Characterization of PD-L1 expression within clinically/radiologically negative but microscopically tumor positive sentinel lymph nodes (SLN) is important to our understanding of the relevance of this immune checkpoint pathway for adjuvant therapy.Patients included had primary cutaneous melanoma, Breslow thickness of 2.01–4.0 or >4 mm with or without tumor ulceration . All patients had microscopically tumor positive SLN. Hematoxylin and eosin (H&E) staining was performed, followed by PD-L1 immunohistochemical (IHC) staining using a preliminary IHC assay with anti-PD-L1 antibody clone 22C3. The slides were separately evaluated by two pathologists (JY and CG). Samples containing metastatic melanoma lesions were scored separately for PD-L1 expression in intratumoral and peritumoral locations, by utilizing two scoring methods.Twenty-four patients where metastatic melanoma presence in the SLN was confirmed by H&E review of the cut sections were included in the final analysis of PD-L1 expression. SLN tumor size ranged from 1 to 2 mm. For three patients, the melanin content was too high to confidently assign a PD-L1 score. For the remaining 21 patients, all had some evidence of either intratumoral or peritumoral PD-L1 expression. The frequency of intratumoral tumor-associated PD-L1 expression was: 0 % of tumor cells ; <1 % ; 1–10 %  and >10 % .Tumor-associated PD-L1 expression is readily detectable within melanoma micrometastases in the SLN of the majority of patients. These results support the testing of a therapeutic role for PD1/PD-L1 inhibition in the adjuvant setting, targeting melanoma micrometastases. PD-1 is an immune-inhibitory receptor belonging to CD28/CTLA4 receptor family that is expressed on activated T cells, B cells and monocytes –5. The rBRAF V600 mutation positive). The overall tumor response rates were 32 and 11 % in favor of nivolumab [Pembrolizumab and nivolumab, both of which target PD-1, have been approved for the treatment of metastatic melanoma. Approval of pembrolizumab was granted based on data from a cohort of a phase I trial (KEYNOTE-002) in which 411 patients with advanced melanoma who were refractory to ipilimumab or were ipilimumab naive received pembrolizumab at 2 or 10 mg/kg every 3 weeks or 10 mg/kg every 2 weeks , 28. Oveivolumab . The useThe significant clinical activity of anti-PD1 antibodies has supported their planned testing as adjuvant therapy in patients with operable melanoma at high risk for relapse and death from melanoma. Adjuvant therapy targets micrometastatic disease which is the source of future mortality from melanoma recurrence and presents an opportunity for curing this disease. We hypothesized that micrometastatic tumors that are the source of future melanoma relapse in high risk patients express PD-L1 making them susceptible to PD1/PD-L1 therapeutic blockade. Characterization of PD-L1 expression within clinically/radiologically negative but microscopically tumor positive sentinel lymph nodes (SLN) is important to our understanding of the relevance of this immune checkpoint pathway for adjuvant therapy. In this report, we present data which shows that tumor-associated PD-L1 expression is readily detectable within melanoma micrometastases in the SLN.Twenty-four patients with primary cutaneous melanoma were included in this study. All patients had a primary tumor Breslow thickness of 2.01–4.00 mm without (T3a) or with ulceration (T3b), or >4 mm without (T4a) or with ulceration (T4b). Patients had known microscopically tumor positive SLN detected during standard SLN biopsy procedures. All patients provided a written informed consent. Table Cut sections (5 µm) were obtained from formalin-fixed, paraffin-embedded (FFPE) SLN tissue from patients enrolled on this study. Slides were first stained with haematoxylin and eosin. PD-L1 immunostaining was performed using a preliminary immunohistochemistry (IHC) assay with anti-PD-L1 antibody clone 22C3. Slides from two patients were also stained using an anti-HMB45/MelA protocol to better ascertain the presence and/or localization of melanoma lesions in the tissue in order to facilitate interpretation of the PD-L1 staining in those samples. All staining was performed on Dako autostainers at Merck Research Laboratories, Palo Alto, CA. The anti-PD-L1 antibody clone 22C3 is a mouse anti-human PD-L1 IgG1k generated through murine immunization with a fusion protein containing the human extra cellular domain of PD-L1 and subsequent hybridoma formation .The slides were separately evaluated by two pathologists. Samples containing metastatic melanoma lesions were scored separately for PD-L1 expression in intratumoral  and peritumoral  locations. PD-L1 positivity was defined as partial or complete membrane staining of a tumor cell using the 22C3 antibody . Two scoDescriptive statistics were used to tabulate and present the study findings.Twenty-four patients where metastatic melanoma presence in the SLN was confirmed by H&E review of the cut sections were included in the final analysis of PD-L1 expression. Table Of the remaining 21 patient samples, 13 (62 %) showed PD-L1 positivity defined as partial or complete membrane staining in ≥1 % (range 1–90 %) of tumor cells using the 22C3 antibody. The majority of patient samples  showed some degree of intratumoral or peritumoral PD-L1 expression. Table In this study, we have reported that melanoma patients with SLN micrometastases detected