. A solution to this problem can be achieved not only through the improvement of the existing techniques, but also through the development of new approaches. One of the most promising candidates for the development of real-time detectors for heavy metals is graphene . Due to 2/g) , high ch2/g)  and exce2/g) , graphenHowever, sensors based on reduced graphene oxide are only well investigated in terms of determination of the concentration limit of heavy metals and improving the response time –26. In pk gate dielectrics. These additional steps can result in the formation of unexpected and uncontrollable interface states, deteriorating the output characteristics of the devices and their sensitivity. A simpler solution is to use Schottky diode sensors, which can be grown more easily, have no gate insulator and a high sensitivity in the reverse and forward diode regimes.In many cases FET devices are considered as effective sensing platforms for heavy metals –31. The I–V curves can be calibrated to measure the concentration of heavy metals. It is important to emphasize that the key factors determining the performance of the graphene-based Schottky barrier diode as sensing platform are the quality of the interface between SiC and graphene (minimization of the density of interface states) as well as the homogeneity of the graphene thickness. Indeed, a non-uniform interface may cause the formation of a great number of surface states, a decrease in Schottky barrier height, Schottky barrier inhomogeneity and an increase in the ideality factor. As a result, different authors reported on a large variation of these parameters for such Schottky diodes based on nominally the same material [During the last decade the thermal decomposition of silicon carbide (SiC) in argon atmosphere was shown to be a reliable and effective approach for the formation of homogenous epitaxial graphene layers with controllable thickness –33. Combmaterial –49. The From Here we report on the fabrication of epitaxial graphene/Si-face-4H-SiC Schottky barrier diodes with improved barrier height uniformity, formed on uniform 1 ML graphene. Based on density functional theory (DFT) calculations and experimental findings we propose a strategy for development of a sensing platform for detection of the toxic heavy metals Cd, Hg and Pb.n-type (nitrogen-doped) 4H-SiC (0001) substrates. A study of the grown samples by reflectance mapping and Raman characterization provides evidence for the formation of monolayered graphene [The top-down sublimation growth process in an inductively heated furnace at 2000 °C under an argon pressure of 1 atm  was usedgraphene . The 1 MI–V) characteristics of the fabricated devices were measured and analyzed in order to extract the Schottky barrier height value, ideality factor, saturation current and series resistance. In addition, we investigated the electrical properties of the fabricated structure in the simple-resistor regime. In other words, we measured the current–voltage characteristics of the graphene resistor between two Pd metallic contacts. In fact, the detection of heavy metals can also be realized in the resistor regime, since a change in conductivity of the graphene channel after metal adsorption is expected. I–V characterization was performed in an electrical probe station .To avoid the necessity to etch parts of the graphene coverage in order to form the ohmic contacts to SiC, we did not utilize the lateral Schottky diode structure and mainly focused on designing vertical devices. It might be expected that the vertical structure has some advantages over the lateral device, since it offers a simpler design and a higher breakdown voltage (because of larger area efficiency). Palladium contacts (110 nm) to graphene were formed on the top side of the structure by a conventional thermal evaporation technique. Electrodes were patterned into 1 × 1 mm square shapes by the use of a shadow mask. For comparison to the diodes with Pd contacts, we have fabricated several diodes using a layer of conducting silver glue deposited on the Pd contact. As a back ohmic contact to SiC a Pd metallic electrode was also used. Assuming that the electron transport through the graphene/4H-SiC vertical structure is dominated by thermionic emission, one can write the following Richardson relation between the applied voltage and the current :IS is the zero-bias saturation current, A is the electrically active diode area, A* is the effective Richardson constant, which is equal to 146 A·cm−2·K−2 for n-type 4H-SiC [q is the elementary charge, kB is the Boltzman constant, T is the absolute temperature, V is the applied voltage, φB0 is the zero-bias Schottky barrier height and η is the ideality factor. To define directly the saturation current and ideality factor from experimental I–V curves, it is convenient to re-write where y-axis intercept of the straight line can be used to determine ideality factor and saturation current, respectively. Knowing the value of the saturation current one can easily estimate the zero-bias Schottky barrier height [Once the semi-log plot is constructed, the slope and the r height :RS, the Richardson equation must be written as [In order to obtain the series resistance itten as :The slope of the straight line fitting this plot can be used to determine the series resistance.The practical realization of sensors based on a graphene/SiC Schottky junction for the detection of heavy metals requires a deep understanding of how heavy metals  behave on the graphene surface and how they modify the graphene properties, such as Fermi level, work function, density of states, C–С bond lengths. Once we know the influence of heavy metals on the work function of graphene, we can predict the values of the Schottky barrier height and output characteristics of the devices (for example current–voltage characteristics). The shift of these curves can be an indicator of the presence of heavy metals. Since each of the heavy metals can be expected to behave in a unique manner on the graphene surface, e.g., different response kinetics, different shape of the response curve, careful data analysis could make it possible to draw conclusions about the selectivity of the graphene-based sensors towards different heavy metals.30H14 cluster). To study the concentration dependence we also used the 2 × 2 (C16H10) cluster. It should be pointed out that the geometry optimization of interacting systems  was done at the Becke3LYP level of density functional theory with a 6-31G basis set on carbon and a basis set of Stuttgart-Dresden SDD effective core potentials [An important step is to study the interaction between heavy metals and the graphene surface by DFT calculations. Furthermore, in order to establish the most favorable (from the energetic and thermodynamic point of view) geometrical configuration of incoming adsorbates, we consider the adsorption of Cd, Hg and Pb at different high-symmetry positions on the graphene surface, such as on-top site, hollow site and bridge site. For simulation of graphene, we used a graphene model of 30 carbon atoms with edges terminated by 14 hydrogen atoms , the binding energies were calculated by means of counterpoise method [where e method .The most important parameter that was extracted from our calculation, the work function of graphene before and after interaction with heavy metals, was calculated by using the following equation :IP is the ionization potential of the graphene supercell and EHOMO-LUMO is the energy difference between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). It should be mentioned that in reality where where χ is the electron affinity of silicon carbide.To make the theoretical analysis tractable, three simplifying assumptions were used. First, we consider small H-terminated clusters as a graphene model. It is generally accepted that the small clusters behave themselves as conventional semiconductors and have an energy gap. This energy gap depends on the size of the hydrogenated graphene clusters and significantly decreases with increasing cluster size . FurtherI–V curves of devices after applying the heavy metals were performed with consideration of the fact that the graphene distortion, which is induced by interaction of the heavy metals with graphene, can cause the appearance of an inhomogeneous graphene/SiC interface and, as a consequence, a deviation from ideal diode behavior. In this case, an ideality factor above unity might be expected. Third, we neglect the role of the SiC substrate in the chemical interaction to reduce the software execution time of Gaussian 09, although it is well known that electron charge transfer from SiC to the graphene layer is responsible for n-type doping in graphene. As reported earlier by Deretzis and La Magna [n-type charge carriers at the surface of the graphene/SiC structure to study the adsorption of metals. Nevertheless, we believe that our calculations will be useful to uncover the common trends in nature of the interaction between planar structures of carbon-containing flakes and Cd, Hg and Pb.Second, the theoretical calculations of the La Magna , graphenI–V characteristics of the graphene/SiC vertical devices display a rectifying behavior. The depicted curves correspond to different measurements of the same device with six Pd contacts. In fact, more than six Pd contacts were deposited on the graphene surface and we chose these six ones as representatives. It is indeed necessary to get information about the uniformity distribution of the electrical parameters  that are suited to the fabricated graphene/SiC Schottky vertical diodes. Furthermore, the measurement of the I–V characteristics was repeated six times for each of the six contacts. It allowed us to extract the mean value of the corresponding electrical parameter and its standard deviation.As can be seen in n-type 4H–SiC  to 1.03 and 1.76·10−3 A (for the Ag/Pd contact), respectively. Such a difference in output characteristics between vertical structures with single layer Pd contact and double layered Ag/Pd contacts can be explained by the modification of the work function of the layered contact and/or inhomogeneous surface of the contact.Naturally, the question arises whether we have the Schottky barrier between graphene and 4H-SiC or between palladium and 4H-SiC. Taking into account the fact that the 1 ML coverage is approximately 99% and values for palladium Schottky contacts on –0.89 eV ), it is  results , one caner paste . For thiThe difference between I–V curves presented in 30H14 cluster play the role of electron acceptors.The second part of this paper is devoted to the discussion of the results of DFT calculations and the implementation of these results to predict the behavior of the graphene/SiC Schottky diode after interaction of graphene with heavy metals. Generally, the mechanism underlying the sensing of heavy metals is governed by the nature of the bonding between the metals and graphene. The nature of bonding is definitely depending on the geometrical position of the metal on the graphene (adsorption site) and the unique chemical properties of each metal . Therefore, our main goals are to illuminate the specific nature of interaction and to understand the sequence of the binding strength for heavy metals. According to our findings, the hollow site  is the most preferred site for adsorption of cadmium and mercury species, whereas the bridge site (the center of the C–C bond) is the most favorable site for the adsorption of a lead atom. The corresponding optimized