The ionic liquid-based electrolyte containing ring-like Pyr1,2O1TFSI and chain-like DMS exhibits good overall performance in lithium-ion batteries. N-methoxyethyl-N-methylpyrrolidinium bis(trifluoromethanesulfonyl)-imide  and lithium difluoroborate (LiODFB) lithium salt, with ethylene sulphite (ES) or dimethyl sulphite (DMS) as a cosolvent. The mixed electrolyte system exhibits good ion transport properties (a conductivity of 8.163 mS cm–1), a wide electrochemical window (5.2 V), non-flammability, the ability to form films to protect the anode and a large operating temperature range (–40 °C to 60 °C). We compare the performance and function of the new mixed electrolyte system with a variety of ionic liquid/cosolvent electrolyte systems developed in previous studies. The ring-chain synergy takes advantage of the availability of both high permittivities based on the ring-like components and low viscosities based on the chain-like components in the mixed electrolyte system and causes the electrolyte to exhibit a good overall performance in safety, ion transport and compatibility with electrodes.Lithium-ion batteries have been attracting much attention which enables the revolution of wireless global communication. Ionic liquids are regarded as promising candidates for lithium-ion battery electrolytes because they can overcome the limitations of high operating temperatures and flammability concerns of traditional electrolytes. However, at low temperatures they suffer from low ionic conductivity and phase transition. In this paper mixed electrolyte systems are described based on  With increasing global concerns about climate change and the environment and a growing scarcity of traditional fossil fuels, substantial efforts have been focused on the development of new and renewable energy sources.–1 at 40 °C.2/Li cells reaches up to 102 mA h g–1 at 10 °C with negligible damage to the safety characteristics.23In lithium-ion batteries, a variety of liquid solvents can be used as electrolytes to produce batteries with excellent ionic conductivity, and the use of organic carbonates as solvents for electrochemical applications is well established.N-methoxyethyl-N-methylpyrrolidinium bis(trifluoromethanesulfonyl)-imide . The ether-functionalised cation  and chain-like dimethyl sulphite (DMS) in electrochemical and physicochemical performance for the electrolyte based on Pyr+ in the electrolyte depends upon the interaction of Li+ and the solvent molecules.1,2O1TFSI  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O become weaker. Consequently, the bond energy of O–S–O becomes stronger and the peak is red shifted to a lower frequency. The O–S–O stretching bands varying in ES (910 cm–1 and 654 cm–1) and DMS (953 cm–1 and 692 cm–1) indicates that the enhancement of Li+ solvation is more obvious in DMS.Solvation of LiI Fig. S1 are show1,2O1TFSI electrolyte with and without sulphites over a temperature range of –40 °C to 80 °C are given in –1) is not yet comparable to that of traditional carbonate electrolytes (5–10 mS cm–1).1,2O1TFSI is mixed with sulphites, a significant increase (3–4 times) in the ionic conductivity is observed. LiODFB–Pyr1,2O1TFSI/DMS possesses an ionic conductivity at room temperature of 8.163 mS cm–1 with a relative standard departure of 4.14% among permitted error bars after testing 5 times .1,2O1TFSI exhibits a large exothermal crystallisation peak (at –43 °C) due to incomplete crystallization of the sample prior to the DSC heating scan. It becomes lower (–51 °C) when DMS is introduced, which suggests that the addition of DMS makes the electrolyte useful at low temperatures.The ionic conductivity and the Arrhenius plots of the ionic conductivity of the LiODFB–Pyrs Fig. S2. The conA is assumed to be proportional to the number of carrier ions and T0 is the temperature at which the configurational entropy becomes zero. The VTF parameters for this system are presented in B relating to the apparent activation energy shows the energy to overcome association among the ions of the ionic liquid to become free conductive ions. The modest ion association in the ionic liquid owing to the resultant effect of electrostatic forces, hydrogen bonds and van der Waals force brings about ion pairs and ion networks causing high viscosity and low conductivity. The large van der Waals force due to the long side-chain in Pyr1,2O1+ and strong hydrogen bond resulting from TFSI– with a big size leads to the high apparent activation energy. The addition of sulphites with good dielectric properties and a low viscosity contributes to the dissociation between the positive and negative ions in the ionic liquid, resulting in B with relatively low value. The T0 of LiODFB–Pyr1,2O1TFSI/DMS is –125 °C, which is lower than that of ionic liquid electrolytes with carbonates (–103 °C),The relationship between temperature and ionic conductivity is described by the Vogel–Tamman–Fulcher (VTF) equation,1,2O1TFSI becomes significant, suggesting oxidative decomposition at a potential of 5.2 V (as compared to Li/Li+). The addition of sulphites only has a small influence on the oxidation potentials of the electrolytes; for all cases the oxidation potential exceeded 5 V (vs. Li/Li+). On the basis of molecular orbital theory, the difference between the calculated energies of the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) determine the vertical electron affinity (VEA) and the vertical ionisation potential (VIP).To clarify the influence of the sulphites on electrochemical stability, the cyclic voltammograms (CVs) determining the oxidation/reduction potentials of the electrolytes are shown in The flammability of the electrolytes based on either ionic liquids or carbonates was tested, and the results are shown in 1,2O1TFSI do not feature a stable plateau, and the CV  and (c)A mesocarbon microbead (MCMB) is chosen as the electrode to evaluate the electrochemical properties of cells with the electrolytes because it is abundant, inexpensive and has a high specific energy. In V Fig. S5 without 1,2O1TFSI exhibit a limited reversible charge capacity below 100 mA h g–1. The initial discharge of cells with LiODFB–Pyr1,2O1TFSI/ES are about 313.9 mA h g–1. Compared with ES, the cells upon addition of DMS acquire a higher charge capacity of 376.2 mA h g–1 in the first cycle and retain a value of 345.0 mA h g–1 after 50 cycles. The coulombic efficiency is 99.6%, which is higher than that of cells containing electrolytes of disiloxane-functionalised phosphonium,1,2O1TFSI/DMS because DMS is beneficial for SEI forming on the surface of the MCMB.The cycling performances of the Li/MCMB cells containing the electrolytes are demonstrated in Rb), the resistance to Li+ migration through the SEI layer (Rsei), and the charge transfer resistance (Rct).Rb and Rct values are much smaller for electrolytes with sulphites than for those of LiODFB–Pyr1,2O1TFSI. The Rct value of cells containing LiODFB–Pyr1,2O1TFSI/ES is higher than that of cells containing LiODFB–Pyr1,2O1TFSI/DMS, which might be attributed to the excessively thick SEI layer formed in ES and the consequent difficulty for Li+ to transfer through the electrode–electrolyte interface.The Nyquist plots of AC impedance spectra of cells containing different electrolytes are shown in Fig. S6.2B[ox]– anions may contribute to the formation of the inorganic inner layer of the SEI:45A combination of density functional theory (DFT) modelling, electrochemical testing and scanning electron microscopy (SEM) analysis reveals the film-forming mechanisms of the mixed electrolytes. LiODFB can facilitate the formation of a SEI by electrochemical redox reactions.2 gas which is noxious and causes the swelling of cells is negligible because the reactions are endergonic to produce the SO2 gas.The [ox]B˙ radical can react with sulphite molecules, and the reactions with the energy change obtained by DFT calculations are shown in 1,2O1TFSI. Moreover, the film grown in the electrolyte containing ES is thicker and has a lower S content than in DMS  and schematic illustration of the synthesis route to the SEI layer with prime reactions in the electrolytes are shown in  Table S1, which i4 has been developed with a low toxicity, low cost and high safety4 containing the traditional electrolyte, cannot operate at low temperatures below –20 °C according to observations reported by Wang Y.464 cells in LiODFB–Pyr1,2O1TFSI/ES, LiODFB–Pyr1,2O1TFSI/DMS, the traditional electrolyte (1 M LiPF6–EC/DEC (1 : 1 by volume)) and the 1 M LiODFB–EC/DEC (1 : 1 by volume) at temperatures ranging from –40 °C to 60 °C. The cells with the traditional electrolyte exhibit poor low temperature performance. LiODFB provides some improvement in the electrochemical performance of the lithium-ion cells at low temperatures,4 cells containing LiODFB–Pyr1,2O1TFSI/DMS are 354.2 mA h g–1 and 130.3 mA h g–1 at –10 °C with average voltages of 0.306 V and 3.283 V, respectively. Additionally, these cells are able to operate with acceptable electrochemical performance at temperatures as low as –40 °C.The olivine LiFePO2 has been widely used in lithium-ion batteries as a cathode material. To ensure the effect of the developed electrolyte on the electrochemical performance of lithium-ion cells, the rate capability of the LiODFB–Pyr1,2O1TFSI/DMS electrolyte in Li/LiFePO4, Li/LiCoO2 and Li/MCMB cells are shown in 4 and Li/LiCoO2 cells containing LiODFB–Pyr1,2O1TFSI/DMS at 1C (150 mA g–1) are around 129 and 106.7 mA h g–1, respectively, and the charge capacity for the Li/MCMB cells at 0.5C (150 mA g–1) is about 206.5 mA h g–1. A good rate performance depends on both satisfactory ion transport and acceptable interfacial reaction resistance in the cells.LiCoO1,2O1TFSI offer some support for this relationship.To clarify if the ring-chain synergy in the carbonate electrolytes used in lithium-ion batteries is also applicable for the ionic liquid electrolytes, a summary of representative electrolyte systems of ionic liquids with organic cosolvents for lithium-ion batteries and the corresponding characteristics and properties are given in Generally, a safe electrolyte should satisfy several requirements such as non-flammability, good ion transport and compatibility with both the cathode and anode in lithium-ion batteries. The electrolytes based on imidazolium are electrochemically unstable at high potential.Safety is related to the flammability of the electrolyte and can be improved using combined ionic liquid/cosolvent electrolyte systems. For the electrolyte systems investigated in the present work, the non-flammability is retained when the amount of cosolvent added is kept below approximately 40 wt%. The electrolytes containing chain-like components require higher concentrations of ionic liquid, as the chain-like cosolvent is more thermally unstable.80+ conductivity greater than 10–1 mS cm–1.–1 at room temperature. To reach this target, the amount of ring-like and chain-like cosolvents introduced into the ring-like ionic liquid pyrrolidinium or piperidinium should be approximately 25–35 wt% or 15–25 wt%, respectively. The most conventional tetra-alkylammonium cations are seldom selected to form electrolytes for lithium batteries, mainly because of their high melting temperatures.