by SLN biopsy demonstrate evidence of tumor associated PD-L1 expression. The majority of patients were found to have tumor associated PD-L1 expression estimated by two scoring methods, including the percentage estimates method utilized in prior studies in patients with metastatic melanoma . InhibitThe findings of this study further support the investigation of PD-1/PD-L1 blockade in the adjuvant setting in melanoma patients with high risk for relapse.Previous studies have shown that increased tumor associated expression of PD-L1 is associated with a higher likelihood of clinical response to PD-1/PD-L1 blockade –42. KeffScoring of non-tumor associated PD-L1 expression in in the SLN was complicated by the fact that historical PD-L1 scoring has consistently centered on expression localized to and immediately surrounding tumor tissue. Normal or reactive lymphoid tissue is not typically included in scoring, so no experiential base exists for this type of analysis. In order to try to provide maximal information however, attempts were made to provide scores of PD-L1 expression in non-tumor infiltrated regions of lymph nodes where metastatic lesions were identified. PD-L1 expression in these areas could be classified as falling into two basic categories: (1) expression by histiocytes in the sinuses of the node and (2) expression observed in non-sinus lymphoid tissue with morphologic characteristics consistent with antigen presenting cell populations (dendritic cells and macrophages). Each of these categories was scored separately using a 0–5 semi-quantitative system, focused on signal prevalence. Given the very different representation of tissue from sample to sample and the lack of historical experience with this type of non-tumor associated scoring by the evaluating pathologists, this data should be interpreted as extremely exploratory, and not suitable for drawing conclusions.In conclusion, tumor-associated PD-L1 expression is readily detectable within melanoma micrometastases in the SLN of the majority of patients included in this study. These results support the testing of a therapeutic role for PD1/PD-L1 inhibition in the adjuvant therapy setting, targeting melanoma micrometastases. The testing of PD-L1 expression as a predictive biomarker for therapeutic benefit from anti-PD1 therapy is also warranted and to the best of our knowledge is planned in the context of ongoing adjuvant trials.


The SnO2/graphene composite was characterized by X-ray diffraction, thermogravimetric analysis, Fourier-transform infrared spectroscopy, Raman spectroscopy, scanning electron microscope, X-ray photoelectron spectroscopy, transmission electron microscopy and high resolution transmission electron microscopy. The size of SnO2 grains deposited on graphene sheets is less than 3.5 nm. The SnO2/graphene composite exhibits high capacity and excellent electrochemical performance in lithium-ion batteries. The first discharge and charge capacities at a current density of 100 mA g−1 are 2213 and 1402 mA h g−1 with coulomb efficiencies of 63.35%. The discharge specific capacities remains 1359, 1228, 1090 and 1005 mA h g−1 after 100 cycles at current densities of 100, 300, 500 and 700 mA g−1, respectively. Even at a high current density of 1000 mA g−1, the first discharge and charge capacities are 1502 and 876 mA h g−1, and the discharge specific capacities remains 1057 and 677 mA h g−1 after 420 and 1000 cycles, respectively. The SnO2/graphene composite demonstrates a stable cycle performance and high reversible capacity for lithium storage.SnO Therefore, the development of new alternative anode materials with higher performance is desired. Graphene, a one-atom-thick planar sheet of sp2-bonded carbon with a theoretical lithium storage capacity of 744 mA h g−1, has been widely studied for potential application in LIBs because of its unique properties, such as superior electronic conductivity, high theoretical specific surface area exceeding 2600 m2 g−1, and excellent mechanical properties12O/graphene, CoO/graphenen, Co3O4/graphene, Fe2O3/graphene, Fe3O4/graphene, Mn3O4/graphene, NiO/graphene, SnO2/graphene, TiO2/graphene, VO2/graphene, and V3O7 nanowire templated graphene scrolls34567891011121314152 is considered as one of the most promising anode material substitutes due to its high theoretical specific capacity (782 mA h g−1) and low potential for lithium alloying171819202/graphene composites used as an anode material has been conducted. Various approaches to fabrication of SnO2/graphene composites were reported, such as gas-liquid interfacial reaction, co-precipitation, in situ chemical synthesis, in situ oxidation route, hydrothermal, laser irradiation, microwave and ultrasonication methods222324et al.2/graphene composite by a gas-liquid interface reaction that exhibited a high reversible specific capacity when used as an anode material for LIBs. Zhao et al.2 composite by the bivalent tin ion-assisted reduction method. Electrochemical measurement results showed that the composite could deliver a reversible capacity of 775.3 mA h g−1 and capacity retention of 98% after 50 cycles. Zhu et al.