distances of cadmium, mercury and lead are 3.540 Å, 3.506 Å and 2.611 Å, respectively. Thus, the maximal charge transfer might be expected between lead and graphene. To be more specific, the order of the binding strength between neutral atoms of heavy metals (located at the most favorable places) and graphene follows the sequence of Pb > Cd > Hg. It can be explained by the difference in electron affinity and ionization potential between different heavy metals. On one hand, the electron affinity of lead is approximately equal to 35.1 kJ/mol. This value is significantly higher than the electron affinity of mercury and cadmium . On the other hand, the ionization potential of the lead has the smallest value (7.416 eV) among all considered heavy metals . Higher electron affinity indicates that an atom more easily accepts electrons , whereas a lower ionization potential implies stronger charge transfer from metal to graphene . In order to elucidate the nature of charge transfer between the analyte and sensing material, we also carried out a natural bond population analysis. According to our findings, the atomic charges on the heavy metals for their preferred adsorption sites were calculated to be Cd (+0.043), Hg (+0.058), and Pb (+0.284). It means that the atoms act as electron donor. At the same time, the ionic species of all considered heavy metals on the C30H14 cluster is about 4.81 eV. The situation is far more complicated in the case of the adsorption of heavy metals on graphene. Upon interaction with neutral atoms of heavy metals, its energy gap and, as a consequence work function, are changed. After adsorption of Cd and Hg, the work function increases to 5.44 eV and 5.46 eV, respectively. While adding of a Pb atom to the graphene causes a decrease of the work function from 4.81 eV to 4.08 eV. Remembering from the previous discussion that a small H-terminated cluster is considered as graphene model, we would like to emphasize that the predicted value of the work function is not quantitatively exact and differs from the well-known literature data for graphene (ca. 4.2 eV). However, trends of the work function of graphene interacting with adsorbates are expected to be correctly predicted.It should be pointed out that adsorption of the heavy metals on the graphene surface drastically affects energy gap and work function of the graphene cluster. The work function of pristine C30H14 are strongly localized at the cadmium atom, whereas the HOMO is mainly distributed on the graphene sheet. Due to the chemical nature of the interaction between the graphene and lead atom, hybridization of the molecular orbitals has occurred and thus the HOMO is shared by lead adsorbate and graphene sheet. On the other hand, the LUMO of Pb@C30H14 is delocalized over the graphene sheet. In principle, such features of electron density distribution depending on the interaction of the C30H14 with heavy metals are correlated with the values of the HOMO–LUMO gap. The original HOMO–LUMO gap for a pristine graphene-like cluster estimated from the DFT calculations is approximately 2.230 eV. To our surprise, the interaction of graphene with cadmium and mercury atoms does not influence significantly the energy gap of the graphene cluster . Meanwhile, a drastic change in molecular orbitals and HOMO–LUMO gap compared to the pristine graphene cluster has occurred after adsorption of Pb on the graphene surface. An energy gap of 0.772 eV was calculated for the Pb@C30H14 cluster. The order of the HOMO–LUMO gap follows the sequence of Cd@C30H14 ≈ Hg@C30H14 ≈ C30H14 > Pb@C30H14. This trend is accompanied by changes in the degree of hybridization. Indeed, the smallest value of the HOMO–LUMO gap (ca. 0.772 eV) is observed for the Pb@C30H14 cluster because of hybridization of the HOMO orbitals. Based on the derived parameters we estimate that the Schottky barrier height in the case of pristine graphene, before and after interaction with heavy metals, is connected with silicon carbide into one system. According to our estimations, the Schottky barrier height reaches a value of 1.11 eV for the pristine graphene/SiC junction and increases to values of 1.76 and 1.74 eV for Hg@C30H14/SiC and Cd@C30H14/SiC structures, respectively. At the same time, the barrier height of the junction after applying the Pb atom attains a value of 0.384 eV.Let us address the issue of electron density distributions for HOMO and LUMO as well as the energy gap of the graphene cluster before and after interaction with heavy metals. The drastic effect of the heavy metals on the electronic properties of the graphene cluster is evident from the comparison between pristine graphene and graphene interacting with Cd, Hg and Pb see . In partBecause of the general tendency of some metals to cluster it is very important to investigate the dependence of the sensing parameters on the concentration of the heavy metals, since these dependences may provide useful information concerning the detection limit. With this aim, we simulated the adsorption processes on a 2 × 2 graphene flake. At the first stage, one heavy metal atom was placed on the graphene surface. We then optimized the resulting structure. At the second stage, we added a second heavy metal atom to this relaxed structure and predicted the most stable geometry. The same procedure was performed to simulate the adsorption of the third and fourth heavy metal atoms. By doing so, the role of metal–metal interaction in adsorption processes involving heavy metals on graphene can be elucidated.16H10-related molecular orbitals occurs. As can be seen from Further increase of the lead concentration causes a decrease in the degree of chemical interaction between Pb and graphene. This can be explained by the fact that the Pb–Pb attraction plays a more important role in binding than the interaction between Pb and graphene. Thus, the Pb clusters consisting of three and four Pb atoms are much more stable than the isolated Pb atoms on the graphene flake. The electronic gap of the graphene flake is also dependent on the amount of atoms of heavy metals . From FiI–V characteristics shift.The calculated current–voltage characteristics of our device before and after interaction with heavy metals are shown in The sensitivity of the proposed device was determined from the normalized change in the initial current at a fixed voltage of 1 V. The current, following stabilization after adding heavy metals, changed depending on the type of heavy metals. A histogram of the sensitivity of the graphene/SiC structure to different heavy metals is presented in Because of the high sensitivity of the work function of graphene to adsorbates , as the sensitivity criterion we choose the ratio between the work function of the graphene flake after interaction with heavy metals and the work function of the pristine graphene cluster. In fact, this index is proportional to the ratio between the initial current and the current following stabilization after adding the heavy metals. Therefore, this parameter can be effectively used to predict the sensitive characteristics of the proposed Schottky-based sensing platform. As shown in It is necessary to emphasize that the presence of the substrate can modify the interaction between the heavy metals and graphene. Thus, a response of a graphene system to a change from an equilibrium state can differ from theoretically predicted behavior. This difference can be related to the change in the absolute value of adsorption energy. At the same time, we believe that the general trend in adsorption of heavy metals by graphene surface will not be changed. Another important aspect is the recovery time. According to the transition state theory , the recT is the temperature, kB is Boltzmann’s constant and ν0 is the frequency of the desorption event, and Eads is the adsorption energy. An increase in adsorption energy will cause an increased recovery time. In fact, in the case of strong adsorption, the recovery time will be too long for the sensor signal to return to its initial value in a reasonable time. This is not applicable for real-time sensors. Our findings suggest that the recovery time of the sensor for Pb detection will be longer than that of the sensors for determination of Hg and Cd. This is mainly associated with the difference in the binding energy of different heavy metals with graphene. Anyway, a trade-off must be reached between the adsorption energy on one side and the recovery time on the other side.where Eg and conductivity σ is valid [The graphene/SiC interface (working in resistor regime) can also function as an effective sensor for the detection of the heavy metals. The key issue is that the heavy metal adsorption leads to band-structure reorganization and changing of the energy gap of the graphene clusters (as was discussed before). Bandgap opening may also be expected in epitaxial monolayer graphene on SiC due to the interaction with heavy metals. It is generally accepted that for many materials the fundamental relationship between the energy gap is valid :It is reasonable to assume that the decrease of the energy band after the adsorption event induces a change in the conductivity. Since the Pb adsorption leads to a decrease in the HOMO–LUMO gap of the graphene cluster, the electrical conductivity of Pb@graphene is enhanced in comparison with the pristine graphene flake and Hg/Cd@graphene and a better sensitivity towards Pb is expected.The results presented herein exhibit the possibility to use a vertical graphene/SiC junction as a sensing platform for the simultaneous detection of toxic heavy metals such as cadmium, mercury and lead. Our investigations on the graphene/SiC structure have shown that the high-temperature thermal decomposition of the Si-face 4H-SiC substrate in argon atmosphere is a good strategy to grow monolayered epitaxial graphene with high thickness uniformity and low fluctuations in barrier heights. As a result of the presence of a homogeneous interface between graphene and SiC, analysis of the current–voltage characteristics by thermionic emission approach yields a Schottky barrier height of 0.488 eV ± 0.013 eV and an ideality factor of 1.0180 ± 0.0049. These values are in good agreement with theory and suggest that the fabricated graphene/SiC Schottky diodes are characterized by stable rectifying behavior and a high degree of barrier height homogeneity. With first-principles density functional theory calculations, we demonstrated the features of the interaction of graphene with cadmium, mercury and lead atoms and the strong sensitivity of the electronic properties of pristine graphene  to incoming adsorbates. The binding strength order predicted by DFT follows the sequence Pb > Cd > Hg. The maximal charge transfer is expected between Pb and graphene and implies the largest change in Schottky barrier height and current flowing through the barrier. For this reason, the proposed sensing platform was characterized by high selectivity towards lead atoms. It was found that an increased concentration of heavy metals leads to a weakening of the chemical interaction with graphene due to enhanced metal–metal attraction. Pb atoms tend to form more stable metallic clusters. The proposed sensing platform can selectively detect Pb atoms in the concentration range up to 67.2 wt % (2 Pb atoms per 16 carbon atoms) with a high sensitivity. Our findings expectedly offer a new reliable strategy to develop portable and real-time environmental sensors based on uniform self-organized graphene/SiC Schottky diodes.


Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease characterized by progressive loss of lung function and poor prognosis. The so-called acute exacerbation of IPF (AE-IPF) may lead to severe hypoxemia requiring mechanical ventilation in the intensive care unit (ICU). AE-IPF shares several pathophysiological features with acute respiratory distress syndrome (ARDS), a very severe condition commonly treated in this setting.A review of the literature has been conducted to underline similarities and differences in the management of patients with AE-IPF and ARDS.During AE-IPF, diffuse alveolar damage and massive loss of aeration occurs, similar to what is observed in patients with ARDS. Differently from ARDS, no studies have yet concluded on the optimal ventilatory strategy and management in AE-IPF patients admitted to the ICU. Notwithstanding, a protective ventilation strategy with low tidal volume and low driving pressure could be recommended similarly to ARDS. The beneficial effect of high levels of positive end-expiratory pressure and prone positioning has still to be elucidated in AE-IPF patients, as well as the precise role of other types of respiratory assistance  or innovative therapies . The use of systemic drugs such as steroids or immunosuppressive agents in AE-IPF is controversial and potentially associated with an increased risk of serious adverse reactions.Common pathophysiological abnormalities and similar clinical needs suggest translating to AE-IPF the lessons learned from the management of ARDS patients. Studies focused on specific therapeutic strategies during AE-IPF are warranted. Idiopathic pulmonary fibrosis (IPF) is a chronic disease of unknown etiology characterized by a deterioration of the structure of lung parenchyma, thus resulting in a progressive decline of respiratory function and early mortality .In the course of the disease, patients suffering from IPF may develop acute exacerbations of respiratory function impairment, referred to as AE-IPF , which cAlthough patients with AE-IPF receive mechanical ventilation in the intensive care unit (ICU), few studies report their inhospital mortality risk compared to ARDS . MoreoveThe purpose of this narrative review is to discuss the pathophysiological similarities and differences between AE-IPF and ARDS and to analyze the evidence on treatments currently proposed for AE-IPF, including mechanical ventilation strategies and other therapies.noxae, including sepsis, pneumonia, and exposure to high oxygen concentration. DAD is also the histologic hallmark of ARDS, although this feature can only be found at biopsy in about half of patients meeting the clinical criteria for ARDS diagnosis [The typical pathological feature of AE-IPF is the presence of diffuse alveolar damage (DAD) superimposed on the usual interstitial pneumonia (UIP) pattern . The teriagnosis . In thisiagnosis . Data onA retrospective analysis in patients with ARDS who underwent open lung biopsy showed a significant increase in hospital mortality in patients with DAD compared to those without DAD (71.9% vs 45.5%) . DespiteSome evidence shows that the clinical features and prognosis of AE-IPF according to the mentioned definition are very similar to the exacerbation of IPF with known cause such as pneumonia or aspiration . Since eDuring the course of AE-IPF, the percentage of neutrophils in bronchoalveolar lavage (BAL) fluid is significantly increased compared with stable chronic IPF, while lymphocytes and macrophages are reduced . This ceIn AE-IPF, the upregulation of M1 macrophage activation chemokines such as IL-8 and CXCL1 results in neutrophil chemoattraction. Interestingly, in animal models, the increased expression of CXC chemokines and their interaction with the CXCR2 receptor are involved in the lung sequestration of neutrophils following mechanical stress due to ventilation, thus suggesting a role in the development of VILI . FurtherThe alternative M2 macrophage activation pathway was also observed in AE-IPF, playing a determinant role in damage healing , 17. A dTherefore, both ARDS and AE-IPF share an overexpression of proinflammatory cytokines produced by alveolar macrophages with chemotaxis of neutrophils. However, overexpression of anti-inflammatory M2 cytokines with a profibrotic role is simultaneously present only in AE-IPF see Fig. .Fig. 1DuIn patients with ARDS, several studies have documented changes in lung mechanics, describing the role of mechanical ventilation in the development of VILI and the consequent increased risk of death . Much leT/EELV) applied to the lung tissue are the physical forces responsible for the development of VILI [Over the past 20 years, following the awareness that VILI can highly contribute to mortality, the goal of mechanical ventilation in ARDS has changed from improving gas exchange to protecting the lung from the damage induced by mechanical ventilation , 25. Unp of VILI , 27.Defining a threshold of safety for stress and strain remains a challenge . Transpu2O and set a VT less than 6 ml/kg of predicted body weight [T are parameters that are easy to monitor during mechanical ventilation but inadequate to truly represent the stress and the strain applied to the lung [Since stress and strain are not measured routinely, the plateau pressure and the tidal volume are considered surrogates of stress and strain, respectively, and are monitored closely in clinical practice when setting the ventilator in patients with ARDS. Currently, it is recommended to maintain an airway plateau pressure below 30 cmHy weight . Notwiththe lung .dynamic stress, representing the ratio between VT and the compliance of the respiratory system. It is reasonable to assume that compliance and end-expiratory lung volume (EELV), both being associated with the severity of lung injury, are correlated: under this assumption, ΔP would also reflect VT/EELV . Thus, the ΔP of the respiratory system or the lung represents a simple and promising tool at the bedside to monitor the injury caused by ventilation.Recently, interest has grown toward the variation of airway pressure achieved during tidal breath . ΔP equals plateau pressure minus positive end-expiratory pressure and can be considered the L) and the absolute level of transpulmonary pressure at end inspiration depend on the ratio between lung elastance (EL) and the total elastance of the respiratory system (ETOT = EL + ECW) according to the following equation [The transpulmonary pressure  was reported, mainly due to an abnormal lung elastance (46 cmH2O/L) with a normal chest wall elastance (5 cmH2O/L) and an EL/ETOT ratio around 0.9: In this case, the application of a plateau pressure of 30 cmH2O at a PEEP of 4 cmH2O, which are elevated pressures often seen in AE-IPF patients, causes a ∆P of 30 – 4 = 26 cmH2O, with an absolute end-inspiratory transpulmonary pressure of 30 × 0.9 = 27 cmH2O. Both values are above acceptable levels. If feasible in terms of gas exchange, a reduction of plateau pressure and driving pressure should be warranted.All of these features and concepts specifically refer to ARDS, and much less is known for AE-IPF. In a single study evaluating the respiratory mechanics of mechanically ventilated patients with end-stage IPF , a markeFurthermore, alveolar collapse and consolidation, that are responsible for permanent derecruitment, are present in IPF and do not improve with the application of positive pressure to the airways. Collapse induration is characterized by septal wall thickening and alveolar epithelial hyperplasia, with obliteration of alveoli due to enlargement and overgrowth of epithelial type II cells . It is tTherefore, despite some similarities with ARDS, the lung in AE-IPF is characterized by some unique pathophysiological properties  that might make it more susceptible to VILI. Figure Few studies have evaluated the outcome of patients with AE-IPF receiving mechanical ventilation in the ICU, and in addition they all share important limitations .2O [Pressure-controlled or volume-controlled invasive ventilation is the most widely applied mode of respiratory support for AE-IPF. As learned from ARDS, even in AE-IPF the main objective of ventilation should be lung protection, avoiding VILI (see earlier) while ensuring an acceptable, but not necessarily optimal, gas exchange. As reported in the literature, it is reasonable to apply a tidal volume even lower than 6 ml/kg of ideal body weight to target a plateau pressure lower than 30 cmH2O . Moreove2O), taking into account the intrinsic low recruitability potential, with high risk of hyperinflation. Indeed, poor survival with high end-expiratory pressure applied has been documented in a cohort of patients with interstitial lung disease [open lung approach, with recruitment maneuvers, recently questioned even in the early phase of ARDS [Despite the lack of studies about the usage of neuromuscular blockade in IPF patients, we could hypothesize that complete muscle paralysis at the early onset of severe AE could help in reducing the lung stress and strain, and avoiding a deleterious patient–ventilator asynchrony . Positiv disease . An open of ARDS , has no L level [2O in homogeneous lungs or below 12 cmH2O in inhomogeneous lungs, as is the case in AE-IPF [In patients with AE-IPF with high plateau pressure, the measurement of esophageal pressure (Pes) as a surrogate marker of pleural pressure may allow one to identify the lung stress and the risk of injury from ventilation by calculating the inspiratory transpulmonary pressure. To date, only experimental models suggest setting the protective mechanical ventilation to a ‘probably safe’ PL level , namely n AE-IPF .2/FiO2 < 150 mmHg) [Prone position (PP) has been used since the 1970s as a rescue therapy for severe hypoxemia in patients with ARDS . An imprOnly one study has evaluated the effect of PP on gas exchange in pulmonary fibrosis by comparison with both hydrostatic pulmonary edema and ARDS . In patiSpontaneous assisted breathing can have beneficial effects on shunt reduction and improvement in oxygenation, maintaining diaphragmatic tone and increasing dependent lung ventilation, in moderate but not severe acute respiratory failure , 54. Non2O. This means that, during assisted ventilation, this is the major contribution to the total transpulmonary pressure, also when airway pressure is apparently low. Even at comparable flow and volume conditions, spontaneous breathing can be more injurious when patients present a high respiratory drive [During assisted spontaneous breathing, inspiratory muscle activity leads to negativity of the pleural pressure, and thoracic structures are subject to inward forces. Patients with AE-IPF have a significant hyperactivation of the respiratory drive with a pleural swing that can even reach −30 to −40 cmHry drive .T [pendelluft effect, namely