–1 is lower than that in ring-like pyrrolidinium or piperidinium.In this study, the ionic conductivity of the electrolyte with DMS is higher than that with ES because the chain-like DMS possesses a lower viscosity, which is beneficial for ionic mobility. A satisfactory electrolyte for a battery cell should have a LiThe electrochemical window is an important index related to the compatibility of the electrolyte with the electrodes in a battery cell. A high-voltage cathode material requires an electrolyte with a high oxidation potential, which restricts the composition of the electrolyte systems. As shown in + to transfer through the electrode–electrolyte interface. Moreover, the calculated [ox]B˙ radical releases more energy when reacting with DMS than with ES, so DMS is more prone to form an SEI. Meanwhile, a larger mass of product is introduced by the reduction of ES, making the SEI thicker. These results confirm that in electrolytes containing a ring-like ionic liquid, the chain-like DMS is more beneficial as the cosolvent than the ring-like ES. An optimal proportion of cosolvents should be chosen such that they form a SEI layer that is neither too thick (leading to high resistance in the battery) nor too thin (providing no protection for the electrodes).Carbon is widely used as an anode which requires cosolvents with a suitable reduction potential to form the SEI layer. In this study, both higher capacities and better cycle stability are obtained in Li/MCMB cells with an electrolyte containing DMS rather than ES. This could be due to one of two reasons: (1) the reduction peak corresponding to the formation of an SEI with the electrolyte containing DMS is weaker, indicating a thinner SEI layer; (2) the electrolyte with DMS has a low viscosity and high separator wettability. An excessively thick SEI layer would result in a high resistance, making it difficult for Li–1. The amount of cosolvents added depends on the nature of the specific components. For systems exhibited in the present and previous studies, it should be more than 25–35 wt% (purple zone).4, LiCoO2, LiNi1/3Mn1/3Co1/3O2, and Li1.2Ni0.2Mn0.6O2. Besides, the contents of the cosolvents should be chosen to form a suitable SEI layer to protect the carbonaceous anodes, which is about 20 wt% (blue zone) in accordance with the previous studies. The performances of the electrolyte systems containing “ring-like ionic liquid and chain-like cosolvent”, “chain-like ionic liquid and ring-like cosolvent” and “chain-like ionic liquid and chain-like cosolvent” are analogously summed up to search for the optimal proportion of the electrolyte components on the basis of this work and the reported studies.General performances of the electrolytes based on ionic liquids with cosolvents are divided into four factors: safety, conductivity, oxidation potential and SEI forming ability. They depend on the nature of the ionic liquids and the cosolvents. We tried to suggest how the electrochemical performances of the electrolyte systems varied with the contents of the cosolvents through analyzing research data from this work and previous studies and the results are shown in 1,2O1TFSI with ES and DMS reveals that the sulphites improve the electrolyte conductivity, compatibility with MCMB electrodes and cell performance at low temperatures. DMS with a lower viscosity and melting point can better enhance the cell performance than ES in the electrolyte with the main component of Pyr1,2O1TFSI. The conductivity of the LiODFB–Pyr1,2O1TFSI/DMS electrolyte was 8.163 × 10–3 S cm–1. The Li/MCMB and Li/LiFePO4 cells containing this electrolyte had good cycle performance and a normal charge curve in the temperature range of –40 °C to 60 °C.Study of the electrolytes based on the ring-like PyrFurthermore, this research identifies the relationship between ionic liquid/cosolvent electrolyte performances and the component structures. According to this work and previous studies, ionic liquid electrolyte systems consisting of both a ring-like component and a chain-like component in a specific ratio can offer good overall battery performance – measured in terms of safety, ionic conductivity and compatibility with cathode and anode materials. The ring-chain synergy that takes advantage of the availability of the two structures might offer a useful starting point to design mixed electrolytes in lithium-ion batteries. In addition, electrolytes with both ring-like components and chain-like components might be developed for applications under particular conditions due to their outstanding electrochemical window and ionic conductivity, respectively. The insight into the ring-chain synergy will allow further anticipation of the ionic liquid/cosolvent electrolytes. It is noteworthy that only the chemical structures of the cosolvent and the cations of ionic liquid were taken into consideration here. In order to gain a better understanding of a broader range of systems and to achieve an even better performance, further study should explore functional modifications and the effect of anions, which might further improve the properties of the electrolyte systems.Supplementary informationClick here for additional data file.


The method of TBLI gives the distribution of periodic dissociation energies for oxygen functional groups, which are capable to reduce the graphene oxide to hierarchical graphene nanostructures, which holds great promise for gaseous molecular adsorption. The fabricated reduced graphene oxide(RGO) sensor enhanced sensing response in NO2 and accelerated response/recovery rates. It is seen that, for 20 ppm NO2, the response (Ra/Rg) of the sensor based on RGO hierarchical nanostructures is 1.27, which is higher than that of GO (1.06) and thermal reduced RGO (1.04). The response time and recovery time of the sensor based on laser reduced RGO are 10 s and 7 s, which are much shorter than those of GO (34 s and 45 s), indicating that the sensing performances for NO2 sensor at room temperature have been enhanced by introduction of nanostructures. This mask-free and large-area approach to the production of hierarchical graphene micro-nanostructures, could lead to the implementation of future graphene-based sensors.We report on the fabrication of a NO Therefore, the development of high performance gas sensor to detect the gas of NO2 in an economic way is crucially important, for not only health protection but also environmental applications. Since the ability to detect individual gas molecules by graphene was demonstrated1, considerable numbers of research activities have been ignited for graphene-based gas sensors7, including flexible gas sensors for wearable sensing applications9, and NO2 detecting sensors13, because graphene and its derivatives exhibit great electronic conductivity, flexibility, low noise and good thermal stability, it has been generally considered as a promising gas-sensing material for various highly sensitive detections. But interaction between intrinsic graphene and gas molecules is weak, in contrast, the surface of graphene oxide contains a large number of chemically active defects, showing remarkably improvement in adsorption capacity and large-scale preparation18. However, the surface area is reduced due to the sheet stacking, which seriously restricts the development of graphene-based gas sensors. In order to make the interior sheet fully contact to the test gas, it is necessary to prepared micro-nano hierarchical structure on the film to expand the specific surface area of material, and then, to improve the performance of the device. Researchers have developed nanostructured materials such as a 3-dimensional (3D) graphene foam network and a graphene nanomesh to overcome low-level sensitivity and slow response of graphene-based gas sensors23. Han et al. reported porous grapheme oxide network for chemical sensing via steam etching20. Yavari et al. using the method of CVD fabricated 3-D grapheme foam network for gas detection21. Paul et al. fabricatied graphene nanomesh using reactive-ion-etching for NO2 and NH3 sensing22. Yun et al. presented 3D nanostructured RGO scaffold using method of electrostatic self-assembly23. The method of freeze drying was also reported for the fabricating of 3D graphene/SnO2 structure24 and 3D SnO2/RGO structure25. Lupan et al. reported a low-powered sensor based on a microtube network26. These methods are limited by restricted temperature, mask, requirement of special equipment and substrate transfer or difficulty in mass production. Here we propose a strategy on the regulation of surface characteristics of graphene oxide using laser micro-nanofabrication technology, to build a high specific surface area of micro-nanostructures and fabricate graphene-based NO2 sensor. Because GO has good solution process compatibility, the device can be implemented on any substrate, this method does not require a substrate transfer process. Laser micro-nanofabrication can be carried out in the air at room temperature, and it has the advantage of rapid preparation of large areas. By Two-beam-laser Interference method, surface area of the film is increased, the sensitivity of the device towards NO2 has been enhanced, meanwhile, the response and recovery time is reduced to varying degrees.Nitrogen dioxide . The variety of response range is caused by two factors together, the increased specific surface area and the reduced oxygen containing groups. We can speculate that in the case of functional groups are invariant, the hierarchical nanostructures would improve the sensitivity of the device to a greater extent. As shown in Fig. 2, indicating the reliability of our RGO NO2 sensor. Figure 2 of the sensor based on GO and RGO hierarchical nanostructures at room temperature. The resistances of the devices were decreased upon exposure to NO2 gas, indicating NO2 doped the RGO film with holes, as RGO film exhibited the electrical behavior of a p-type semiconductor28. It is seen that the response of the sensor based on RGO hierarchical nanostructures is 1.27, which is higher than that of GO (1.06), indicating the enhanced sensing response by laser reduction of GO nanostructures. The response time and recovery time of the sensor based on nanostructured RGO are 10 s and 7 s, respectively, which are much shorter than that of GO (34 s and 45 s). Figure 2 for 15 days. It is seen that the response of the sensor floats slightly. The observed results reveal that RGO sensor exhibits good stability. The selectivity of the RGO sensor towards NO2 is also examined, as shown in Fig. 2 is 1.2, which is much larger than those of the sensor to 4 ppm other gases, such as CO2, CH4, H2 and CO, indicating that the NO2 sensor exhibits good selectivity and can be used for selective detection of NO2. The sensing performances of the sensor were also compared with the previously reported sensors based on micro-nano structured graphene materials, as shown in Table Figure 2, the value of Ra/Rg is 1.04, lower than that of reduced RGO by laser, which confirms the importance of nanostructures in the NO2 sensor. Based on the above results, the fabricated RGO sensor shows improved sensing response in NO2 and accelerated response/recovery rates. We also tested the response of gas sensors based on GO and RGO ethanol gas. When expose thermal reduced RGO to 20 ppm NOasFigure . Differe2 molecule and graphene is 78 meV, which is smaller than the interaction of NO2 and hydroxyl groups of graphene oxide. For epoxy group, a negative binding energy of −144 meV indicating a coulomb repulsion between epoxy and NO2 will push the molecules away from the GO sheet. Overall, according to other studies29, the oxygen functional groups on GO would hold significantly larger ability to absorb NO2 molecules than pure graphene. In this work, the RGO film shows a stronger response than GO film under the influence of the formation of nanostructure and the reduction of oxygen-containing functional groups. However, as shown in Figure To get further insight into the different mechanisms of the gas sensors, first principle study was carried out to give an essential explanation Figure  and S4. 2 sensor has been successfully fabricated by TBLI reduction and nanostructuring of GO film. The presence of nanostructure increases the surface area and thus significantly improves the sensitivity of the device towards NO2, meanwhile, the response and recovery time is reduced to varying degrees at room temperature, compared to GO and thermal reduced RGO, this method was proved to be a novel approach to simultaneous reduction and nanostructuring of GO in a surfactant-free, mask-free and large-area manner. Our present study shows great potential for fabrication of high-performance room temperature graphene gas sensors.A graphene-based NOGO was produced via the Hummers method from natural graphite . The GO films were prepared by spin-coating GO solution on ceramic substrates with interdigital electrodes, at 1000 rpm for 30 s, dried at room temperature. The contacts are interdigital Ag-Pd electrodes as shown in the Figure a/Rg) of the sensor resistance in air (Ra) to that in the NO2 (or ethanol) gas (Rg). The time taken by the sensor to achieve 90% of the total resistance change was defined as the response time in the case of adsorption and recovery time in the case of desorption. X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB 250 spectrometer. Scanning electron microscope (SEM) experiments were performed on a Hitachi S-4800 electron microscope. Optical microscope images were obtained from a Motic BE400 microscope. Raman spectra were measured with a Renishaw Raman microscope using 514 nm wavelength laser.The dilute tested gas was air. Gas sensing properties were measured using a static test system. Saturated target vapor was injected into a test chamber (about 1 L in volume) by a microinjector through a rubber plug. After fully mixed with air (relative humidity was about 25%), the sensor was put into the test chamber. When the response reached a constant value, the sensor was taken out to recover in air. The electrical properties of the sensor were measured by CGS-8 intelligent test meter  <250 mA. The response of a sensor was defined as the ratio  decorated with oxygen group is used to simulate the graphene oxide. In order to avoid the interaction with neighboring images, a vaccum layer of 20 Å is used. Cutoff energy with 500 eV for plane wave expansion and 2 × 2 × 1 Monkhorst-Pack mesh grid for Brillouin zone sampling are carried out for the calculations.Supporting Information