−1 at 100 mA g−1 for the first discharge and charge, respectively. Moreover, the composite exhibited good capacity retention with 649 mA h g−1 after 30 cycles. Yao et al.2/graphene composite with a reversible capacity of 765 mA h g−1 for the first cycle. Recently, several varieties of graphene/SnO2 nanoparticle composites have been reported as anode materials for LIBs, such as flower-like SnO2, SnO2 nanorods, SnO2 hollow nanosphere composites, ternary hybrids of graphene/SnO2/Au, SnO2-graphene-carbon nanotube mixtures and graphene/carbon nanosphere composites2425262728292/graphene hybrids used as anode for LIBs had different structures and exhibited excellent electrochemical performance. However, the cycling stability performance for LIBs at high current densities need to be improved. In addition, most of the preparation methods of these materials used dried neutral graphene oxide as a precursor material and required auxiliary reagents, which result in complex and time-consuming processes. Therefore, an easy and efficient method for synthesizing graphene/metal oxide composites with high capacity for LIBs should be developed.Lithium-ion batteries (LIBs), as power sources for portable electronic devices, mobile communication devices and electric/hybrid vehicles, have attracted tremendous attention due to their high energy density, high working voltage and excellent cycle life. Graphite is the most widely used commercial anode material. However, with a theoretical specific capacity of 372 mA h g2/graphene composite with a microwave reaction system. This method features are quick heating, easily controlled pressure and temperature, high yield rate, and good homogeneity. In reaction process, a precursor material of liquid graphene oxide is reduced in situ, and a bivalent tin ion is oxidized without auxiliary reagents. The uniform composite shows high specific capacity and excellent cycling stability performance for LIBs at high current densities.In this study, we report a one-step microwave-hydrothermal method for the synthesis of SnO2 nanoparticles are clearly distinguishable. All strong diffraction peaks are consistent with a tetragonal crystalline SnO2 phase (JCPDS card no. 41–1445). No diffraction peaks of graphene and graphite oxide are observed in the SnO2/graphene composite, indicating that graphite oxide is reduced to graphene and the graphene layer is exfoliated completely14252 in the SnO2/graphene composite, thermogravimetric analysis (TGA) was carried out in air. The sample was heated from 25 to 800°C at a rate of 20°C min−1. The TGA curve of the SnO2/graphene composite is shown in 2 in the composite is 73.28%. The Fourier transform infrared (FT-IR) spectra of graphite oxide, graphene and SnO2/graphene is presented in −1 can be attributed to O-H stretching vibrations of adsorbed water molecules and structural −OH groups. In the graphite oxide spectrum, the peak at 1628 cm−1 can be attributed to O-H bending vibrations. Carboxyl and epoxy functional groups can also be detected at approximately 1732, 1225, and 1052 cm−1, respectively. Compares to the FT-IR spectrum of graphite oxide, those of graphene and SnO2/graphene show that carboxyl group peaks at 1732 cm−1 decrease significantly, while carboxyl group peaks at 1225 cm−1 and those of epoxy functional groups at 1052 cm−1 disappeared. Two strong peaks of SnO2/graphene are detected at 581 and 1565 cm−1 that corresponds to Sn-O-Sn antisymmetric vibrations and skeletal vibrations of the graphene sheets, respectively2/graphene are shown in −1 (G band) corresponds to the E2g mode of graphite, which is related to the vibration of sp2-bonded carbon atoms in a 2-dimensional hexagonal lattice. The peak at approximately 1346 cm−1 (D band) is an indication of defects associated with vacancies, grain boundaries and amorphous carbon speciesID/IG) of the D band to the G band is related to the extent of disorder degree and average size of the sp2 domainsID/IG of SnO2/graphene is lower than those of graphite oxide and graphene sheets, indicating that bivalent tin can increase the order degree of graphene layers.The structures of materials were systematically research. The X-ray diffraction (XRD) patterns of the prepared products are illustrated in 2/graphene composite obtained from the X-ray photoelectron spectroscopy (XPS) is presented in 2/graphene, which reveals the presence of carbon, oxygen and tin, and no other elements are detected. The peaks of Sn 3d, 4d, 3p, 4p and 4s from SnO2 are observed. The peak of C 1s is attributed mainly to graphene. The Sn 3d spectrum, as shown in 5/2 and Sn 3d3/2 spin-orbit peaks of SnO2, confirming the formation of SnO2 nanoparticles on the surface of graphene sheets. 2/graphene. The C 1s region of graphite oxide gives five components at around 284.6, 285.5, 286.8, 287.4 and 288.6 eV, which can be generally assigned to the C-C, C-OH, C-O, C = O and O-C = O components, respectively2/graphene exhibits dramatically decreased intensities of the C 1s components associated with carbon-oxygen bond, which indicates that most of the oxygenated functional groups on graphene oxide have been removed during the microwave-hydrothermal process. A few residual functional groups are reported to be useful in obtaining stable, highly dispersed SnO2 particlesThe surface chemistry of the SnO2 and the SnO2/graphene composite are observed by scanning electron microscope (SEM), transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM). 