the fast exchange of gas volume that occurs during strong effort between different regions of the lung before starting VT, with deflation of nondependent regions and gas swing toward the dependent regions, which leads to increased local stretch: this regional inflation–deflation pattern is considered one of the causes of injury. Third, patient–ventilator asynchrony may increase the risk of lung injury [2 < 120 mmHg) may counteract these potentially detrimental effects of assisted breathing, resulting in improved survival.The reason for this effect on pulmonary stress depends on several factors. First, airway pressure can fall under end-expiratory pressure during spontaneous breathing, when performing vigorous inspiratory efforts . In thisT , 59. Thig injury . Early u01), esophageal pressure, and VT during spontaneous breathing could therefore be helpful in identifying patients at risk of self-inflicted lung injury (SILI) and to verify favorable changes when invasive pressure support ventilation is applied [In patients with AE-IPF, monitoring the respiratory drive with occlusion pressure  is a method of spontaneous breathing support not requiring endotracheal intubation, potentially reducing the risk of ventilator-associated pneumonia (VAP). Retrospective studies that have analyzed the effectiveness of NIV in AE-IPF reported a mortality rate between 45 and 75%, always related to the worsening of respiratory failure –40. OverMore recently, an observational study on a large cohort of patients with AE-IPF who underwent mechanical ventilation showed a lower mortality rate when NIV was applied 30.9%) as compared to conventional mechanical ventilation (51.6%) [0.9% as cAlso, high-flow oxygen delivered through nasal cannulae (HFNO) has proven efficacy in the management of nonhypercapnic acute respiratory failure . To dateExtracorporeal life support is a salvage strategy increasingly applied in ARDS with severe hypoxemia. Venovenous extracorporeal membrane oxygenation (ECMO) is able to provide adequate gas exchange beyond mechanical ventilation, and can potentially reduce the injurious effects of positive pressure ventilation . The besThere is evidence that patients who received mechanical ventilation in the pretransplant period have a significantly higher posttransplant mortality than nonventilated patients, suggesting that a bridge treatment with ECMO should be provided as early as possible . Indeed,Thus, it can be argued that ECMO might be a promising strategy to bridge lung transplantation in severe patients developing AE-IPF. Notwithstanding, reduced availability and high costs may limit its use in this condition.At present, no randomized controlled trials on drug treatments in AE-IPF are available; therefore, recommendations of international consensus are based on weak evidence, as is the case for systemic glucocorticoids (GC) .Considering again ARDS as a model, perpetuated DAD can be assumed as a dysregulated systemic and pulmonary inflammatory condition, where massive elevation of inflammatory cytokines in blood and BAL fluid correlates with a worse prognosis .GCs are able to block nuclear translocation of NF-κB, the main pathway of inflammatory cytokine synthesis, through their interaction with the glucocorticoid receptor (GR). Despite this rationale, the use of steroids in ARDS is not recommended routinely, as clinical trials have demonstrated improvements in oxygenation and lung mechanics but not in survival .Studies on the correlation between systemic inflammation and prognosis in AE-IPF are lacking. However, one study in IPF showed that ST2 serum levels, a protein expressed in T-helper type 2 cells and induced by proinflammatory stimuli, were higher during AE compared to the stable phase or in healthy controls, suggesting that systemic inflammation is a hallmark of AE-IPF , thus opGuidelines in France also support the use of intravenous cyclophosphamide, in addition to steroids, as an immunosuppressive agent . A retroMoreover, despite the use of many other cytotoxic agents  being reported anecdotally in other case series of AE-IPF, there is no robust evidence to suggest their use . FinallyTable 2/FIO2 ratio in patients with acute exacerbation of interstitial pneumonia of different etiology [Polymyxin-B (PMX-B) is a polypeptide antibiotic with bactericidal activity toward Gram-negative bacteria that binds circulating endotoxin . In patietiology .Enomoto et al.  comparedIn end-stage IPF, lung transplantation may offer better life expectancy with an overall 5-year survival rate around 50% . PatientAE-IPF shares several pathophysiological features with ARDS, and while the optimal ventilation strategy in these patients has not yet been defined, the extreme fragility of fibrotic lungs suggests adopting a protective ventilation strategy, which seems to positively impact inhospital survival. NIV should only be considered as an early measure, while monitoring the level of respiratory drive activation. ECMO has a role to bridge lung transplantation in severe patients with AE-IPF, but should be started early. Systemic steroids and immunosuppressive agents provide no clear evidence on their ability to change prognosis in AE-IPF.Taking all of the available evidence into account, it seems that applying the lesson so far learned from ARDS would be the best option to optimally manage AE-IPF as a critical clinical condition affecting the lungs.


Graphene oxide was functionalized with simultaneous reduction to produce phosphonated reduced graphene oxide in a novel, fully scalable, one-pot method. The phosphonic derivative of graphene was obtained through the reaction of graphene oxide with phosphorus trichloride in water. The newly synthesized reduced graphene oxide derivative was fully characterized by using spectroscopic methods along with thermal analysis. The morphology of the samples was examined by electron microscopy. The electrical studies revealed that the functionalized graphene derivative behaves in a way similar to chemically or thermally reduced graphene oxide, with an activation energy of 0.014 eV. Alternatively, some coupling agents as N,N'-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are used for the synthesis of GO amides or esters. Some other known approaches for GO functionalization via carboxylic groups are based on isocyanate or diisocyanate compounds. Many functionalization protocols of GO were described, but only few papers reported the introduction of phosphorous-containing groups into GO planes.Graphene oxide (GO) with its multifunctionality attracts interest in different fields of science. The chemical nature and reactivity of GO have been studied extensively. Many comprehensive reviews concerning GO chemistry can be found in the literature –3. Sever3 and H3PO3 [3 and water), which coupled with its simplicity makes it attractive for syntheses. Here, GO was functionalized with a simultaneous reduction to produce phosphonated rGO. The spectroscopic and thermogravimetric analysis confirmed the successful functionalization and simultaneous reduction of GO. The electrical studies showed a substantial increase in conductivity after functionalization. The measured energy of activation equal to 0.014 eV is in accordance with literature reports for reduced graphene oxide (rGO).Recently, Kim and Jeon described a ball milling process to efficiently functionalize and exfoliate pristine graphite directly into graphene phosphonic acid. In the first step, graphite is ball-milled for 48 h with red phosphorus to produce a derivative that is edge-functionalized with phosphorus. Then, upon exposure to air moisture, the resultant derivative of graphite is spontaneously oxidized forming phosphonic groups, with simultaneous exfoliation to produce phosphonic acid graphene . Phosphond H3PO3 . In thisThe promising uses of graphene derivatives in different fields are hindered by complicated and expensive functionalization methods, suitable only for laboratory scale. Effective functionalization by fully scalable, low-cost methods and detailed characterization of the synthesized nanomaterials is the first step towards utilizing the whole spectrum of applications. Functionalization of carbonaceous nanomaterials with phosphonic groups offers great potential for various applications, e.g., in energy conversion and storage ,10, flam3PO3), so if an excess of PCl3 is used, the reaction mixture is eventually PCl3/H3PO3. If GO is introduced into this reaction mixture the carboxylic groups react with PCl3 to produce an intermediate product, which is then converted into the respective phosphonate derivative in the reaction with H3PO3. The scheme of the reaction is presented in The phosphonic derivative of graphene (GO-P) was obtained through the reaction of GO with phosphorus trichloride in water. Phosphorous trichloride reacts with water to produce phosphorous(III) acid  can be ascribed to C-O vibrations in carboxyl, phenol and/or epoxide functionalities.Different spectroscopic methods were used to examine the functionalized GO. FTIR spectroscopy was used to determine the type of chemical groups in the analyzed samples. −1), which were well visible in the GO spectrum, disappeared in the GO-P spectrum. The small band visible in the GO-P spectrum in this region (ca. 1722 cm−1) may be due to the –OH vibrations in O=P(OH)2, which are of low intensity. Other bands at 1570 cm−1 and 1429 cm−1 can be ascribed to aromatic C–C bonds. Intensive bands at 1167, 1112 and 1060 cm−1 coming from P=O and two P–OH, respectively, confirm the presence of phosphonate groups in the analyzed sample [−1 refers to –OH groups in adsorbed water. The new broad and weak band at 2800 cm−1 additionally gives evidence that phosphonate groups tend to form hydrogen bonds (within themselves or water molecules) as this band it connected with associated PO–H stretching.Significant differences can be observed in the spectrum of GO-P. Firstly, the bands in the region of carbonyls vibrations  further confirmed the presence of phosphorous in the GO-P sample. The XPS spectra of GO and GO-P are presented in 3, producing hazardous waste. Finally, after ball milling for two days the resultant carbonaceous material is rather high-surface-area graphite than graphene.The high P content, together with the simplicity of the reaction makes the proposed method attractive compared to previously published protocols. For example, Liu et al.  reportedThermogravimetric (TG) analysis with differential scanning calorimetry (DSC) provided information about thermal stability of GO and GO-P and allowed for an estimation of the functionalization degree. As can be seen in The DSC analysis also revealed differences in thermal properties of the studied samples. The DSC profile of GO and functionalized GO is not frequently reported. 