We studied the spin-transfer torque (STT) in graphene based normal-metal/topological-insulator/ferromagnet heterostructures (N/TI/F), which is induced by the helical spin-polarized current in the quantum spin Hall insulator. We found that the STT is comparable in magnitude to the STT in ferromagnetic-normal- ferromagnetic graphene junction, while not requiring additional ferromagnetic layer with fixed magnetization, which makes it advantageous for the manipulation of magnetic devices in spintronics. More interestingly, the STT is very robust in our proposed nanostructure, as it is immune to changes in the geometry due to an asymmetrically notch or the presence of random nanopores in the quantum spin Hall insulator. Our theoretical prediction suggests that graphene based quantum spin Hall insulator could be used for very efficient magnetization manipulation for magnetic materials. The two-dimensional topological insulator, known as the quantum spin Hall insulator, was first predicted by Kane and Mele12 in graphene. The electrons can be transported in the gapless edge states while the bulk is insulating, and spin-polarized dissipationless currents are transported in the transport channels confined at the two edges without backscattering. However, the quantum spin Hall insulator can not be realized in graphene owing to the exceedingly weak spin-orbit interaction16. Recently, theoretical and experimental work suggested that spin-orbit interaction can be enhanced by appropriate substrates or adatom deposition25 which leads to the transition into the topological non-trivial phase. It is therefore useful to investigate how the topological non-trivial phase can be used in spintronic applications.The topological insulator is a new quantum state of matter characterized by an insulating bulk gap and gapless edge states topologically protected, which has been intensively studied owing to the new physical properties and potential technological applications26. When spin current flows into a ferromagnetic layer, the magnetization of the ferromagnetic layer exerts a torque on the electron spins and the spin current exerts an equal but opposite torque on the ferromagnetic layer, so the orientation of magnetization is modified27. This spintronic effect was predicted independently by Slonczewski28 and Berger29, and it has been confirmed in many experiments36. In spintronics, it is of great importance to reorient the magnetizations in magnetic memory and logic devices, and the STT effect offers us an efficient method to control magnetizations through current. The STT effect can be used to manipulate the magnetic device elements without external magnetic fields.Spin-transfer torque (STT) is an important spintronic phenomenon, in which a spin current injected into a ferromagnetic layer exerts a torque on the magnetic layer and may change its magnetization orientation39 and the nonlocal electrical spin transport at room temperature was first demonstrated in graphene40. Some work has been done to study the STT generation in graphene based F/N/F structure. Ding et al.41 investigated theoretically the current-induced STT in ferromagnetic-normal-ferromagnetic graphene nanoribbon junction, and Yokoyama et al.42 also considered the STT generation in ferromagnetic-normal-ferromagnetic bulk graphene junctions.Graphene has been proven to be a promising candidate for future practical applications in spintronics owing to the long spin relaxation time and length30, we theoretically study the generation of the STT in graphene nanoribbon through a quantum spin Hall insulator. It is found that reliable STT can be generated by a graphene based N/TI/F heterostructures, and the STT can not be affected by asymmetrically notched the ribbon or nanopores.Motivated by the successful idea of realizing quantum spin Hall insulator in graphene and the recent measurements of the STT induced by a topological insulatorHN, HTI and HF are Hamiltonians for the normal graphene, graphene based topological insulator and ferromagnetic graphene respectively. In the tight-binding approximation, the Hamiltonians for the three different regions can be written asε is the onsite energy, λSO is the spin-orbit interaction, M is the magnetization of the ferromagnetic electrode, θ and φ describes the direction of the magnetization, vij = +1(−1) if the electron makes a counterclockwise (clockwise) turn, with respect to the positive z axis, in going from site j to site i. The coupling between the leads and the TI region is represented by the operatorThe schematic view of the device under consideration is presented in Fig. 43,For a small bias voltage V and zero temperature, the STT per unit of the bias voltage can be obtained using the nonequilibrium Green’s function method asThere is an out-of-plane torque which is perpendicular to the plane defined by the z-axis and the magnetization, which is given by the expression4445. Kwant is a Python package which can be used to calculate quantities, such as the Green’s function and the scattering matrix, and determine the transport properties of tight-binding models. The technical details about how to use Kwant can be found in ref.45. Here we give a brief description of the steps we used to find the Green’s function and the self-energies using Kwant. First of all, we constructed the tight-binding model of the scattering region using the Kwant package by specifying the atomic sites of the scattering region, the site energies of the sites and the hopping strengths between sties. This is carried out in the form of a small Python program. The spin degree of freedom of the atomic sites is represented by 2 × 2 matrices. For example, the spin-independent site energy and neighboring sites hopping are represented by the two 2 × 2 matrices These expressions describe the STT exerted on the magnetization of the ferromagnetic electrode by the electrons injected from N region and spin polarized when travelling through the topological insulator region. Using the same noequilibrium Green’s function (NEGF) formalism, the conductance can be obtained within the Landauer-Buttiker framework, and expressed ase as Eq. . All num12 that graphene exhibits a quantum spin Hall effect in the presence of intrinsic spin-orbit interaction. The spin-orbit interaction can be described by a second neighbor tight-binding model . When the Fermi energy moves away from 0, the STT oscillates with the spin conductances. Zero STT is obtained when the Fermi energy coincides with a subband edge in the FM lead. The oscillation amplitude of the STT decreases with the Fermi energy, because the spin conductances decrease with increasing Fermi energy when there are more subbands in the FM lead into which the electrons can be scattered.Figure EF > 0.25t or EF > −0.25t), it can be noted in Fig. When the Fermi energy is outside the gap of TI  which is within this range. We have also considered other two values of strength within the range (280 meV and 56 meV). The results are similar to the present at most of the energies.According to ref.In Fig. Ly) of the square notch for the graphene based topological insulator, and the inset is a schematic illustration of the devices in our numerical calculation. Ly is the width of the square notch, and we have an ideal ribbon when Ly = 0. In our calculation, the width of the ribbon is chosen to be Ny = 200 which is around 21.2 nm. Here, the width of the square notch changes from 0 to 20.8 nm. As shown in the figure, the STT does not change much with the width of the notch until it is very close to the ribbon width. To be more specific, we calculate the STT for a fixed notched square Ly = 10.2 nm, and it is plotted as a function of the Fermi energy .We have also considered structures with armchair edges and random edges, which consist of random combination of armchair and zigzag edges with random lengths. The random edges are created by cutting triangles of random sizes from a zigzag edged structure. The STT obtained in these structures are close to the zigzag edged structure for low Eθ) angular dependence and the out-of-plane STT is very small in Fig. t, the in-plane STT does not reach its peak at θ = π/4 anymore, and there is a small deviation. We found that the amplitude of the in-plane STT increases with an increase in the magnetization intensity. For example, the amplitude for M = 0.005t is around 0.7, but is increased to be around 0.9 for the amplitude M = 0.003t. Figure φ. It is clear that the out-of-plane STT is small for these values of φ and the magnitude of the in-plane STT decrease with the increase in φ and then increase again but with a reverse in direction. So the in-plane STT has oppositie directions for φ = 0 and φ = π/2, while the magnitude are very similar.The angular dependence of the STT is shown in Fig. λSO. In Fig. λSO < 0.05t, because with this SOI strength the Fermi energy is not in the bandgap. When electrons move from the left lead to the right lead, there are electron scatterings, which cause the oscillations in conductance and STT. It is noted that both the STT and the conductance have very negligible changes when the Fermi energy is in the bandgap. The electrons can only move at the edges of the ribbon, and the spin-polarized dispationless currents induce a stable STT.It is known that the spin-orbit interaction can generate an energy gap in graphene, changing it into a quantum spin Hall insulator. The bulk bandgap is dependent on the value of the spin-orbit interaction, which is described by the formula Δ = 6√3e/4π which is comparable in magnitude to the conventional F/N/F junctions. The features of the STT can be explained through the analysis of the band structure of the right ferromagnetic graphene electrode. It is found that the STT obtained in our proposed nanostructure is immune to the changes in the geometry of the quantum spin Hall insulator, and it can not be changed by considering an asymmetric square notch or random nanopores in the quantum spin hall insulator. This offers us an efficient method to change the magnetization direction of magnetic materials without the help of a ferromagnetic layer.In summary, we have investigated the current induced STT in graphene based N/TI/F heterostructures using the nonequilibrium Green’s function method. It is found that the charge current becomes spin polarized, when it flows from the left lead (N) across the quantum spin Hall insulator layer (TI), and induced STT on the magnetization of the ferromagnetic layer (F). The STT per unit of the bias voltage can reach 0.8