2 nanoparticles prepared using the microwave-hydrothermal method aggregated into large particles, and the average particle diameter of SnO2 nanoparticles is 13 nm. These large particles can be pulverized easily owing to an asymmetric volume change during the Li+ insertion/extraction process372/graphene in 2 nanoparticles and the diameter of the SnO2 grains is less than 3.5 nm, indicating that the graphene sheets can prevent the SnO2 nanoparticles from growing effectively. From the HRTEM images in 2 nanoparticles, respectively. According to electron diffraction patterns 2 is oxidized to SnO2 by oxygen from the oxygenated functional groups of graphene oxide in the Teflon vessels at 200°C. The precursor liquid graphene oxide loses oxygen atoms and is reduced to graphene sheets in situ. In sealed high pressure Teflon vessels of the microwave reaction system, when microwave radiates graphene oxide and SnCl2·2H2O mixed solution, graphene oxide and water molecules transform their orientation quickly with the microwave frequency. At the same time, Sn2+ and hydrated ion of graphene oxide solution transfer back and forth, and strike with adjacent molecules under the function of electric field force. The total energy of the molecules is increased due to molecules rotate and collisions of friction. Therefore the solvent can be brought to a temperature well above its boiling point by the increase of pressure resulting from heating, which is beneficial to the rapid nucleation of nanoparticles. In addition, overheated supercritical water can also play the role of reducing agent and offers a green chemistry alternative to organic solvents during this reaction. The physiochemical properties of overheated supercritical water can be widely changed with changes in high pressure and high temperature, which allow the catalysis of a variety of heterolytic (ionic) bond cleavage reactions in water2 nanoparticles can distribute on graphene sheets uniformly and form nanoporous composite with a large number of void spaces. The large number of void spaces can buffer large volume changes of SnO2 nanoparticles during the lithium ion insertion/extraction process12182 nanoparticles, preventing the aggregation of these nanoparticles to a certain extent222 nanoparticles on the surface of graphene can also prevent the graphene from stacking into multilayers.2/graphene composite as an anode material were tested. 2 and SnO2/graphene at a current density of 100 mA g−1 in the voltage range of 3.0 ~ 0.0 V vs. Li+/Li. In the first cycle, the discharge/charge capacities of SnO2/graphene and SnO2 are approximately 2213/1402 and 1607/833 mA h g−1 with coulomb efficiencies of 66.74 and 51.84%, respectively. The initial small plateau in the potential range of 1.2 to 0.8 V corresponds to a classical conversion reaction between SnO2 and Li+ and resulted in the formation of Sn and Li2O in the first discharge of SnO2/graphene, which corresponds to the cathodic peak at approximately 0.85 V. The plateau nearly disappeared during the second cycle, demonstrating that a large amount of Li2O is formed during the first cycle. Because of this irreversible reaction, as well as the solid electrolyte interface (SEI) formed on the anodes43−1, and the discharge capacity of SnO2 dropped more significantly.In order to study the lithium storage performances and mechanism of electrode material, the LIBs of SnO2/grapehen is shown in 2 to Sn, and the synchronous formation of Li2O  curves of SnO of Li2O 1114. Then alloys 23. In thlloys  and graphene (744 mA h g−1), and all the discharge specific capacities at different current densities begin to increase after the 19th cycle. In order to examine the rate performance of SnO2/graphene electrode towards different current densities, the SnO2/graphene electrode was cycled at different current densities  after 100 cycles at 100 mA g−1, and reversed back to low current density of 100 mA g−1. We can see from −1, the capacity almost recover to the original value. This indicates that the SnO2/graphene is quite suitable for large current charge and discharge. The cycling performance and coulomb efficiency of SnO2/graphene at 1000 mA g−1 are shown in −1 is 778 mA h g−1 after 100 cycles. The first coulomb efficiency is 58.3%, and then ~ 99.5% in the following cycles. It should be noted that the capacity increases with increasing cycles of discharge and charge until 1057 mA h g−1 at 420th cycling, and then decreases gradually. The discharge specific capacity is stability and remains 677 mA h g−1 after 1000 cycles.2/graphene before and after 1, 2, 10, 50, 100 charge/discharge cycles. The high-frequency semicircle is attributed to the constant phase element of the SEI film and contact resistance. The semicircle in the medium-frequency region is assigned to charge-transfer impedance and the constant phase element of the electrode/electrolyte interface. The inclined line is associated with Warburg impedance corresponding to the lithium-diffusion process. The diameters of the semicircles in both high and medium frequency areas become noticeably smaller evidently after one cycle, indicating that the first several cycles are the electrode activation process and the decrease in the impedance value of SnO2/graphene electrode. The resistances of SnO2/graphene decrease gradually due to the size of Sn nanoparticles decrease gradually after 10, 50 and 100 cycles. The decreased resistance can enhance ionic conductivity in the composite, which is beneficial for Li+ insertion/extraction into the anodes2330462/graphene during the first 19 cycles can be attributed to the pulverization of original SnO2 and in situ formed Sn nanoparticles during Li insertion and extraction