6 Ω·cm. The temperature-dependent electrical conductivity (σ) of GO-P was investigated in a temperature range from 25 to 70 °C. The functionalization with simultaneous reduction resulted in a pronounced increase of conductivity compared to pristine GO (−2 S·cm−2 to 4.2 × 10−2 S·cm−2 in the measured temperature range) and increases with increasing temperature. Moreover, an exponential temperature dependence of conductivity was noticed. The red line presented in −1)). The good correlation between applied model and experimental results (R2 ≈ 0.998) indicates that the electrical conductivity at higher temperatures of the analyzed GO-P sample can be well described by an Arrhenius temperature dependence model for semiconductors.Finally, a measurement of the electrical properties of GO-P materials was carried out. Before reduction, the GO layers exhibited insulating behavior, with a resistivity higher than 10stine GO . As can The results presented in 3The development of industrially applicable methods for the production of graphene derivatives will pave the way for a wider spectrum of applications. In this paper, a simple one-pot synthesis approach for the functionalization of a graphene derivative was presented. The method benefits from the easiness, fastness, the usage of inexpensive and readily available chemicals and it is fully scalable. The resultant phosphonated graphene was demonstrated to form stable suspensions in various polar solvents including water, which is the preferable solvent for numerous applications. The obtained material was fully characterized and the obtained results confirmed the successful functionalization with simultaneous reduction of GO. FTIR and XPS spectroscopy confirmed the successful introduction of phosphonate groups into GO-P. The phosphorous content calculated from XPS results was 2.15%. Raman spectroscopy, along with UV–vis and XPS spectroscopy results proved the simultaneous restoration of conjugated double bonds in the graphene plane. The functionalized material revealed an electrical conductivity consistent with values reported for chemically or thermally reduced GO. The conducting graphene derivative with polar multiprotic phosphonate groups can find its place in, for example, conducting composites, ion exchangers and proton carriers.3 was purchased from POCh . Spectral grade KBr was purchased from Sigma-Aldrich. Deionized water was used in the experiments.PClDuring the experiments a magnetic stirrer Heidolph MR hei-standard with hot plate was used and the centrifugation for all samples was done using a Chemland model P3032 centrifuge at a speed 15000 rpm for 10 min.−1 in the range of 500–4000 cm−1.The FTIR spectra were recorded using the KBr pellet method on a Perkin Elmer Frontier spectrophotometer with resolution of 2 cmUV–vis spectra were measured using a Perkin Elmer Lambda 10 with 1 cm quartz cuvette.−1 in the range of 100–3200 cm−1.Raman spectra were recorded using a Renishaw InVia spectroscope with argon ion laser operating at 514.5 nm focused through a 50× objective. The collected light was dispersed through a triple monochromator and detected with a charge-coupled device. The spectra were collected in the dark, with a resolution of 2 cmThe morphology of the samples was observed with a scanning electron microscopy .−8 mbar. The photoelectrons were excited with an Mg Kα X-ray source. The X-ray anode was operated at 15 keV and 300 W. An Omicron Argus hemispherical electron analyser with round aperture of 4 mm was used for analyzing the emitted photoelectrons. The binding energies were corrected using the background C 1s line (285.0 eV) as a reference [XPS analyses were carried out with an X-ray photoelectron spectrometer (Omicron NanoTechnology) with 128-channel collector. The measurements were performed with constant energy mode (CAE) with energy pass equal to 50 eV. The samples were pressed before analysis. XPS measurements were performed at room temperature under ultra-high vacuum conditions, below 1.1 × 10eference . XPS spe−6 kg of sample was used, and Al2O3 crucibles with lids were employed. The total uncertainty associated with measurement was 0.005% by weight of the sample and was included in the final result. The first derivatives of TGA and DSC, denoted as DTG and DDSC, respectively, were calculated using Netzsch Proteus Thermal Analysis program.Simultaneous thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were performed in synthetic air with a heating rate of 5 °C/min from 40 to 900 °C using a Netzsch STA 449 F1. The STA449 F1 has a vertical sample carrier and in order to account for buoyancy effects, a correction curve with empty crucibles was first obtained and then subtracted from the experimental results. To avoid heat and mass transfer limitations, approximately 8 × 10In order to investigate the electrical properties of obtained GO structures, the total surface conductivity (σ) was measured by a conventional DC four-wire method with a Keysight 34970A multimeter. The measurements were performed in a temperature range of 25–70 °C in air atmosphere. The sample was prepared as a thin film of rectangular shape.3 was added dropwise under simultaneous agitation. After 5 h of heating at 65 °C, the excess of PCl3 was distilled off and the residue was diluted with water and stirred for 1 h at 50 °C. Then, the solids were separated by centrifugation, washed with deionized water till a neutral pH value and dried overnight under reduced pressure  to obtain GO-P.GO was synthesized by using the improved Hummer’s method proposed by Tour’s group and fully characterized as described previously –12. To aFile 1Additional experimental data.


Odorrana livida. This novel peptide exhibited a dose-dependent contractile property on rat bladder and rat ileum, and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations; it also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the analogue RAP-L1, T6, L8-BK completely abolished these effects on selected rat smooth muscle tissues, whilst it showed inhibition effect on bradykinin-induced rat tail artery relaxation. By using canonical antagonist for bradykinin B1 or B2 type receptors, we found that RAP-L1, T6-BK -induced relaxation of the arterial smooth muscle was very likely to be modulated by B2 receptors. The analogue RAP-L1, T6, L8-BK further enhanced the bradykinin inhibitory activity only under the condition of co-administration with HOE140 on rat tail artery, suggesting a synergistic inhibition mechanism by which targeting B2 type receptors.Bradykinin-related peptides (BRPs) are one of the most extensively studied frog secretions-derived peptide families identified from many amphibian species. The diverse primary structures of BRPs have been proven essential for providing valuable information in understanding basic mechanisms associated with drug modification. Here, we isolated, identified and characterized a dodeca-BRP , with primary structure RAPLPPGFTPFR, from the skin secretions of Chinese large odorous frogs,  Rana temporaria [C/N-terminal extension, were isolated and identified from the Ascaphidae, Hylidae, Ranidae and Bombinatatoridae families. Of note is that mammals, reptiles, birds, and even fish were found to produce structurally discrete BRPs [Bradykinin (BK) is a nonapeptide hormone first discovered in 1949 by Rocha et al. upon the basis of its inherent capability to dilate vessels ,3. Sinceete BRPs . The resete BRPs . Followiete BRPs .BK triggers its effect mainly through two types of receptors, B1 and B2, which all belong to the G protein-coupled receptors family. Compared with B2 receptors, which are generally expressed in multiple tissues, B1 receptors are only constitutively expressed in certain tissues, such as spinal cord, brain, and artery endothelial cells in the aorta and lung . While uStimulation or inhibition of B1 or B2 receptors is thought to be related with many pathophysiological disorders. Therefore, development of agonists or antagonists for either B1 or B2 receptors or even both would provide researchers significant implications no matter in the fields of pharmacology or clinical therapy.Odorrana livida by using 3′RACE and 5′RACE “shotgun” cloning technique. The primary sequence was further confirmed via the MS/MS fragmentation sequencing approach. The synthetic replicates of this novel peptide and its analogue RAP- L1, T6, L8-BK, were characterized by multiple organ-bath based ex vivo rat smooth muscle tissue probes. The wild-type RAP-L1, T6-BK was found to stimulate rat bladder, ileum, uterus contractile and tail artery relaxant responses. By contrast, the analogue RAP-L1, T6, L8-BK completely abrogated these functions, and it showed strong inhibition effect upon the BK-induced rat tail artery relaxation. Further pharmacological analysis revealed that BK B2 receptors are highly likely to be involved in the rat tail artery related effects caused by this novel BRP and its analogue.Here, we reported a novel BRP, RAP-L1, T6-BK, which was isolated and identified from the skin secretion of the Chinese large odorous frog Odorrana livida skin secretion-constructed cDNA library, the open-reading frame of this novel BRP precursor consists of 61 amino acids, and the architecture of translated open-reading frame can be divided into four domains. The 5′ N-terminus begins with a putative signal peptide with 22 amino acid residues followed by a 21 acidic residue-rich spacer, the putative 12-mer mature peptide is preceded by a propeptide convertase processing site -VK-, and it was followed by a C-terminal extension peptide  with the BRPs precursor sequences from Amolops species. The highly-conserved domain includes the first residue Arginine of N-terminal extension and -PPGFTPFR- in the canonical BRP section  on line portal, revealed that the full length open reading frame of novel RAP-L1, T6-BK (OL SBN54116) peptide displayed relative high amino acid sequence identity  in this assay  were used to evaluate inherent cytotoxicity of two BRPs in this study. After incubation for 24 h, the viability of cells was evaluated using the MTT assay. The synthetic replicates of the BRPs exhibited no significant cytotoxic effects at the concentrations employed  on tissue responsiveness between BK-induced artery relaxation and L-NIO pre-administrated BK-induced artery relaxation, which confirmed that the NO was not likely to be involved in the effects of vasodilation under such rat tail artery smooth muscle preparation circumstances   a. For raectively b. On rat 1.05 nM c. For ra 2.35 nM d.p = 0.0002, n = 5), whereas specific BK B1 receptor antagonist R715 did not show any attenuation effect on RAP-L1, T6-BK induced rat tail artery relaxation . In addition, the combined pre-treatment of HOE 140 and R715 exhibited no significant difference with the singular application of HOE 140   were pre-treated alone or in combination with