A major challenge of agriculture is to increase food production to meet the needs of the growing world population, without damaging the environment. In current agricultural practices, the control of pests is often accomplished by means of the excessive use of agrochemicals, which can result in environmental pollution and the development of resistant pests. In this context, biopesticides can offer a better alternative to synthetic pesticides, enabling safer control of pest populations. However, limitations of biopesticides, including short shelf life, photosensitivity, and volatilization, make it difficult to use them on a large scale. Here, we review the potential use of neem oil in crop protection, considering the gaps and obstacles associated with the development of sustainable agriculture in the not too distant future. Attention is increasingly being paid to the use of natural compounds  as a promising option to replace agrochemicals in agricultural pest control. These odoriferous substances are extracted from various aromatic plants, which are rich sources of biologically active secondary metabolites such as alkaloids, phenolics, and terpenoids , using eAzadirachta indica Juss., a member of the Meliaceae family that originates from the Indian subcontinent and is now valued worldwide as an important source of phytochemicals for use in human health and pest control. Azadirachta is a fast-growing small-to-medium sized evergreen tree, with wide and spreading branches. It can tolerate high temperatures as well as poor or degraded soil. The young leaves are reddish to purple, while the mature leaves are bright green, consisting of petiole, lamina, and the base that attaches the leaf to the stem and may bear two small lateral leaf-like structures known as stipules , which appears to cause 90% of the effect on most pests. The compound has a melting point of 160°C and molecular weight of 720 g/mol. Other components present include meliantriol, nimbin, nimbidin, nimbinin, nimbolides, fatty acids , and salannin. The main neem product is the oil extracted from the seeds by different techniques. The other parts of the neem tree contain less azadirachtin, but are also used for oil extraction  Lepidoptera: antifeeding effect and increased larvae mortality ; (ii) HeFor centuries, neem has been used in folk medicine for the treatment of conditions such as malaria, ulcers, cardiovascular disease, and skin problems. Despite the limited existence of clinical trials to support therapeutic claims, the use of neem has expanded over time, and it is an important component of Ayurvedic medicine . In the cosmetics and hygiene sector, neem is used in the composition of face masks, lotions, sunscreens, soaps, and toothpastes  can be used as a biofertilizer, providing the macronutrients essential for plant growth .Nitrogen is one of the main nutrients required by plants for their development, and urea is the main source of nitrogen fertilizer used worldwide to supply the nitrogen demand of crops. The control of urea hydrolysis and nitrification is one of the principal strategies employed to avoid nitrogen losses in agriculture . Neem haDue to their compositional complexity, neem-based products can act as antifeedants, growth regulators, sterilants, anti-oviposition agents, and repellents . Other fThese features of neem support its contribution to organic agricultural production systems that are more sustainable and do not generate chemical residues . This method also helps to maintain soil productivity, ensuring longer production times. Organic agriculture can be a viable alternative production method for farmers, but there are numerous challenges to be overcome. A key to success is to be open to new approaches, and in this respect neem products can effectively contribute to organic agriculture, being used as organic pesticides and as soil fertilizers. In addition, growing concerns about conventional agriculture and the demand for products that do not generate waste justify increased adoption of the use of biopesticides by farmers, which contributes to the growth of organic agriculture .Neem has acquired commercial recognition due to its various beneficial properties, which have been extensively investigated over time. Compared to conventional chemicals, which are generally persistent in the environment and highly toxic, botanical pesticides are biodegradable and leave no harmful residues. Most botanical pesticides are non-phytotoxic and are also more selective toward the target pest. In terms of commercial applications, biopesticides can provide substantial economic advantages, since the infrastructure required is inexpensive, compared to conventional pesticides .This has resulted in the publication of numerous scientific research articles and books, as well as the organization of international conferences to discuss the benefits of the plant .Table 1 lists some of the main commercial products based on neem.Several patents related to processes and products based on neem have been deposited in the United States, India, Japan, Australia, and elsewhere. Many of the products derived from neem are manufactured by crushing the seeds and other plant parts, followed by the use of solvents to extract the active ingredients possessing pesticide activity. The different methods and techniques employed to obtain neem products can result in different concentrations of the active compounds, as well as different biological effectiveness . Table 1Despite its many promising properties, there are limitations that hinder effective large-scale use of neem. These impediments must be overcome and many uncertainties clarified so that the full potential of neem can be exploited. One of the main problems facing the commercial development of neem is a lack of industrial interest, largely due to the difficulty of patenting natural products, as well as a shortage of scientific evidence to support claims regarding the benefits of these substances. As a results, the products are not widely publicized in the farming community and elsewhere .Disadvantages of neem are its low stability under field conditions, due mainly to a high rate of photodegradation, as well as a short residence time and slow killing rates, compared to conventional pesticides . GeneticNeem oil contains a group of active ingredients with different chemical characteristics. It was therefore believed that the development of insect resistance would be virtually impossible. However, as studies have progressed, it has been observed that due to the low residual power of botanical insecticides, multiple applications are required in order to control pests, which can increase selection pressure on the pest population, possibly leading to resistance .Myzus persicae, which were exposed to pure azadirachtin or to refined neem seed extract at the same concentration as azadirachtin. It was found that after forty generations, the line treated with azadirachtin had developed ninefold greater resistance to azadirachtin, compared to a control line, whereas the line treated with the extract did not show resistance.Currently, most of the botanical insecticides that are being studied and that are effective against many pests are those with feeding deterrent action, so their indiscriminate use could result in the development of resistance . Feng anBiological control is defined as the action of natural enemies on a population of pests in order to keep it at a population density that does not cause economic damage to crops . NaturalThe biological control of insects and mites in agriculture can be achieved using small wasps or flies, known as parasitoids, which parasitize eggs, small caterpillars, and even adults. It can also be performed using predators such as ladybugs, bugs, predatory mites, and spiders, as well as parasitism by entomopathogenic microorganisms including fungi, bacteria, and viruses . AlthougBeauveria bassiana, Isaria fumosoroseus, and Lecanicillium lecanii, and the results showed that these entomopathogenic fungi were compatible with most products tested. There has recently been increased interest in the application of plant-based materials , such as neem oil, in pest control. Although these products are safer for the management of pests, compared to synthetic chemicals, their effects in IPM must be evaluated. Several studies have investigated the relationships between botanical insecticides and natural enemies of agricultural pests . SahayarThe integrated use of botanical insecticides associated with biological control (synergism) in IPM is becoming increasingly widespread in the farming and research communities. The advantage of this approach is that it offers the potential to control agricultural pests, without serious impacts on the environment, non-target organisms, and animal and human health.in vitro), due to the instability of this substance under field conditions. From these studies, it is not possible to draw firm conclusions concerning the in vivo biological efficacy of the formulations, due to the effects of numerous environmental variables.Botanical insecticides must meet the same criteria as conventional insecticides. In other words, they must be selective for the target pest and provide sufficient residual activity to protect the plant during the period of vulnerability. Over the past decade, there has been a significant increase in the number of publications concerning the use of neem oil to control agricultural pests . HoweverIn order to overcome the above-mentioned limitations, nanotechnology has emerged as a novel tool to address the problems of agricultural sustainability and food security . Many stFrom the point of view of sustainable agriculture, nanotechnology can help in the development of environmentally friendly agricultural inputs, improving the safety and stability of active agents, enhancing their activity in pest control, and, consequently, increasing their acceptance by producers . The useAlthough studies have demonstrated the beneficial effects of nanoencapsulation of neem oil, some issues need to be resolved so that the synergistic effect of nanoparticles associated with this botanical insecticide can significantly contribute to the control of insect pests. These issues include the need for: (a) regulation of the use of nanomaterials in agriculture; (b) nanoformulations that are easily scalable; (c) comparative studies employing neem formulations available commercially to prove the cost/benefit of nanoformulations; (d) detailed studies of the degradation and behavior of these nanopesticides in the environment; and (e) evaluation of toxicity toward non-target organisms .Given the importance of neem oil and its worldwide use for combating numerous pests in different crops, the nanoencapsulation of this oil should enable the production of more stable formulations for the control of insects that damage crops, especially those that are essential for human consumption. In addition, the use of nanotechnology is an excellent way to combat the development of resistance in insects due to the indiscriminate use of neem oil.EC, JdO, and MP wrote the manuscript. LF and RdL contributed to the discussion and revised the manuscript. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Systematic first-principles calculations were performed to investigate the adsorption and diffusion of Li on different graphene layers with B/N-doping and/or C-vacancy, so as to understand why doping heteroatoms in graphene anode could significantly improve the performance of lithium-ion batteries. We found that the formation of single or double carbon vacancies in graphene are critical for the adsorption of Li atoms. While the N-doping facilitates the formation of vacancies, it introduces over binding issue and hinders the Li diffusion. The presence of B takes the excessive electrons from Li and N and reduces the energy barrier of Li diffusion on substrates. We perceive that these clear insights are crucial for the further development of graphene based anode materials for lithium-ion batteries. His/her work confirmed that the N-decorated graphene with single and double vacancies could greatly improve the reversible lithium capacityet al. found that the substitutional B atoms cause slight electron deficiency, which makes the adsorption of Li on the C3B monolayer easyMany approaches have been pursued to functionalize graphene as an anode material and doping graphene with nitrogen (N) and/or boron (B) atoms is one of the most effective ways. For example, Reddy C-decorated graphene geometries, as well as the Li adsorption and diffusion on them. We sorted out the roles of doping and vacancy decoration on the improvement of Li-adsorption on graphene, and identified several structures that have good adsorption energy, low diffusion barrier and easy preparation in experiments. Our theoretical results provide instructive guidelines for the further development of high performance C-based anode materials for LIBs.In the present work, we performed systematic density functional theory (DFT) calculations for the structures and energetics of different B/N/Vx N-atoms and y VC, G_NxVy (x = 0–4 and y = 0–2), corresponding to a N-concentration of ~1.4–5.6%, comparable with the experimental range for graphene samples synthesized via the thermal reaction between graphene oxide and NH3 at high temperaturey = 0, i.e., N-substituted graphene without VC, N atoms prefer to stay far away from each other to minimize the perturbation to the graphene π-electron bonds, as depicted in y = 1 and 2, i.e., N-doped graphene with VC, the N dopants prefer to connect with two C atoms and form pyridine-like structure, which introduces a strongly localized donor states near the Fermi level34We first studied the adsorption configurations and energies of one Li atom on the decorated graphene cells with Ead, are shown in Ead of Li on the hollow site (H) of a 6 × 6 graphene supercell, 0.27 eV, is lower than that on the top site (T), 0.57 eV. The distance between Li and substrate, dLG, of the H-site, 1.72 Å, is also smaller than that of the T-site, 1.92 Å. These results indicate a single Li adatom prefers the hollow site on pristine graphene but Li atoms are more likely to form Li clusters since the value of Ead is positive, in consistent with previous results241V0 and G_N2V0 systems, we found that Li prefers to stay on the H-site of graphene rather than any places near the N atoms. The Ead of Li on G_N1V0 (G_N2V0) systems, 0.39 (0.34) eV, are also positive, and the corresponding values of dLG are 1.75(1.76) Å, indicating that pure N-dopings in graphene don’t improve the lithium adsorption. Strikingly with the presence of VC on pristine graphene, the Ead decreases to negative values of −1.36 eV for single VC and −0.73 eV for double VCs, as shown in 0V1 have no obvious changes compared with perfect graphene due to the weak intraplanar relaxation0V2 with decreased C-C bond lengths near vacancies from 2.46 Å to 1.81 Å, as depicted in the insets in Ead values decrease further for Li on the N and VC-codoped graphene sheets. For example, Ead becomes −3.12 eV for Li/G_N3V1, which implies a strong lithium adsorption on this substrate, as also demonstrated in previous experimental and theoretical studies31C number doesn’t significantly change the Ead value, and the lowest Ead is −3.59 eV, which occurs in Li/G_N4V2. The detailed analyses of adsorption structures show that the vertical dLG is 1.43 Å for Li/G_N3V1, and decreases to 0.10 Å for Li/G_N4V2, implying that the Li atom is in the hole position surrounded by four pyridine N atoms and almost in the same plane with the N-doped graphene. Overall, the red curve in Ead of Li on different graphene cells suggests that the carbon vacancies play the key role in the adsorption of Li on graphene.For each system, we explored different Li adsorption sites cf.  in SM anC on graphene, Efor(VC), shown in Efor(VC) drops to 4.69 eV on N-doped graphene, and the trend of decrease continues with the further increase of N atoms, as shown in the red line in Efor(VC) of G_N3V1 has a small negative value, indicating the easy formation of the N-VC structure as a group. Similar trend appears for the formation of double-vacancies in graphene, with a very low Efor(VC) of −2.89 eV for G_N4V2. The formation energy of single N-dopant on graphene, Efor(N), is 0.91 eV, comparable with the previous DFT studies of 0.79 eVEfor(N) of single N-dopant on the G_NxV0 substrates (shown in the insets) that were used in Efor calculations of single C-vacancy, and the error bar illustrates the Efor(N) window for different doping configurations of N on G_NxV0 , changes from −5.30 eV under B-rich conditions to +8.09 eV under oxidation conditions, as shown in Efor(B) under typical experimental conditions, ranging from −0.23 eV with B4C precursor to +1.47 eV with NH3BH3 precursor, are a little higher than Efor(N), the calculated Efor(VC) of single C-vacancy on G_B3V1 is comparable with that of G_N3V1, and interestingly, its value for the B/N co-doped graphene, G_BxNx3-V1 (x = 0–3), is even lower than the corresponding value for the pure N-doped graphene by ~1.20 eV, as presented by the green stars in As we mentioned above, the graphite-like B/C/N layered materials prepared using CVD or other methods showed good performances as the anode matrix of LIBs3940Ead for Li on several adsorption sites of G_BxNx3-V1 substrates, as depicted in 1N2V1, we found that Li in the ground state is located at the vacancy site, close to the B dopant with a dLG of 1.65 Å. A similar adsorption structure was obtained for Li/G_B2N1V1, but the Li atom is much closer to the N dopant instead of B. Strikingly, the most stable adsorption site of Li on G_B3V1 substrate is on the hollow site of the hexagonal ring adjacent to the B atom. Overall, the B and N co-doped graphene substrates give negative adsorption energies, and the values of Ead of one Li atom on G_BxNx3−V1 are in the order: G_N3V1 (−3.12 eV) < G_B2N1V1 (−1.77 eV) < G_B1N2V1 (−1.20 eV) < G_B3V1 (−0.99 eV). Obviously, all of them are more active toward the adsorption of Li, compared with pristine graphene. Using G_B2N1V0 as an example, we also studied the x-dependence of Ead  − Ead(H). For each adsorption site, we fixed the lateral coordinates of Li and optimized all the other atomic positions. The energy barrier for the movement of one Li atom on G_B3V1 is only 0.41 eV, and the largest difference in dLG is about 0.27 Å. The low energy barrier and the small change in dLG indicate a relatively flat potential energy surface of G_B3V1 for the Li movement. Similarly, we studied the diffusion of Li on G_B2N1V1 and obtained ΔEad of 0.80 eV and 0.48 eV along two different pathways, and the corresponding difference in dLG of 0.83 Å and 0.56 Å, respectively . The adsorption of Li atom keeps the DOS profile almost unchanged, but the Ef moves to the conduction states due to the addition of 1e from Li. As was well known, the Ef of B(N)-doped graphene is shifted to the valance(conduction) band, as shown in the middle panels in 1(G_N1) is a hole (electron)-rich material. The adsorption of Li on G_B1 substrate compensates the hole attraction and the Dirac state of Li/G_B1 moves back to Ef, making the system stable with a negative Ead of −1.4 eV1, we see an obvious peak existing on the Ef, coming from the N-pz orbital. For these three systems, there is little hybridization between Li and substrate/dopant. For example, the B-px,y states in G_B1 locate at ~−2.9 eV below the Ef, but the Li-s and -pz states appear at ~2.1 eV above the Ef. The attractive/repulsive electrostatic interactions between Li and substrates lead to a negative/positive Ead values. Strikingly, for G_V1 substrate, as shown in the lower panel in Ef caused by the hybridization of Li-px,y and C-px,y in-plane orbitals, and the adjacent peak at −0.7 eV below the Ef is composed of the Li-s and C-px,y orbitals. Thus the interaction between Li and G_V1 substrate is very strong, leading to a low Ead value.We performed detailed analyses on the electronic properties of various graphene substrates with/without Li adsorption, so as to understand the role of doping and/or vacancy on the Li adsorption. The total and partial density of states (DOS) of pristine graphene (G), G_B3V1 substrate in 1, with peaks of B-pz orbital near the Ef. The adsorption of Li makes the B-pz states shift to lower energies, but the hybridization of Li-s,p and B-p states is still relatively weak near the Ef. Similar with G_V1, the DOS of G_N3V1 also has a big peak right at the Ef, coming from the dangling bonds at the N atom sites. The orbital hybridizations of N-p and Li-s,p orbitals can be observed around −1.8 eV and −3.5 eV, which are responsible for the lower Ead of G_N3V1 than G_B3V1. The interaction between Li and substrate can be described more clearly by the electron redistribution, rn = nLi/sub(r) − nsub(r) − nLi(r), obtained from electron densities of Li on substrates, pure substrates and Li atom. As displayed in the insets in 3V1 and G_N3V1 manifest through the influence of N extending to the second nearest neighbors of C atoms due to the symmetry breaking of the π-electron system, whereas B appears to only affect the nearest neighbor C atoms.Interestingly, we found that the DOS profile of G_BC sites (<3% in this study) rather than the dopants serve as attractive centers for Li adsorption on graphene. While the formation energy of one VC on graphene is as high as 8.01 eV, the N-doping drastically decreases this value to as low as −0.14 eV for G_N3V1. The co-doping of B keeps negative adsorption energies for Li, and significantly reduces the energy barrier of Li diffusion, e.g., 0.41 eV for Li/G_B3V1 and 0.48 eV for Li/G_B2N1V1. The electronic structure analyses show that the interaction between Li and B states is rather weak, whereas Li has a strong orbital hybridization with N-p states, causing a high diffusion barrier. Our theoretical studies provide clear insights for the understanding of the individual roles of doping and vacancy decorations for the performance of enhancement of N(B)-doped graphene as electrode materials in LIBs, and provide guidelines for the design of new battery materials.In summary, we performed systematic first-principles calculations to study the adsorption and diffusion of Li on decorated graphene with B and/or N dopants and C vacancies. Our results indicate that Vk-points sampling in the Brillouin zone (BZ). The crystal constant and positions of the ions were fully relaxed until the final force on each atom is smaller than 0.01 eV/Å. The optimized in-plane lattice constant of graphene primitive cell is 2.47 Å, in good agreement with the experimental values of 2.46 Å derived from the X-ray crystallography of AB graphite and with previous theoretical predictionsOur density functional theory calculations were performed using the Vienna Ab initio Simulation Package (VASP)4245Ead) of a Li adatom as followsTo assess the stability of Li adsorbed on graphene, we calculate the adsorption energy  of defects, including N-doping and C-vacancy, on graphene are calculated as follows:The formation energies .