process, which lead to loss of electrical connectivity between neighboring particles+ was extracted. With Li insertion and extraction, the formed Sn nanoparticles became smaller and smaller due to electrochemical milling effects and strong attached to the graphene sheets. Therefore, the conversion reaction of SnO2 in the SnO2/graphene composite, SnO2 + 4Li+ + 4e− → 2Li2O + Sn, would be reversible to a certain extent due to the very small Sn nanoparticles. Ahn et al. claimed that the decrease of reagent particle size could reduce the activation energy for solid-state double decomposition reaction, thus boosting the conversation reaction and contributing to the reversible capacityThe reversible capacities only decrease in the first 19 cycles and then exhibit a slight increase with additional discharge/charge. The decay of reversible capacities of the SnO2/graphene composites, especially at high current densities. The excellent reversible capacities are also attributed to the critical size of SnO2 nanoparticles2, where smaller particle size can help to prevent gradual aggregation of Sn into large clusters2 shows no aggregation upon cycling with cubic Sn formation2/graphene composite, the morphologies of electrode materials after cycling performance testing were studied. 2/graphene composite after 15, 100, 400 and 1000 cycles of discharge/charge test at 1000 mA g−1. As can be seen from 2 nanoparticles are uniformly distributed on the graphene sheets, indicating that SnO2/graphene composite can maintain its structure after 15 cycles. After 100 cycles, the average size of nanoparticles become small, but the boundaries of SnO2 nanoparticles are still clear  The critical size of SnO2 nanoparticles ensures the isolation of nanoparticles with cubic Sn formation and is beneficial for the suppression of their aggregation during electrochemical discharge/charge process, resulting in improved cycling stability. (2) The uniform SnO2 nanoparticles in the as-prepared SnO2/graphene composite can reduce the path length for Li+ transport and supply large specific surface, which is suitable for large current density charge and discharge. (3) The strong contact between SnO2 nanoparticles and graphene sheets can guarantee the stability of the SnO2/graphene composite, which can suppress peel off of SnO2 nanoparticles and increase the electronic conductivity. (4) The porous network structure of the SnO2/graphene composite can increase theoretical lithium storage capacity and abundant active sites for full utilization of active materials, also can facilitate liquid electrolyte diffusion into the electrode material, which is beneficial for achieve high reversible capacity. The results of electrochemical measurement show that the SnO2/graphene composite synthesized by the one-step microwave-hydrothermal method is a promising anode material for LIBs.In summary, a SnO2O2 (30%) was added to the mixture and continuously stirred for 1 h. The mixture was filtered and washed with 10% HCl and 1% H2O2 solutions until the pH was 5. Graphite oxide of 1 mg mL−1 as a precursor material was exfoliated by high power ultrasonication for 30 min.Graphene oxide solution was synthesized by a modification to Hummer's method. Briefly, 2 g expanded graphite was added to a 500 mL beaker, followed by 150 mL concentrated sulfuric acid; the beaker was then placed in an ice-water bath and stirred for 10 min. Potassium permanganate (8 g) was added slowly and stirred for 30 min, then the beaker was placed in a water bath at 35°C and stirred with a mechanical stirrer for 24 h. Deionized water (100 mL) was added to the beaker at 98°C and stirred for 10 min. Then 40 mL H2/graphene composite was prepared by a one-step microwave-hydrothermal method. In a typical reaction, 35 mL 0.04 mol L−1 SnCl2·2H2O solution (0.3159 g SnCl2·2H2O and 35 mL deionized water) was added to 120 mL 1 mg mL−1 graphene oxide aqueous solution, followed by ultrasonic treatment for 10 min. The brown supernatant solution was transferred to high pressure Teflon vessels of the microwave reaction system (Anton Paar Synthos 3000). The system power, temperature, pressure and reaction time were 1000 W, 200°C, 20 bar and 30 min, respectively. The black as-synthesized product was cleaned several times by centrifugation with ethanol and deionized water.The SnO2·2H2O. Pure SnO2 was prepared by a microwave-hydrothermal method, then 15 mL polyethylene glycol (MW = 400) was added to 30 mL SnCl4·5H2O solution with concentration of 0.04 mol L−1; the pH was adjusted by ammonia. The autoclave was sealed and placed in a microwave reaction system at 200°C for 60 min. The hoary solid product was annealed under 500°C for 2 h.As comparison, graphene sheets were prepared with a one-step microwave-hydrothermal method under the same parameters without the addition of SnCl−1 with a PerkinElmer spectrometer. XPS (VG Scientific ESCALAB 2201XL) was carried out using Al Kα X-ray radiation and fixed analyzer transmission mode.The structure and morphology of the as-prepared materials were characterized by SEM (QUANTA 200F), TEM (FEI TECNAI G2 F20), TGA , and XRD (Bruker D8 Advance with Cu Kα radiation) operated at 40 kV and 40 mA. Raman (Renishaw RM-1000) were recorded in a plus laser Raman spectrometer with an excitation laser beam wavelength of 514.5 nm. FT-IR analysis was carried out using pressed KBr disks in the range 4000–400 cm−2. The films were then dried at 120°C in a