RAP-L1, T6-BK in rat tail artery smooth muscle preparations. BK B2 receptor antagonist, HOE-140, significantly inhibited the RAP-L1, T6-BK induced relaxation of rat tail artery  . These d−11 to 1 × 10−5 M , although the IC50 values were 1.20 ± 0.08 nM vs. 0.50 ± 0.56 nM. Similar to RAP-L1, T6, L8-BK, HOE140 pre-treatment largely antagonize BK-induced relaxation, whereas this effect was not detected under R715 pre-treatment , RAP-L1, T6, L8-BK and HOE140 significantly attenuated rat tail artery dilation induced by BK . Co-administration of R715 with RAP-L1, T6, L8-BK did not further enhance the BK attenuation effect, while co-administration of HOE140 with RAP-L1, T6, L8-BK exhibited a synergetic inhibition activity to almost block BK-induced rat tail artery relaxation   .Frog skin secretions contain large number of bioactive components, including antimicrobial, protease inhibitory, and pharmacological relevant peptides. As we stated in the introduction, discrete BRP may have unique ligand-receptor specificity and potency, which is vital to provide valuable structure-interaction relationship information for agonistic and antagonistic research. Therefore, we decided to focus on “shotgun cloning” novel BRP by using BK-derived degenerate primer in this project. In addition, BRPs from amphibian secretions and venoms are considered to be homologous counterparts of endogenous mammalian polypeptide hormones. Therefore, study on their physiological and pathological relevance is essential for us to understand metabolic homeostasis in vivo, which could provide us novel insight into cardiac, vascular, renal and inflammatory associated disorders ,11,12,13Odorrana livida, through “shotgun” cloning of a biosynthetic precursor-encoding cDNA from a lyophilised skin secretion-derived cDNA library  through a common downstream signalling, and NO is widely accepted as the final substance that induces smooth muscle relaxation and inflammatory status . The eNO−7 M, RAP-L1, T6-BK have similar vasodilate capacity with BK, and when the concentration raised to 10−6 M and 10−5 M, RAP-L1, T6-BK dramatically enhanced the vasodilation on rat tail artery smooth muscle compared to BK, which indicated that the threshold concentration for RAP-L1, T6-BK and BK to induce a significant rat tail artery relax is differentiated, BK is more potency at lower concentration (10−9 M–10−7 M), but RAP-L1, T6-BK is more effective at higher concentration (10−6 M–10−5 M). In consideration of this, the primary structure similarity between them proposed us the hypothesis that RAP-L1, T6-BK mediates these effects through the cognate G protein-coupled receptors, and thus, we examined the arterial smooth muscle relaxing property modulated by RAP-L1, T6-BK in the presence of HOE140, a well-established BK B2 antagonist and R715, a selective BK B1 antagonist. Surprisingly, we observed a significant attenuation (nearly 60%) in HOE140 treatment condition, while R715 barely antagonize RAP-L1, T6-BK, which suggests a BK B2 receptors-related mechanism. Further experiments are still needed to address this issue thoroughly. Additionally, stimulation of neurokinin receptors in rat bladder, muscarinic receptors in ileal and endothelin receptors in uterine smooth muscle, which leads to intracellular calcium concentration, are reported to be involved in the contractions of these tissues [The myotropic potency of this novel identified RAP-L1, T6-BK peptide is relatively weak compared to BK, not only in contraction of rat bladder, ileum and uterus, but also in dilating vessels. Whilst, the novel peptide seemed to exhibited higher efficacy for the receptors compared to BK, which could be deduced from the  tissues ,23,24,25−8 M–10−5 M. These data suggested that although this novel BRP has a three residues -RAP- extension and leucine 1, threonine 6 substitutions, the leucine 8 still plays the essential and dominant role in reversing the BK’s effect. To investigate the precise manner how RAP-L1, T6, L8-BK exert BK antagonistic effect, again, we pre-treated rat tail artery smooth muscle with specific BK B1 receptors and/or B2 receptors inhibitors. When the tissues pre-treated with R157, BK-induced artery relaxation has a trend to be attenuated, but no statistical differences were observed. In contrast, when the tissues were pre-treated with HOE140, BK-induced artery relaxation was largely impaired. This probably due to that B1 receptors are rare expressed in rat tail artery or B2 receptor-mediated signalling is mainly responsible for BK-induced relaxation in rat tail artery, further examination by pre-treating the tissues with RAP-L1, T6, L8-BK along or together with R157 showed similar BK inhibition potency, which implies that the peptide might exert the effect in a B2 receptors-dependent manner. While RAP-L1, T6, L8-BK pre-treatment along with HOE140 almost abolished BK-induced relaxation suggested a novel synergistic inhibition mechanism via the BK B2 receptors might be involved compared with HOE140. In terms of this additive effect, others receptors, rather than BK receptors, activated by RAP-L1, T6, L8-BK is also a feasible option. Therefore, the primary structure initiated ligands-receptor interaction study of BRPs could provide researchers valuable information for developing novel molecular therapeutics.The analogue peptide RAP-L1, T6, L8-BK completely abrogated those bioactivities. Of interest is that only the leucine residue substitution at position 8 of BK would cause such an astonishing function loss. Leucine 1 is a common substitution occurred in BRPs, compared with valine substation at position 1, it normally exhibits moderate agonistic effect of BK . As the Odorrana livida, which presented distinct bio-functionalities, have unique structure/activity relationship, and continuously provides virtues for drug design.Taken together, due to the inherent characteristics of the physiopathological significance of diverse BRPs for their potential to treat multiple vascular, cardiac diseases, they have been focused on for many years. In this project, we reported a novel BRP and its analogue isolated from Chinese large frog Odorrana livida  were obtained from the mountain areas of Fujian province, People’s Republic of China. The gentle electrical stimulation   was performed to induce the secretion from the glands of the frog dorsal surface. After secretion harvesting, the frogs were released. This method was non-lethal to frogs. The distilled deionized water was used to rinse the frog skin surface for secretions collection. The secretions were then subjected to snap frozen with liquid nitrogen, lyophilized and stored at −20 °C before use. Sampling of skin secretion was performed by Mei Zhou under UK Animal (Scientific Procedures) Act 1986, project license PPL 2694, issued by the Department of Health, Social Services and Public Safety, Northern Ireland. Procedures had been vetted by the IACUC of Queen’s University Belfast, and approved on 1 March 2011.Adult Chinese Large Odorous frogs, Odorrana livida skin secretion was dissolved in lysis/Binding Buffer. The extraction of polyadenylated mRNA was performed by using Dynabeads® mRNA DIRECT™ Kit . The cDNA library was obtained in reverse transcription reaction. Both 3′-RACE and 5′-RACE PCRs were employed to obtain the novel full-length prepro-BRP nucleic acid sequence by using SMART-RACE kit . Briefly, the 3′-RACE PCR reaction employed a specific sense primer (5′-CCRVCNGGGTTYASSCCWTTY-3′)  that was complementary to the nucleic acid sequence encoding the common -PPGFSPF- and -PPGFTPF- internal amino acid sequences of ranid frog skin BRPs and a UPM primer (supplied with the kit). The 3′-RACE reactions were purified and cloned into pGEM®-T Easy Vector  and sequenced using an ABI 3100 automated capillary sequencer. The 5′-RACE PCR employed an antisense primer (AS: 5′-CCGTATGTCATTTAGCTAAATGATGA-3′), which was designed to a highly conserved domain located within the 3′ non-translated region of the obtained 3′-RACE transcripts, and a UPM primer. In addition, the 5′-RACE products were gel-purified, cloned, and sequenced.Five milligrams of lyophilized Odorrana livida skin secretion was dissolved in 1 mL trifluoroacetic acid (TFA)/water , followed by centrifugation to clarify the sample solution. The clear supernatant was decanted and pumped into an analytical reverse phase HPLC column  for fractionation by using a Cecil Adept 4200 HPLC system . The linear gradient elute method was used from TFA/water  to TFA/water/Acetonitrile  over 240 min at a flow rate of 1 mL/min. The fractions were collected automatically at one-minute intervals; the effluent absorbance was continuously monitored at 214 nm. Fractions were lyophilized and stored at −20 °C prior to being subjected to the smooth muscle assay. Meanwhile, the molecular masses of polypeptides in the collected fractions were analysed through matrix-assisted, laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) on a linear time-of-flight Voyager DE mass spectrometer . The sample was analysed in positive detection mode using α-cyano-4-hydroxycinnamic acid as the matrix. Internal instrument calibration was performed by using standard peptides of established molecular masses, which provides the determined accuracy of ±0.1%. Fractions with the masses coincidence with those deduced mature peptides from the molecular cloning were subjected to MS/MS fragmentation sequencing analysis by using a Thermoquest gradient reversed phase HPLC system , fitted with an analytical column (C-18), and interfaced with a Thermoquest LCQ™ Deca electrospray ion-trap mass spectrometer .An aliquoted sample of five milligrams lyophilized ® Peptide Synthesizer . The analogue RAP-L1, T6, L8-BK was also synthesized with the same method. All synthetic peptides were purified with reverse phase HPLC. The authenticity of their primary structures and purity were confirmed via MALDI-TOF MS and LCQ MS/MS fragmentation sequencing.Once the unequivocal primary structure of the novel bradykinin-related peptide had been determined through MS/MS fragmentation sequencing and molecular cloning of its biosynthetic precursor-encoding cDNA, Fmoc chemistry based solid phase peptide synthesis was performed to produced RAP-L1, T6-BK peptide replicates by using Tribute−11 M to 10−5 M for 24 h. Cell viabilities were assessed by MTT assay, as described previously [Prior to the smooth muscle assay for evaluating the pharmacological actions of BRPs, the MTT cell viability assay was performed. Cultured human microvessel endothelial cells (HMECs) were used to assess the possible influence of inherent cytotoxicity caused by the synthetic novel BRPs. In detail, the HMECs were seeded in 96-well plates with the density 5000 cells/well. After overnight incubation, HMECs were subjected to 6 h starvation prior to being treated with synthetic