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Eutreptiella pomquetensis, and the spread and peculiarities of introns.Over the last few years multiple studies have been published showing a great diversity in size of chloroplast genomes (cpGenomes), and in the arrangement of gene clusters, in the Euglenales. However, while these genomes provided important insights into the evolution of cpGenomes across the Euglenales and within their genera, only two genomes were analyzed in regard to genomic variability between and within Euglenales and Eutreptiales. To better understand the dynamics of chloroplast genome evolution in early evolving Eutreptiales, this study focused on the cpGenome of Etl. pomquetensis cpGenome was sequenced, annotated and afterwards examined in structure, size, gene order and intron content. These features were compared with other euglenoid cpGenomes as well as those of prasinophyte green algae, including Pyramimonas parkeae.The Etl. pomquetensis, a basal taxon in the phototrophic euglenoids, was considerably larger than the two other Eutreptiales cpGenomes sequenced so far. Although the detected quadripartite structure resembled most green algae and plant chloroplast genomes, the gene content of the single copy regions in Etl. pomquetensis was completely different from those observed in green algae and plants. The gene composition of Etl. pomquetensis was extensively changed and turned out to be almost identical to other Eutreptiales and Euglenales, and not to P. parkeae. Furthermore, the cpGenome of Etl. pomquetensis was unexpectedly permeated by a high number of introns, which led to a substantially larger genome. The 51 identified introns of Etl. pomquetensis showed two major unique features: (i) more than half of the introns displayed a high level of pairwise identities; (ii) no group III introns could be identified in the protein coding genes. These findings support the hypothesis that group III introns are degenerated group II introns and evolved later.With about 130,561 bp the chloroplast genome of  Euglena, Monomorphina, Euglenaformis, Colacium, Strombomonas and recently Phacus  in addition to small cpGenomes with low intron numbers like M. aenigmatica  have been largely used to retrace evolutionary steps of phototrophic euglenoids. Members of the genera y Phacus  cpGenomeectively . Fittingectively . This hydetected . Howeverigmatica . AlthougEutreptiella pomquetensis  Marin & Melkonian in In the present study Eutreptiella da Cunha, with ten described species, and Eutreptia Perty, with eight known species. They are regarded as basal phototrophic euglenoids in aspects of morphology  Marin & Melkonian in Three-hundred mL of cell culture were harvested by centrifugation and submitted to cell cleaning and chloroplast isolation protocol as described in n homology search  and a Long Amp Taq DNA Polymerase .Using a BLASTy search  the fourThe final annotation of the chloroplast sequence was performed with Geneious 9 Pro ” and Emboss Sixpack Sequence translation (EMBL- EBI 2015) “Codon Table” set at “Bacterial” and added to the annotation. The introns within protein coding genes were analyzed for the presence of potential twintrons as described in abcdef (3–8 nucleotides) f′e′d′A∗c′b′a′ (four nucleotides)). Afterwards, all 51 introns were searched for the conserved 5′ insertion sequence GUGYG. RNA secondary structure for group II introns was created by RNA folding via Mfold web server using default settings  for being a previously identified chloroplast protein-coding gene were included in the annotation. ORFs were named according to the number of amino acids in the coding region. To evaluate the proportion of short repeated sequences the variable number of tandem repeats was scanned with the online version of REPuter  from the beginning of the 16S to the rpl32 gene. Primers were created manually by Primer3Plus  and C. vesiculosum  were found for each protein coding gene, except rpoA, where an alternative start codon (ATA) was accepted. Four protein coding genes were annotated with alternative start codons in Etl. gymnastica and Et. viridis (P. parkeae (Etl. gymnastica (59), where psaI was missing. Et. viridis lacked psaM, ycf12 (psb30) and ycf65 and hence counted only 57 protein coding genes. No mat5 or mat2 genes have been identified in Etl. pomquetensis, but mat1 (ycf13) was detected, as was expected from results in other Eutreptiales  to 51 introns in Etl. pomquetensis , but additionally to an increased intergenic space in Etl. pomquetensis. The intergenic space of Etl. pomquetensis comprised more than 23 kb, which was more than twice in that of Etl. gymnastica. While most of the blocks in Etl. gymnastica were quite compact with little intergenic or intron space in blocks C, E and G, all of the identified clusters showed heavily fragmented blocks in Etl. pomquetensis, except A and B . As the phototrophic euglenoids have a reduced amount of protein coding genes in contrast to the green algae, this high number of clusters was expected.Three additional Mauve analyses using O. tauri . A compaEtl. pomquetensis. A BLASTp analysis was performed against the NCBI nonredundant protein sequences (nr) database to determine whether any of the ORFs had functional similarity to previously sequenced genes (psbD gene of Etl. pomquetensis contained two ORFs (orf585 and orf532). The intron encoded orf585 of Etl. pomquetensis psbD I2 shared strong similarity with the orf583 of atpB I1 in the chloroplast genome of Pycnococcus provasolii  were found in ed genes . The psbovasolii , with anindracea . The opete 2.4.0  and a maThere is no evidence of a VNTR (variable number of tandem repeat) sequence, though this could be a result of our inability to circularize the genome.psaA contained the highest count with six introns (Et. viridis (27), nearly eight times higher than found in Etl. gymnastica (7), and consequently constitutes the highest intron number known in the Eutreptiales  with the presumed “branch-point” *A for splicing at position eight in domain VI, where the first transesterification takes place  in Etl. pomquetensis with high pairwise identity were closely related and arose from a single ancestor proliferating via retrotransposition and moved horizontally into DNA target sequences, which resembled the homing site. According to Etl. pomquetensis could be explained.We assume that all of these 28 introns of Etl. pomquetensis used host encoded proteins to promote splicing, reverse splicing and mobility, which is typical for most mitochondrial and plant chloroplast group II introns (Chlamydomonas reinhardtii even utilized nuclear-encoded maturases for splicing of the trans-spliced group II introns (Still the question remained of how these introns could be spliced without an ORF including maturase activity in domain IV. One possibility was that they rely on trans-acting RNAs or proteins with two feasible splicing mechanisms: (1) The introns of  introns . Chlamyd introns .orf1311 in Oedocladium (Chlorophyceae), with an intron encoded maturase, could function as promoter for splicing the ORF-less group II introns. G. planctonica without ORFs, which may reflect an evolutionary pressure for a smaller and more compact intron structure enabling increased efficiency of splicing and mobility, when maturase activity is provided from elsewhere. Furthermore, it might be assumed that an early event in the Etl. pomquetensis cpGenome was the deletion of an intron encoded ORF, which appeared to have occurred prior to the spreading of introns across the genome and that other group II introns with encoded IEPs or freestanding ORFs acted in trans to promote splicing and mobility of ORF-free introns . The gene psaA contained the most of these introns, and five of six introns contained high similarity.(2) All these introns could be spliced by a single IEP (Intron-Encoded Protein) that could either be free-standing or located in a functional intron. This would provide an accessible splicing apparatus and allow all but one intron to lose its own IEP . Brouard introns . To gainrocesses , thus heEtl. gymnastica, Et. viridis and P. parkeae and all other euglenoid cpGenomes did not reveal any sequential or positional homology. Insertion sites found in Etl. pomquetensis were unique to that taxon.A search for related introns in E. gracilis var. bacillaris with 56.7%, but only for three small 97 bp long group III introns . Also, outside of euglenoid chloroplast introns, very few species showed high pairwise similarity. For instance, Oedocladium carolinianum with high levels of nucleotide identities, which displayed over 80% pairwise identity. As well ycf3 and psbH in Gloeotilopsis sarcinoides were 85.6% identical. To our knowledge, Etl. pomquetensis is the first organism with more than 50 introns within protein coding genes and half of those sharing a pairwise identity of 90%.The highest pairwise identity of introns was found in Etl. pomquetensis is the absence of group III introns. Group III introns are believed to be the descendants of group II introns which only retained domains DI and DVI (abcdef (3–8)f′e′d′A∗c′b′a′ . Furthermore, the intron size was even larger than group III twintrons (group III introns within group III introns), which were found in the chloroplast genome of E. gracilis . It is clearly a group II intron/ twintron in all Eutreptiales, but a group III twintron in E. gracilis with an open reading frame  within the internal group III intron  that is at least three times larger than the group III twintron (I4) including mat1 of E. gracilis  act in trans to promote splicing and mobility of ORF-less introns.Summarizing, we presume that group II introns appeared first in an intron-less ancestral genome and gave rise to group III introns and from there on degeneration went on independently in different lineages. Further on, either the petB (I1) and that this intron/twintron may be a synapomorphy for at least the Euglenaceae. petB I1 of P. orbicularis and discussed this intron/ twintron as a putative synapomorphy for the order Euglenales. However, in the cpGenome of Etl. pomquetensis two introns were detected in petB. The first was found at the identical insertion site, but nearly two times larger than that of E. gracilis strain Z and five times larger than that of P. orbicularis. All petB I1 introns started with a typical group II 5′ -GUGYG  with only one 3′ motif and only one 5′ -GUGYG prior to the identified 3′ motif. Two of these potential group II twintrons (rpoB I1 and psbD I4) were those which share strong nucleotide identity with half of the introns detected in Etl. pomquetensis. We assume that all 28 introns (petG I1 and psaC I2) with equal intron organization  are potential twintrons with an external and internal group II intron  near the base of the stem of domain V were detectable, but the secondary structure showed a slightly altered terminal loop and no branch-point A∗ was detectable in domain VI  shared 88% pairwise identity with the other 27 potential twintrons . It is ln search for the external intron of psaC I2 (psbC I2 (containing a still unspecified maturase) of Etl. gymnastica. Secondary structure analysis of domains V and VI of psbC I2 from Etl. gymnastica, realigned by psaC I2 in Etl. pomquetensis revealed highly conserved structures of domains V , Pycnococcus provasolii (B) and Ostreococcus tauri (C). In the Mauve alignment the repeat regions of rRNA were not included, because Mauve will not align repeat regions, which have multiple matches on both genomes.Each box represents a cluster of homologous genes between Click here for additional data file.10.7717/peerj.3725/supp-3Figure S3Etl. pomquetensis based on the model proposed by Michel, Umesono & Ozeki, (1989) and on comparative analysis of other euglenoid group II introns  with branch-point A* at position 8 of domain VI and conserved three base pairs (5’- …AGC …-3’) near the base of the stem of domain V (red box). Domain V and VI of r po B I1 of Etl. gymnastica (B) with slightly altered base pairs (5’- …AGUC …-3’) of domain V (red box) but without branch-point A* at position 8 of domain VI.Domain V and VI of Click here for additional data file.10.7717/peerj.3725/supp-5Figure S5rpo B I1 of Etl. pomquetensis (A) with conserved three base pairs (5’- …AGC …-3’) near the base of the stem of domain V (red box). Internal intron of psb D I4 of Etl. pomquetensis (B) with conserved three base pairs (5’- …AGC …-3’) near the base of the stem of domain V (red box).Internal introns of Click here for additional data file.10.7717/peerj.3725/supp-6Figure S6psa C I2 external intron of Etl. pomquetensis (A) with branch-point A* at position 7 of domain VI and conserved three base pairs (5’- …AGC …-3’) near the base of the stem of domain V (red box). Secondary structure model of putative domain V and VI of psb C I2 of Etl. gymnastica (B) with slightly altered branch-point AU* at positions 7 and 8 of domain VI and conserved three base pairs (5’- …AGC …-3’) near the base of the stem of domain V (red box).Secondary structure model of putative domain V and VI of Click here for additional data file.10.7717/peerj.3725/supp-7Table S1* Chloroplast circle not closed. a Twintrons were counted as single insertion sites. b Including rRNA repeats and intermediate tRNAs. c Includes the identified 5S, for S. acuminata the two identified 5S. d Includes the two introns in rps18. e Includes the one intercistronic intron rps4-rps11. f First exon could not be identified, so gene length is a minimum. g Realigned, but with alternative start codon. h Start codon not determined, due to undetermined exon1 i: New intron start after re-analysesClick here for additional data file.10.7717/peerj.3725/supp-8Table S2Click here for additional data file.10.7717/peerj.3725/supp-9Supplemental Information 1Click here for additional data file.10.7717/peerj.3725/supp-10Supplemental Information 2Click here for additional data file.