vacuum oven for 3 h and used as anodes for the coin cells. The weight of working electrode materials is ~ 1.15 mg, and the weight of SnO2/graphene composite in a coin cell for testing is 0.9124 mg. The coin cells were assembled inside a glove box filled with pure argon, using lithium metal as the counter/reference electrode and Celgard2325 as the separator. The electrolyte was 1 M LiPF6 dissolved in a mixture of dimethyl carbonate, diethyl carbonate and ethylene carbonate (1:1:1 by weight). Galvanostatic charge-discharge curves of the cells were recorded by a Battery Testing System  at various current densities from 100 to 1000 mA h g−1 with the voltage between 3.0 and 0.01 V versus Li+/Li at room temperature. CV curves were measured from 0.01 to 3.0 V at a scanning rate of 0.1 mV s−1, and EIS data were obtained by applying an AC voltage of 5 mV in the frequency range of 0.01–100 kHz using an electrochemistry working station (AUTOLAB PGSTAT302).Electrochemical measurements were carried out using CR2025 coin-type cells. The working electrode was prepared by coating slurries consisting of the active material, polyvinylidene fluoride and acetylene black with a weight ratio of 80:10:10 in N-methyl-pyrrolidone solvent. The slurries were uniformly pasted on a copper foil thin film and dried at 120°C in a vacuum oven for 12 h. The thin film on copper foil was cut into round disks with diameter of 12 mm and pressed under a pressure of approximately 200 kg cmM.Z.A. originated the work and designed the experiments. L.L.L. performed the experiments, analyzed the data and wrote the manuscript. P.X.Y. performed XPS and XRD measurement and data analysis. J.Q.Z. carried out cyclic voltammetry and electrochemical impedance spectroscopy measurement and data analysis. All authors reviewed the manuscript.


Complementary experimental techniques such as electrical transport, high resolution transmission electron microscopy and Raman spectroscopy conclusively demonstrate the unforseen stability of this transparent conductor to a relative humidity up to 100% at room temperature for 25 days, to a temperature up to 150°C in atmosphere and to a temperature as high as 620°C in vacuum, that is more than twice higher than the temperature at which the intercalation is conducted. The stability of FeCl3 intercalated few-layer graphene together with its unique values of low square resistance and high optical transparency, makes this material an attractive transparent conductor in future flexible electronic applications.We present the first systematic study of the stability of the structure and electrical properties of FeCl Intuition might suggest that FeCl3 functionalized graphene would not be stable in air, with the humidity present in air rapidly diminishing the integrity and properties of the material.The chemical functionalization of graphene with atoms or molecules provides a valuable way to engineer the desired combination of physical properties needed to enable the aforementioned novel technologies1011123 intercalated few-layer graphene (FeCl3-FLG) is an unexpected highly stable form of functionalized graphene. This conclusion is based on a systematic comparative study of the evolution of the Raman spectra, high resolution transmission electron microscopy (HRTEM), and electrical transport characteristics upon exposing FeCl3-FLG to high relative humidity (H > 90% and up to 100%) at room temperature, to a temperature up to 150°C in atmosphere and up to a temperature as high as 620°C in vacuum for HRTEM. After three weeks continual exposure to a relative humidity ranging from 95 to 100% at room temperature, we observe that the square resistance and the Raman spectra are unchanged. HRTEM measurements up to a temperature of 620°C in vacuum, that is more than twice higher than the temperature at which intercalation is conducted, show no measurable change in the structure of the material. The stability of FeCl3-FLG to high humidity and temperatures widens considerably the range of potential applications targetted by graphene materials.Here we demonstrate that FeCl2 substrates and transmission electron microscopy (TEM) grids. The intercalation with FeCl3 is conducted using an established vapour transport method in a two-zone furnace1215in situ while controlling the humidity in a closed chamber and (2) in air after heating the samples using a hotplate.Few layer graphene are prepared by micromechanical cleavage of natural graphite on standard Si/SiO3-FLG after exposing the sample to an atmosphere with relative humidity H > 95% for various days as indicated in the graph. In contrast to the case of pristine graphene for which a single G-peak−1 (G0), in the intercalated material this peak shifts to 1615 cm−1 (G1) and 1625 cm−1 (G2) can be observed. Both G1 and G2 are the consequence of stiffening of the E2g phonon mode173 to graphene. More specifically, G1 is characteristic of a graphene layer being doped by only one adjacent FeCl3 layer whereas G2 corresponds to a graphene layer sandwiched between two layers of FeCl3  and after (bottom graphs) heating the sample for 1h on a hotplate in atmosphere at 100°C. It is apparent that Raman shifts of just a few cm−1 are measured, corresponding to the accuracy of the spectrometer. This is more clearly seen when comparing the corresponding histograms of the Raman shifts for G1 and G2 peaks before  Raman spectroscopy, (2) high resolution transmission electron