BRPs  ranging from 10eviously .3, 1.15 mM NaH2PO4, 2.5 mM CaCl2, 1.1 mM MgCl2 and 5.6 mM glucose), and equilibrated with a mixed gas (95% CO2 and 5% O2). The tissues were dissected and mounted as shown in the table to obtain maximum viability and to achieve the best response (Wistar rats (200–250 g) were euthanized by asphyxiation with carbon dioxide in accordance with institutional animal experimentation ethics and UK animal research guidelines. The rats were placed on their dorsal surfaces, an incision was made along the mid ventral line towards the bottom of the abdomen and subcutaneous fat was carefully removed. The smooth muscle tissues of bladder, ileum, uterus, and tail artery were dissected gently followed by being placed in ice-cold Krebs’ solution immediately  at 37 °C and constantly bubbling 95% O2 and 5% CO2 gas mixture. The prepared tissues were attached to the force transducer in order to detect the tension changes. The responses of tension change were recorded and amplified through pressure transducers connected to a PowerLab System . The peptide solutions of RAP-L1, T6-BK and RAP-L1, T6, L8-BK were prepared in the range of 10−11 M to 10−5 M with fresh Krebs’ solutions to construct the dose–response curves. Each concentration of individual peptide was applied to a minimum of six muscle strips.After dissection, small strips of particular tissues were connected to a triangular hook. Then, the removed tissues were mounted to the force transducers in a 2 mL organ bath which were perfused with Krebs’ solution  for 10 min to achieve constriction plateaux. To further exclude the possible vasorelaxant effect caused by endothelial-derived nitric oxide (NO), tail artery smooth muscles were pre-treated with/without 0.5 µM eNOS (endothelium nitric oxide synthase) inhibitor N5-(1-Iminoethyl)-l-ornithine dihydrochloride  for 20 min, prior to performing a BK sequential concentration (10−11 M to 10−5 M) -induced vasodilation.For the rat tail artery preparations, endothelial linings were essentially removed from the dissected smooth muscle, a basal tension of 0.5 g was applied to the organ bath incubated tail artery before adding phenylephrine  were applied to artery smooth muscle preparations prior to peptide treatment.To examine what type of BK receptors in rat tail artery smooth muscle were targeted by BRPs, a single 10p value less than 0.05 was considered significant.Data from this study were analysed by one-way ANOVA with Bonferroni’s post-test and two-way ANOVA using GraphPad Prism software . A


The simulated result shows that the proposed absorber can achieve a near-perfect absorption above 90% with a bandwidth of 4.8 Thz. Owing to the flexible tunability of graphene sheet, the state of the absorber can be switched from on (absorption > 90%) to off (reflection > 90%) in the frequencies range of 3–7.8 Thz by controlling the Fermi energy of graphene. Moreover, the absorber is insensitive to the incident angles. The broadband absorption can be maintained over 90% up to 50°. Importantly, the design is scalable to develop broader tunable terahertz absorbers by adding more graphene layers which may have wide applications in imaging, sensors, photodetectors, and modulators.We propose and numerically demonstrate an ultra-broadband graphene-based metamaterial absorber, which consists of multi-layer graphene/dielectric on the SiO In recent years, the terahertz band has become one of the most interesting platform because of the huge application in spectroscopy, medical imaging, modulators, security, and communication –3. The tOwing to the superiority of graphene materials in terahertz absorbers, there are some graphene absorbers that have been proposed and demonstrated –34. TheoIn this paper, we present a tunable graphene-based terahertz absorber composed of multilayer graphene which can achieve an ultra-broadband absorption over 90% in the frequencies range of 3–7.8 Thz. The average absorptivity of the absorber is higher than 96.7%. Besides, the proposed absorber has higher switching intensity, the absorption amplitude can be tuned from near-perfect absorption (> 90%) to high reflection (> 90%) by changing the Fermi energy of graphene layer in the whole bandwidth of 4.8 Thz. When the Fermi energy of graphene is 0 eV, the proposed structure will be a near-perfect reflector with a reflection more than 97% in the high frequency band (about 5.5 Thz later). In addition, the absorber is independent to the incident angles with the absorption more than 90% up to 50°. To our best knowledge, we first propose the two-dimensional multi-layer graphene/dielectric structure to realize an ultra-broadband absorption. The proposed absorber is simple which do not depend on complex patterned graphene, and the design provides great convenience for the fabrication of multilayer graphene structures , 39. Imp2 layer and a thick metallic reflecting plate on the bottom. As shown in Fig. W) is embedded in the dielectric at a certain gap t2 (t2 = 2 μm). The width W of each graphene is 5, 5, 27, 4, 4, 2, 21, 21, and 26 μm, respectively (from top to bottom). Each layer is symmetrical about the z-axis. The distance t1 between the bottom of the graphene layer and the SiO2 layer is 2 μm. The thickness of the dielectric is H1. The middle layer is SiO2 with a thickness of H2. The bottom is a metallic film with a thickness of D. The period of the unit is P. These initial values of the structure parameters are set to H1 = 21 μm, H2 = 7 μm, D = 0.5 μm, P = 32 μm. The bottom metallic material is gold, and its permittivity can be properly represented by the Drude model in terahertz range as follows:ε∞, plasma frequency ωp, and collision frequency γ are set to 1, 1.38 × 1016 rad/s, and 1.23 × 1013 s− 1, respectively. The permittivity of the dielectric material and SiO2 material are set to 3 and 4, respectively.The diagram of the proposed structure is shown in Fig. w is the radian frequency, ε is the energy, kB is the Boltzmann’s constant, τ is the carrier relaxation time, T is the temperature (T = 300 K in our paper), ℏ is the reduced Plank’s constant, and Ef is Fermi energy. The Kubo formula (2) indicates that the complex graphene surface conductivity can be adjusted by Fermi energy Ef. The graphene Fermi energy of each layer can be individually controlled by the biased voltage, the relation between Ef and biased voltage can be written as [In the simulation, graphene is treated as an ultra-thin film embedded in the dielectric. The complex graphene surface conductivity dominated by interband and intraband contributions can be calculated by using the Kubo formula :2\documeitten as , 42:3\dovF = 0.9 × 106m/s is the Femi velocity, V0 is the voltage offset [a0 is the capacitive model of the structure, where ε0 is the permittivity in vacuum. εd is the permittivity of dielectric, d is the height of dielectric, and e is the charge of an electron. Vn(V1~V9), that is, the voltage applied to the graphene can be obtained from the additional circuit of Fig. where e offset , \documetg is the thickness of the graphene, ε0 is the permittivity of vacuum, and σg is the surface conductivity of the graphene. According to Formula (4), the permittivity of graphene can be obtained by the surface conductivity, which can also be obtained by the applied voltage. Therefore, Formula (2–4) indicate that the electromagnetic properties of graphene can be dynamically controlled by the applied voltage, leading to that the absorption characteristics of the structure can also be dynamically controlled.In which z direction with its electric field E along x direction. The Bloch boundary condition is applied to the oblique incidence in the periodic structure. We use 1-R-T to calculate the absorption of the model, where R and T represent reflectivity and transmissivity, respectively. Since the thickness of the metal is much greater than the skin depth of incident light in the metal, the transmissivity T is zero. Thus, we simplify the calculation formula for 1-R.To investigate the absorption performance of the designed structure, we implement the numerical simulations using two-dimensional FDTD. In our simulation, we set the structure to a periodic boundary condition in the x direction. A beam of terahertz plane wave is incident to the model normally along the Ef of the each graphene layer to 0.9, 0.9, 1.1, 0.8, 0.8, 1.1, 1.1, 0.9, and 0.8 eV). As shown in Fig. BW is the bandwidth and f0 is central frequency). We also calculate the average absorptivity of the absorber, which is as high as 96.7%. On the other hand, with the Ef = 0 eV, the proposed structure will be a near-ideal reflector with reflection more than 90% over the whole operation bandwidth, and in the high frequency band (about 5.5 Thz later), the reflection even more than 97%. Of course, we can also tune the voltage of each graphene layer to get the desired amplitude which may have potential applications in some areas.Firstly, we tune the voltage of each graphene layer to achieve perfect absorption .Then, in order to achieve broadband absorption, we need to allow the resonance absorption peaks which achieve the admittance matching close to each other. As the absorption peaks are close enough to merge, a broadband absorption is obtained. Therefore, we add graphene layers to get more resonance absorption peaks. And at the same time, we adjust the parameters that affect the resonance peak, including As shown in Fig. To understand the physical mechanism behind the ultra-broadband near-perfect absorption, we also give a detailed calculation and analysis about the electric field amplitude (|E|) distributions of the proposed structure at different operating frequency. As shown in Fig. In order to better illustrate the stacking effect, for example, according to Fig. As discussed above, the strong coupling between graphene and the dielectric plays a major role in broadband absorption. In the practical applications, we hope that the broadband absorption is insensitive to the incident angles. As shown in Fig. H1 and with different thickness of the SiO2 layer H2, respectively. As shown in Fig. H1 is 21 μm. On this basis, H1 increase or decrease 0.5 μm, the performance of the absorber almost has no change. Even if H1 changes by 1 μm, the absorber still maintains an absorption above 90% in most bands except the band around 7 Thz. As shown in Fig. H1, the absorber is more sensitive to the height of the SiO2H2. Even in this case, in addition to the band around 6 and 7.1 Thz, the absorber also maintains a good performance in the most bands. As discussed above, we can find that although the thickness of the dielectric layer and the SiO2 layer are changed even at the micron scale, the absorber still maintains a good absorption performance in most wavelengths which will greatly improve the robustness of the absorber in the fabrication.Finally, considering the difficulties of multi-layer structure in fabrication, we discuss the effect of the relevant structure parameters on the absorber performance. Figure Ef of different graphene layers. With the Ef = 0 eV, the proposed design will be a near-ideal reflector with reflection more than 90% within the whole operation bandwidth of 3–7.8 Thz. The ultra-broadband absorption is ascribed to the stacking effect of strong resonance absorption at different frequencies excited by localized surface plasmon (LSP) and graphene surface plasmons. In addition, the proposed absorber is insensitive to the incidence angles, and we also find that the thickness of the dielectric layer and the SiO2 layer has little effect on the absorption performance, which is more beneficial to practical applications. Moreover, the proposed absorber is simple which do not depend on complex structured graphene and the bandwidth may be widened by adding more graphene layers. This tunable broadband absorber may have great potential applications in photodetectors, imaging, and modulators.In this paper, we propose an ultra-broadband, tunable graphene-based terahertz absorber consisting of multilayers of graphene/dielectric. The proposed absorber can achieve a broadband absorption over 90% with a bandwidth of 4.8 Thz through altering the Fermi energy