Our results suggest pT3N0M0 ESCC clinical outcomes correlate with CD8+ and FOXP3+ TIL densities and PD-L1 levels. Moreover, an intrinsic mechanism for induction of PD-L1 overexpression may be occurring during early tumor oncogenesis. This information may be useful for stratifying patients and guide the application of checkpoint blockade therapy in ESCC.Data describing relationships between the tumor immune microenvironment and patient outcome are limited for esophageal squamous cell cancer (ESCC). The present study investigated the prognostic values of programmed death-ligand 1 (PD-L1) expression and CD8+ or forkhead box protein 3+ (FOXP3+) tumor-infiltrating lymphocytes (TILs) in 133 pathological T3N0M0 stage ESCC patients who underwent radical resection without neoadjuvant or adjuvant therapy. CD8+ and FOXP3+ TIL densities as well as PD-L1 levels in tumor cells and lymphocytes, were assessed through immunohistochemical staining. Patient survival was not associated with CD8+ or FOXP3+ TILs alone, but PD-L1 expression and the CD8+/FOXP3+ ratio were independent predictors of both disease-free and overall survival. PD-L1 expression correlated with age ( Esophageal squamous cell carcinoma (ESCC) remains the predominant type of esophageal cancer (EC) in the “EC Belt”, which extends from China via central Asia to northern Iran . DespitePatients with pathological T3N0M0 stage (pT3N0M0) disease represent a considerable fraction of middle-stage patients. Although they have the same TNM stage, they are divided into stages IB, IIA, and IIB based on tumor location and histologic grade in the 7th edition of the AJCC manual . Howeveret al. proposed stratifying tumors according to PD-L1 status, and TIL presence or absence in the microenvironment might predict patient response to anti-PD-1 therapy [As an immune checkpoint, the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis facilitates T-cell exhaustion and enables tumors to avoid immunosurveillance . PD-L1-P therapy . However133 patients with pathological T3N0M0 stage ESCC who underwent radical Ivor-Lewis esophagetomies were included in this study  were negatively correlated (PD-L1 showed a predominantly membranous or cytoplasmic (or both) focal or scattered staining pattern , longer tumor length , poorer histological differentiation (p = 0.002) and tumor recurrence (p = 0.037)  Table . There w7) Table .p = 0.013, 0.001 respectively; Figure p = 0.002, 0.001 respectively; Figure p = 0.043 and 0.022, respectively) and OS  compared to patients with negative PD-L1 expression  and OS   but was not an independent prognostic factor in multivariate analyses  for PD-L1 IHC detection (SP142) and validated our results via qRT-PCR in 10 tissue samples . We asseMolecular mechanisms underlying immune avoidance appear to be distinct among tumors. To date, two phenotypes have been identified: T cell-inflamed  and non-T cell-inflamed  . The twoOur study had several limitations. First, while pT3N0M0 stage ESCC patient optimal management strategies are still controversial, surgery alone is not standard treatment for such patients. We analyzed tumor original immune states, which are not affected by chemotherapy and radiotherapy. However, preoperative chemoradiotherapy may alter the tumor immune microenvironment, inducing T cell influx and PD-L1 upregulation, and this requires further study , 36, 37.Within the context of these limitations, our study identified an inverse relationship between CD8+ TILs and PD-L1 expression, suggesting that an intrinsic induction-type mechanism is active in ESCC. Our findings also suggest that differences in TIL density and PD-L1 expression may partly explain the diversity of clinical outcomes observed in patients with similar ESCC stages. These findings may be suggestive of the optimal treatment choice and checkpoint blockade therapy in patients with pT3N0M0 ESCC.The study cohort included 133 patients with pathological T3N0M0 stage thoracic ESCC who had radical esophagectomies in Shandong Cancer Hospital and Institute since June 2005. No patient exhibited evidence of distant metastases in preoperative examination, none had received any prior anticancer treatments or postoperative adjuvant therapy, and all underwent complete macroscopic tumor removal. All tumor tissues were confirmed as ESCC by hematoxylin and eosin (H&E) staining after surgical resection. Only patients who survived more than three mo post-surgery were included in the study. Tumor stage was determined according to the American Joint Committee on Cancer  TNM  staging system . Each tuIHC analyses were performed using the streptavidin-biotin-peroxidase method with postoperative specimens from all patients. 4-μm thin sections were deparaffinized with xylene, rehydrated with graded alcohol solutions, and then exposed to the antigen retrieval system under high pressure for two min. Sections were stained with anti-CD8 , anti-FOXP3  and anti-PD-L1  in a humidified chamber at 37°C for 60 min. Slides were incubated with a secondary anti-rabbit and anti-mouse antibodies (Beijing Zhongshan Golden Bridge Biotechnology Company) at 37°C for 15 min. Diamino-benzidine (DAB) was then added to visualize CD8, FOXP3 and PD-L1 staining and slides were counterstained with hematoxylin.Quantitative evaluations of CD8+ or FOXP3+ TILs were performed by examining at least five non-overlapping high-power fields (40× objective and 10× eyepiece) with the most abundant TILs in each stained section. PD-L1 expression was considered positive if distinct membranous or cytoplasmic staining was observed in tumor or stromal cells. PD-L1 staining intensity was recorded as follows: negative for no staining, positive for membranous or cytoplasmic staining in tumor cells or lymphocytes. Sections were evaluated independently by two experienced pathologists blinded to clinicopathologic information. Variations of > 5% were reassessed and a consensus was reached. As other cell populations can stain with CD8 and FOXP3 antibodies, CD8+ or FOXP3+ cells with apparent different morphological appearances were excluded from cell counts. FOXP3+ and CD8+ TILs were counted both in the cancer cell nest and tumor stroma. The CD8+/FOXP3+ TILs ratio was defined as the number of CD8+ TILs divided by the number of FOXP3+ TILs (Tregs). The mean numbers of CD8+ TILs and FOXP3+ TILs per field and the ratio of CD8+/FOXP3+ TILs were calculated. ESCCs were classified into high and low groups for CD8+ TILs, FOXP3+ TILs and the CD8+/FOXP3+ TILs ratio using a cut-off value defined as the median number of infiltrating cells per field, as described previously Figure .−ΔΔCT) method, and normalized to with the endogenous control, GADPH. The following primers were used in this study: HIF-1α, 5′-GAACGTCGAAAAGAAAAGTCTCG-3′(forward) and 5′ -CCTTATCAAGATGCGAACTCACA-3′ (reverse); PD-L1, 5′ - TTGCTGAACGCCCCATACAA-3′ (forward) and 5′ - TCCAGATGACTTCGGCCTTG-3′ (reverse); and GADPH, 5′ - GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′ - TGGTGAAGACGCCAGTGGA-3′ (reverse).To validate the PD-L1 IHC detection method, PD-L1 mRNA was quantified via qRT-PCR in 10 tissue samples. Total RNA was extracted using the RNA extraction kit spin column method  following the manufacturer's introductions. Total RNA (5 ng) was reverse transcribed with cDNA Reverse Transcription kits  according to the manufacturer's instructions. qPCR was conducted in a SYBR Green PCR Master Mix (ROX). Relative gene expression was calculated using the comparative cycle threshold (CT) . Cumulative survival time was calculated using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate analyses were performed according to the Cox proportional hazards model. Associations among variables were evaluated using Fisher's exact test or the χ


Expression of programmed cell death receptor ligand 1 (PD-L1) has been shown to be up-regulated in some gastric cancer patients and to correlate with the density of tumour infiltrating lymphocytes (TILs). However, conflicting results have been reported regarding TILs and the expression of PD-L1 as a prognostic marker for gastric cancer. We investigated the correlation of PD-L1 and TILs expression with clinicpathological characteristics in 105 well characterized gastric cancer patients. PD-L1 expression and CD3+ and CD8+ TILs were evaluated by fluorescent multiplex immunohistochemistry (mIHC) analysis. PD-L1 positive staining on tumour cells was observed in 35% cases and 48% cases showed PD-L1 expression on immune cells. Up-regulated PD-L1 expression on tumour cells and immune cells was associated with high density of pre-existing tumour infiltrating CD3+ and CD8+. In additional, more than 70% tumor infiltrating CD3+ cells were CD3+CD8+ cells. More than 60% PD-L1+ immune cells were PD-L1+CD3+CD8+ cells. PD-L1 expression in tumour cells was associated with poor prognosis and high density CD3+ and CD8+ TILs indicated improved overall survival in gastric cancer patients. Increased PD-L1 expression with low density CD3+ and CD8+ TILs had the shortest overall survival. In accordingly, PD-L1 absence with high density CD3+ and CD8+ TILs indicated the best prognosis. Combination of PD-L1 with pre-existing TILs may be more precise than PD-L1 alone for predicting survival in gastric cancer. Gastric cancer (GC) is the fourth most common cancer and second leading cause of cancer associated death for men and women worldwide , 2. AlthPD-1 is present on the surface of active T and B cells, and PD-L1 is expressed on many types of immune cells. Several tumour types express PD-L1, including gastric cancer , 9. The There are several reports suggesting that PD-L1 expression is associated with poor prognosis of gastric patients –18. GastWe investigated the expression of PD-L1, CD8 and CD3 in gastric tumour specimens and evaluated the relationship of these factors to clinicopathological characteristics and patient survival. These results may serve as a surrogate marker for PD-L1-positive GCs and may help in validating biomarkers to select patients for immune checkpoint treatment strategies.We examined PD-L1 expression levels in tumour and immune cells in 105 gastric cancer specimens, but not in non-neoplastic gastric epithelium. We observed that PD-L1 was mainly expressed on the membrane and cytoplasm of tumour and immune cells Figure . In the We also evaluated PD-L1 expression on immune cells. PD-L1 staining in ≥1% of immune cells was considered PD-L1 positive. Fifty of 105 cases (48%) were PD-L1 expression Table  and 15 cClinicopathological features and molecular characteristics according to PD-L1 expression on tumour cells and immune cells are summarized in Table n = 105) included 84 males (80%) and 21 females (20%). According to age, patients were classified into <65y (49%) and ≥65y (51%) subsets. Expression of PD-L1 in tumour cells was associated with location (P = 0.012), tumour differentiation (P = 0.016), Ki67 status (P = 0.022) and HER-2 status (P = 0.022). In 30 cases (29%), tumours were located in the gastric cardia and body (17% had PD-L1 positive tumour cells). In 75 cases (71%), tumours were located in the gastric antrum (43% had PD-L1 positive tumour cells). Forty-two cases were moderate to well differentiated (21% had PD-L1 positive tumour cells), and 63 cases were poor differentiated (44% had PD-L1 positive tumour cells). Expression of PD-L1 in tumour cells was significantly associated with the high Ki67 and HER-2 positive cases. In Ki67 high status cases approximately 45% had PD-L1 positive tumour cells. However, in Ki67 low subsets 23% had PD-L1expression in tumour cells. In HER-2 positive cases approximately 58% had PD-L1 positive tumour cells. However, in HER-2 negative subsets less than 30% had PD-L1 positive tumour. Expression of PD-L1 in immune cells was also associated with antral location (P = 0.007) and high Ki67 subtype (P = 0.004). Interestingly, in Ki67 high cases approximately 60% had PD-L1 positive immune cells. However, in Ki67 low cases 32% had PD-L1 positive