microscopy, and (3) electrical transport. The Raman spectra and the square resistance of this material are unchanged upon exposing continually FeCl3-FLG to an atmosphere with relative humidity up to 100% at room temperature for at least 25 days, and to a temperature up to 150°C in atmosphere. At the same time high resolution transmission microscopy confirms that the structure of the material is unaffected by heating FeCl3-FLG up to 620°C in vacuum. The surprising stability of the structure and electrical properties of FeCl3-FLG together with its unique values of low square resistance and high optical transparency, makes this material an attractive replacement for ITO in future transparent and flexible electronic applications.In conclusion, we demonstrate that FeClµm spot size and 1 mW of incident power.The Raman spectra where measured in air and at room temperature with a Renishaw spectrometer using a 532 nm laser wavelength with a 1.5 µA and 11Hz frequency) while the voltage was recorded using phase sensitive lock-in detection.The electrical measurements where conducted in a four terminal geometry using an AC current bias (1 The relative humidity was detected using a HIH-4000 Humidity sensor from Honeywell, and controlled by passing dry nitrogen or ambient air through DI water in a gas bubbler.M.F.C. and S.R. conceived and directed the experiment. D.J.W. conducted the fabrication, Raman and electrical measurements. T.H.B. produced intercalated few-layer graphene and conducted the electrical measurements at high temperature in atmosphere. T.B. conducted the HRTEM measurements. T.B. and P.B. interpreted the HRTEM measurements.HRTEM


As a result, the dc-resistance (R) of graphene is reduced significantly by 60%. Only a negligible amount of defect is created by the irradiation. Also the hole-doped low resistance state of graphene remains robust against external perturbations. This carrier doping is achieved without requiring the bias-gate voltage as is the case for other field effect devices. We make two important observations, (i) occurrence of the doping after the irradiation is turned off (ii) indispensability of the SiO2-layer in the substrate, which leads to a purely electronic mechanism for the doping where electron-hole pair creation and interlayer Coulomb attraction play a major role. A flux-dependent study predicts that an ultrahigh doping may be obtained by longer irradiation. We expect the irradiation doping method could be applied to other atomically thin solids, facilitating the fundamental study and application of the 2d materials.Great effort has been devoted in recent years to improve the electrical conductivity of graphene for use in practical applications. Here, we demonstrate the hole carrier density of CVD graphene on a SiO R ~ 1 kΩ/□ for graphene on SiO2/Si, whereas much lower values of the order of a few hundred Ω/□ are required for industrial touchscreen111213n = 1 ~ 2 × 1012 cm−2 15162/Si. For example, Bae et al. investigated chemical doping by using the molecular adsorption of an acidic gas such as HNO3 onto grapheneet al. synthesized a hybrid device using a ferroelectric polymer and graphene in which the carriers were introduced through a ferroelectric gating mechanismn = 1014 cm−2 − 1015 cm−2 19Graphene holds great promise in the future of electro-optical device applications, thanks to the outstanding transport124578+ ion irradiationIn literature, the idea of irradiating solids with ionized particles has led to important breakthroughs. 2 MeV-proton irradiation induced room-temperature magnetic ordering has been observed in bulk graphite crystal21+) particles. We have irradiated the graphene/SiO2/Si system with 5 MeV proton beam, which leads to an increase of the hole-carrier density by more than an order of magnitude up to n = 3.0 × 1013 cm−2, and a significant decrease of the dc-resistance. We also find that, despite the high energy H+-irradiation, little lattice damage is caused to the graphene crystal structure, suggesting a purely electrical origin for the observed phenomenon. Furthermore, the low resistance remains robust under ambient conditions. To understand the nature of the hole-doping effect, we perform a series of experiments: we measure real-time change of resistance upon turning on, maintaining, and turning off the beam. Also we study whether the doping behavior depends on substrates by employing various materials for the substrate. On the basis of these and other related experiments we will propose mechanism for the irradiation-driven doping as to be described later. We further investigate the relation between the carrier doping and the irradiation time, which suggests that even higher densities could be accomplished by longer irradiation times.In this Article, we report on a new route for carrier doping, by utilizing high energy proton , we deduce the hole density ni = 1.1 × 1012 /cm2 and nf > 7.2 × 1012 cm−2, where i and f stand for before and after irradiation, respectively. To independently confirm the density increase, we measure the optical transmission T(ω) in the far-infrared frequency range for another irradiated sample. T(ω) due to free carriers in graphene. The peak is fit using the Drude conductivity σdc increases from 1.2 × 10−3 (Ω−1) to 3.2 × 10−3 (Ω−1) after irradiation. The width of the Drude peak, γ, represents the carrier scattering rate  which changes from 120 cm−1 to 100 cm−1. γ decreases after the