The role of programmed cell death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC), especially according to histologic type, remains controversial. The purpose of this study was to assess PD-L1 expression and its association with overall survival (OS) and clinicopathologic characteristics in NSCLC.Formalin-fixed paraffin-embedded specimens were obtained from 108 patients with surgically resected primary NSCLC. PD-L1 expression was assessed via immunohistochemistry using a histochemistry score system. The relationship between OS or clinicopathologic characteristics and PD-L1 expression was evaluated via the Kaplan-Meier method and Cox proportional hazards model, respectively.p = 0.003). Patients without PD-L1 expression had remarkably longer OS than those with PD-L1 expression . In the subgroup analysis of non-squamous cell carcinoma, OS was more favorable in those without PD-L1 expression than in those with PD-L1 expression . Multivariate analysis revealed that PD-L1 expression , male sex and higher tumor-node-metastasis stage were significantly correlated with shorter OS.Of 108 NSCLC specimens, 44 had high PD-L1 expression, which was highly associated with histologic type (This study demonstrated that PD-L1 expression is an independent prognostic factor for poor survival in NSCLC patients, especially those with non-squamous NSCLC. Lung cancer is the leading cause of cancer death worldwide . AlthougCytotoxic T-lymphocyte–associated antigen 4 (CTLA-4), a second counter receptor for the B7 family of co-stimulatory molecules, was the first clinically validated checkpoint pathway target. CTLA-4 inhibits T cell activation in initial stages and breaks tolerance of human cancer antigens, many of which are normal self-antigens, yet produces frequent immune-related adverse events –7. More Nonetheless, data on the prognostic role of PD-L1 expression in NSCLC tumor cells and its correlation with clinicopathologic characteristics are conflicting. Two previous studies showed that PD-L1 expression was a favorable prognostic factor for overall survival (OS) in NSCLC , 23. HowIn this study, we used immunohistochemistry (IHC) to assess the prognostic role of PD-L1 in the OS of patients with surgically resected NSCLC and the association between PD-L1 expression and clinicopathologic characteristics, including histologic type, sex, age, and pathologic tumor-node-metastasis (TNM) stage.A total of 108 patients with NSCLC were included in the study. The patients’ characteristics are shown in Table Of the 108 cases of NSCLC, 44 had PD-L1 expression in the tumor cell membrane and cytoplasm. Squamous cell carcinomas and adenocarcinomas with PD-L1 staining are shown in Figures p = 0.003). The percentage of PD-L1-positive cells in squamous cell carcinoma was higher than that in non-squamous cell carcinoma (62.8% vs. 31.1%), whereas, the absence of PD-L1 expression was more common in non-squamous cell carcinoma than in squamous cell carcinoma (68.9% vs. 38.2%). In contrast, PD-L1 expression was not significantly correlated with sex (p = 0.408), age (p = 0.596), TNM stage (p = 0.134), tumor stage (p = 0.240), node metastasis (p = 0.061), or the degree of differentiation (p = 0.065).The correlation of PD-L1 expression with clinicopathologic characteristics is presented in Table p < 0.001).In Figure p = 0.002). Similar results were observed in those who were male, had a higher TNM stage, had a greater degree of node metastasis, and had squamous cell carcinoma (Table p < 0.001), male sex , and higher TNM stage  remained independent risk predictors for poorer OS , 1.355-3.840, p < 0.001) and patients of different age groups , but not among females  , TNM stage III/IV disease , and tumor stage pT3 , but not in patients with tumor stage pT1, pT2, or pT4 or node metastasis (p > 0.05) . However, subgroup analysis of squamous cell carcinoma did not reveal any significant difference in OS between those without PD-L1 expression and those with PD-L1 expression . Similarly, there was no significant difference based on the degree of differentiation (p > 0.05) , an engineered IgG anti-PD-L1 antibody, has also shown activity in ongoing phase III trial in NSCLC. Avelumab (MSB0010718C), a fully human anti-PD-L1 IgG1 antibody, currently being investigated in a phase III head-to-head trial of avelumab versus docetaxel in patients with NSCLC . We antiAlthough NSCLC patients have favorable clinical benefits from blocking the PD-1/PD-L1 pathway, certain issues must still be considered. The most important is that different score systems and different tricks of immunohistochemical technology maybe lead to different results. Furthermore, anti-PD-L1 monoclonal antibodies used for testing PD-L1 expression may lack sensitivity and yield false-negative results, and there is no uniform standard at present. In addition, the expression of PD-L1 is very dynamic, and its uneven distribution in tumor tissue might lead to false-negative results –45. CompA standardized method of detecting PD-L1expression maybe provide support for patients who are initially diagnosed with primary NSCLC by allowing for early anti-PD-1 or anti-PD-L1 monoclonal antibody immunotherapy, thus improving treatment efficacy and managing cost. Although the current method of detecting PD-L1 is not as precise as EGFR test, the prospects of PD-L1 as a prognostic factor for poor survival are encouraging.Efforts were made to conduct sound and rigorous research, but our study had some limitations. First, we obtained several interesting results using samples from 108 patients, but the results will need to be validated with a larger sample size. Second, we retrospectively collected all the information, rather than performing a prospective study. Third, PD-L1 expression was assessed with only IHC, but other detection methods are needed to verify the results.In conclusion, the current study demonstrated that PD-L1 expression is an independent prognostic factor for poor survival in NSCLC patients, especially those with non-squamous NSCLC. We expect to develop an optimized method of detecting PD-L1 expression in tumor cells to validate PD-L1 as an accurate predictor for anti-PD-1/PD-L1 monoclonal antibody treatment. We anticipate that the anti-tumor efficacy of anti-PD-1/PD-L1 monoclonal antibodies will be strengthened and survival in NSCLC patients will be improved.This study included 108 patients who underwent tumor resection or palliative surgery for a primary tumor that was histologically confirmed as primary NSCLC between 2002 and 2012 at the Fifth Affiliated Hospital of Sun Yat-sen University. From their medical records, we retrieved and recorded patients’ clinical data, such as sex, age, histologic type, and TNM stage (using the 7th UICC TNM Staging System of lung cancer). All of the patients were carefully monitored after initial treatment. Expert pathologists at the hospital re-reviewed hematoxylin-eosin–stained slides from all cases, and corresponding formalin-fixed, paraffin-embedded specimens were retrieved, rendering 2-μm-thick pathological sections mounted on glass slides.PD-L1 IHC was performed using the commercial rabbit monoclonal antibody E1L3N . Tissue sections were deparaffinized in xylene and a subsequent ethanol series was used to rehydrate them. The sections were unmasked with antigen retrieval buffer  in an autoclave for 10 min at 120°C. To block endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide for 30 min. and subsequently washed with phosphate-buffered saline. After being washed with phosphate-buffered saline, the sections were incubated with the PD-L1 antibody at a 1:200 dilution overnight at 4°C. The sections were then washed three times with wash buffer for 5 min each time. The secondary antibody of the EliVision Plus kit detection system and the enhanced polymer 3, 3′diaminobenzidine detection kit  were used according to the manufacturer's instructions. After staining, the sections were washed in distilled water and dehydrated in graded alcohol. Finally, the sections were mounted with coverslips.All of the stained sections were scored in five randomly selected areas containing tumor cells, which showed membranous and cytoplasmic staining. The percentage of positive tumor cells was graded on a scale of 0-4: 0 (< 1%), 1 (1-10%); 2 (11-50%); 3, (51-70%); and 4 (> 70%). The intensity of staining was scored as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The H-score, ranging from 0 to 12, was calculated by multiplying the percentage of positive tumor cells by the intensity of staining on the tissue sections. The H-scores were categorized as follows: 0: negative (-), 1-4: weak positive (+), 5-8: moderately positive (+ +), 9-12: strong positive (+ + +).p value of < 0.05 was considered statistically significant. Several variables were unknown; thus, their implications were not assessed. All statistical analyses were performed using SPSS 20 software .Each clinicopathologic characteristic was evaluated using Pearson's chi-squared test or Fisher's exact test . OS was measured from the date of the initial operation until the date of death or last follow-up. The Kaplan-Meier method and log-rank test were applied to assess OS. Univariate Cox regression proportional hazards analysis was used to assess clinicopathologic characteristics significantly related to OS with HRs and 95% CIs. Multivariate Cox proportional hazards analysis was performed to determine whether PD-L1 expression is an independent prognostic factor. A two-sided