immune cells. Unlike in the tumour cells, expression of PD-L1 in immune cells was not significantly associated with the poor differentiation or HER-2 status.The study cohort (Staging was classified according to the tumour-node-metastasis (TNM) classification of the American Joint Committee on Cancer . PD-L1 expression was not significantly associated with age, gender, disease stage or tumour growth pattern on either tumour cells or on immune cells.Increased PD-L1 expression on tumor cells and immune cells both positively correlated with CD3+ and CD8+ cell infiltration in gastric cancer. PD-L1 positive in tumour cell subset had a high density of tumor infiltrating CD3+ cells and CD8+ cells Figure . And morTumor infiltrating CD3+ and CD8+ cells were counted, scored as 1 (<1%), 2 (1–9%), 3 (10–20%), or 4 (>20%) and classified into low and high subsets. The correlation between density of different TIL types and PD-L1 expression was summarized in Table These results demonstrated that PD-L1 expression in tumor cells and immune cells was positively associated with the density of CD3+ and CD8+ TILs.P = 0.025). There was no significant difference (P = 0.138) in cumulative survival time between PD-L1 positive and PD-L1 negative immune cell subsets in the whole population  for 10 min at room temperature to block endogenous enzyme. Then, 5% BSA  was used to block unspecific binding for 20 min at room temperature. For IHC, sections were incubated with 1:250 diluted primary antibody PD-L1  incubated at 4°C over-night. Anti- Rabbit IgG SABC kit  and DAB detection kit  were used. For Fluorescent Multiplex immunohistochemistry (mIHC), firstly, slides were incubated in 1:250 diluted primary antibody CD8  for 60 minutes at room temperature. After wash, slides were incubated in HRP labeled anti-mouse secondary antibody  for 30 minutes at room temperature. Wash slides by TNT buffer . Sections were keep in dark place and incubated with 1:50 diluted TSA Plus Cyanine 5  for 5 minutes at room temperature. Slides were stripped in 10mM sodium citrate buffer, pH 6.0 at a sub-boiling temperature for 10 min by microwave. Secondary, 1:250 diluted primary antibody CD3  were add on the sections and incubated in dark for 60 minutes at room temperature. HRP labeled anti-rabbit secondary antibody  and 1:50 diluted TSA Plus Cyanine 3  were used. After stripping, slides were incubated with 1:250 diluted PD-L1  for 60 minutes at room temperature. HRP labeled anti-rabbit secondary antibody  and 1:50 diluted TSA Plus Fluorescein  were used. Finally, mount sections with coverslips using DAPI . Leica Laser confocal microscopy was used for Multi-Color Fluorescence Imaging.The diagnosis was verified for all samples using H&E staining. For IHC, sections were deparaffinised, rehydrated and incubated in Antigen retrieval Citra solution, pH 6.0  at 95°C-99°C for 20 min, then cooled to room temperature. Slides were incubated in 3% HAll IHC and mIHC analyses were performed by two independent observers (Shen and Huang) who were unaware of the patients’ record of clinicopathological features. PD-L1 expression in tumour cells was graded into three groups: 0 (no staining), 1 (weak staining), 2 (moderate to intense staining). We counted PD-L1 staining cells per 1000 tumor cells. The percentage of PD-L1 expression tumuor cells was determined based on 10 random areas (HPF 400 × magnification) in each section and the values were average for statistical analysis. Cases in group 1 or 2 with ≥5% PD-L1 in tumour cells were considered positive as previously. Immune cells were scored separately. PD-L1expression immune cells  were counted per 1000 immune cells, and the percentage was determined based on 10 random areas (HPF 400 × magnification) in each section and the values were average for statistical analysis. Cases with ≥1% PD-L1 in immune cells were considered positive. The number and percentage of CD3 and CD8 expression TILs were determined as PD-L1. Expression of CD3 and CD8 in TILs was scored as 1 (<1%), 2 (1–9%), 3 (10–20%), or 4 (>20%). Subsequently, the grades were classified into low (scores 1–2) and high (scores 3–4) subsets. The IHC results for Ki67 and HER-2 expression were obtained from the pathology department of the First People’s Hospital of Zhenjiang.t-test was used to analyze the significance of differences between the mean values of two variables. All comparisons were calculated using a two-tailed test. Significant differences were considered at P < 0.05.All statistical analyses were performed using SPSS 22.0 . Pearson’s chi-square test and Fisher’s exact test were performed to evaluate the correlation between PD-L1 expression, density of TILs and other clinicopathological features. Kaplan-Meier methods were used to calculate cumulative survival and 95% confidence intervals (CI) were computed. Log-Rank test was used to determine significant difference between survival curves. Student


Nowadays, many types of materials are elaborated for microwave absorption applications. Carbon-based nanoparticles belong to these types of materials. Among these, graphene presents some distinctive features for electromagnetic radiation absorption and thus microwave isolation applications. In this paper, the dielectric characteristics and microwave absorption properties of epoxy resin loaded with graphene particles are presented from 2 GHz to 18 GHz. The influence of various parameters such as particle size  and weight ratio (from 5% to 25%) are presented, studied, and discussed. The sample loaded with the smallest graphene size (3 µm) and the highest weight ratio (25%) exhibits high loss tangent (tanδ = 0.36) and a middle dielectric constant ε′ = 12–14 in the 8–10 GHz frequency range. As expected, this sample also provides the highest absorption level: from 5 dB/cm at 4 GHz to 16 dB/cm at 18 GHz. It presents interesting thermal, mechanical, and electrical properties  along wider form  and σ = yer form ). Anotheyer form ,6,7,8 anyer form . GraphenThe present paper deals with graphene particles embedded into an epoxy resin for microwave absorption applications, and more specifically for the microwave isolation of closer antennas attached on the same composite panel and working between 2 GHz and 18 GHz. It is organized as follows. In Samples were fabricated from pre-dispersals of graphene particles in epoxy resin and a related hardener (masterbatch). The graphene particles consist of the agglomeration of single-layer graphene flakes, each about 0.34-nm-thick corresponding to the interlayer spacing of graphite from sp2 carbon chemistry . Graphen11 reflection coefficient measured via a commercial dielectric kit (High Temperature probe (19 mm in diameter) associated with the N1500A Material Measurement Suite software, both supplied by the Keysight Technologies company, Les Ulis, France). The set-up consisted in a probe in close contact with the surface sample. After an air/short/water probe calibration, results between 2 GHz and 18 GHz were presented to be consistent with the forthcoming reflection and absorption characterizations. To restrict any implementation error and microwave cable effect, a homemade bench-test was fabricated  were retrieved from the Sbricated a. At lea11 and the transmission S21 coefficients, the material absorption was computed from the linear power conservation Equation (1) expressed as follows (Equation (2) [The second and third studied parameters here are the microwave reflection and absorption of the materials. To this end, 3 pairs of high matching horn antennas (4.65–8 GHz // 8–12 GHz // 12.5–18 GHz) were successively used to measure the S-parameters in a near-field setting. The largest apertures of the horn antennas (109.25 mm × 79 mm) had imposed the sample dimensions  were first studied. Other samples fabricated with different weight ratios and the three particle sizes  were studied . MaximumSamples loaded with the largest particle sizes at the maximum weight ratio exhibit closer dielectric constant and loss tangent values . The use11|2 and the electromagnetic transmitted power |S21|2 by and through the composite material under test, respectively. With Equation (2), it is easy to compute the electromagnetic absorption α of the composite material.The assessment of the electromagnetic absorption was made using an homemade near field free-space bench as described in 11|2 ≈ 0 dB), |S11|2 values of the composite materials remain lower than −3 dB in the 8–18 GHz frequency range, evidencing the penetration of the electromagnetic wave into the materials. Reflected power and complex permittivity are closely interrelated with respect to Equation (3) applied with a normal incident electromagnetic wave in air striking a dielectric slab, as follows [air = 1, tanδair = 0) is the dielectric characteristics of air, and  is the dielectric characteristics of the composite material with thickness d.Electromagnetic absorption of the graphene composite materials depends on different parameters. The influence of the graphene particle size in epoxy resin was first investigated.  follows :(3)|S1111|2 between −0.8 dB and −6.4 dB, as observed in 11|2 ≤ −10 dB is explained by the total reflection of the electromagnetic waves by the composite material, the guide wavelength λg into the composite material ; the sample loaded with a 3-µm graphene particle size presents a resonance peak at 11.7 GHz (n = 1); those with 6-8-µm graphene particle size at 7.6 GHz (n = 1) and 15.0 GHz (n = 2); those with 15 µm at 5.7 GHz (n = 1) and 11.4 GHz (n = 2)—in agreement with the increase of the ε′ value when the graphene particle size increases (The variation of ε′ between 5 and 24 a and tantion (4) :(4)n×λgncreases a.n = 1); those with 20% weight ratio at 10.8 GHz (n = 1). The magnitude peak is damped by the increase of the graphene weight ratio, due to the rising of the related tanδ value  to convert α in dB/cm. In order to check the relevance of the ε′ and tanδ values provided by the dielectric kit method  from :(5)α=A×t method a,b, α vat method , despite11|2 ≤ −10 dB). Samples with a 6–8-µm graphene particle size and 10% weight ratio shows two resonance peaks at 7.6 GHz (n = 1) and 15.0 GHz (n = 2). With the increase in the weight ratio to 15%, these peaks shift at 5.6 GHz (n = 1) and 10.5 GHz (n = 2). This behavior is consistent with that of samples loaded with a 15-µm graphene particle size at 5% and 10% weight ratios. The second point is a behavior similar to these samples. The reflected power of a sample loaded with a 6–8-µm graphene particle size with a 10% weight ratio and that of a sample loaded with a 15-µm graphene particle size with a 5% weight ratio are alike. In the same way, a sample loaded with a 6-8-µm graphene particle size with a 15% weight ratio presents a similar |S11|2 to that loaded with a 15-µm graphene particle size with a 10% weight ratio. We attribute these results to the close dielectric constants and loss tangents of the two sets of samples  and the graphene weight ratio (ranging from 5% to 25%). For these, the dielectric constant and loss tangent , and the microwave absorption α of each sample, were measured using a dielectric kit (from 2 GHz to 18 GHz) and a homemade near field free-space bench (from 4 GHz to 18 GHz), respectively. The epoxy resin loaded with the smallest graphene size (3 µm) and the highest weight ratio (25%) exhibits the highest loss tangent (tanδ = 0.36) and a middle dielectric constant value (ε′ ≃ 12–14) in the 8–10 GHz frequency range. Filling the epoxy resin with the smallest graphene particles promotes high loss tangent at microwaves and thus the microwave absorption of the electromagnetic waves by the composite material. Moreover, a lower dielectric constant will be favored to restrict the impedance mismatch at the interface between air and surface samples and thus to restrict the electromagnetic wave reflectivity at this interface. The composite material then provides a high absorption level: from 5 dB/cm at 4 GHz to 16 dB/cm at 18 GHz.