irradiation due to increased n, i.e., the scattering is reduced because the carriers screen the charge impurity scattering centers more effectively for higher nni = 2.9 × 1012 cm−2 and nf = 3.0 × 1013 cm−2. The final state density nf is ten times larger than the initial density ni. ω = 18 cm−1 and Δω = 14 cm−1. Previous works teach us that the blue shifts of the G and 2D peaks occur when graphene is hole-doped. One can determine the hole density based on the carrier-controlled Raman measurements26ni ~ 2 × 1012 cm−2 and nf ~ 2 × 1013 cm−2. The intensity of the two peaks I(G) and I(2D) increases and decreases respectively, a behavior which again roots in hole-doping. Our transport, infrared and Raman phonon measurement consistently indicate that the hole carrier density has increased 10-fold by the H+-irradiation, which leads to the low-resistance of CVD graphene. The Raman D-peak amplitude increases slightly in the final state suggesting some defects are created by the irradiation. However, the I(D)/I(G) ratio is only about 0.07, corresponding to a very small defect concentration.R-decrease, we perform real-time 4-probe resistance measurements during the irradiation. +-beam is turned on, R rises shortly before strongly dropping until saturation at R = 30Ω, which is remarkably low compared to the initial resistance Ri = 1.7 kΩ. When the beam is turned off, R rises back and then decreases again. This second phase of R-decrease occurs spontaneously and stabilizes at Rf = 665Ω. In overall, the resistance was thus reduced by more than 60%, ΔR/Ri = −0.61.(ΔR≡R(t) − Ri). The strong hole-doping in the final state is taking place during the second phase. The low-R state, which has been monitored for more than one year, remains stable in air without any notable change. We also tested whether this state can be altered by various perturbations. The resistance value remains robust after both annealing the sample at high temperature (T = 250 °C) for 1.5 h and exposing the sample to UV radiation. Additionally, we prove that the system is in a charge neutral state by connecting a copper wire between the graphene sheet and the ground, which did not modify the resistance. +-free R-decay occurs over a much longer time, ~40 hours [To better understand the 40 hours , implyinRf depends on the chosen substrate. A series of graphene samples are prepared on various substrates where the irradiation is performed with the same Ip. The result are summarized in 2O3) substrate, the low-R state is absent. i.e., R decreases spontaneously in Phase-II, but the final Rf is the same as Ri suggesting that perhaps a low-R behavior may occur for SiO2/Si only. SiO2/Si substrates are used with different Si-doping type (p-Si and n-Si) and different Si-doping level . In these systems, the low-R is reproduced with the same magnitude irrespective of doping type. Finally, to resolve the role of SiO2 and Si separately, we transferred graphene on a bare-Si substrate and performed the irradiation. Here R-measurement is not a proper probe because current flows dominantly through the weakly doped Si which obscures precise change of R in graphene. Instead we measure far-IR transmission which detects the Drude change of graphene only. The far-IR transmission shows that the Drude peak for G/Si sample barely changes after the irradiation indicating an absence of hole-doping in contrast with the clear Drude increase for the G/SiO2/Si system. These results suggest that SiO2/Si is essential to achieve the hole-doped low-R state and that SiO2 is playing an important role.As part of the efforts to understand the mechanism behind the strong hole-doping, we investigate how the value of +-irradiation. When the irradiation is turned off, the electron and hole recombine with each other and R returns to the initial resistance Ri. We develop this idea into a quantitative model and use the theoretical result to explain the real-time R-change in Phase-I which is described in detail in Supplementary-I.High energy protons are known to interact primarily with nuclei of the target solid. The excited nuclei relax energy by creating secondary excitations such as phonons, photons, plasmon, etc28292 and Si as well. For graphene and Si they disappear when the beam is turned off through recombination process. However in SiO2 the e-h pair follows a different fate due to strongly asymmetric carrier mobilities: The conduction electron in SiO2 is highly mobile (μe = 20 cm2/V · s) and some of them can move to Siμh ~ 10−6 · μe) and also has larger effective mass than electron. As a result the uncompensated holes are trapped inside SiO2. The hole-trapping and accumulation increase as the irradiation is continued. The trapped hole can diffuse through SiO2 by thermal assistance. When it reaches the G/SiO2 interface the hole binds with the electron in graphene via Coulomb attraction [see 2-glass, Al2O3, and Si-the spatially separation of the e–h pair does not take place and consequently the R-decrease effect is absent.The creation of the electron-hole pair due to irradiation occurs not only in graphene but also in the SiOtion see . ConsequA few subsidiary but important issues of this experiment need to be clarified.μm depth from graphene . To clarify the role of these embedded protons in the R-change, we transferred graphene on a thinner SiO2/100 μm-thick Si substrate for which the H+ beam should completely penetrate through the substrate and avoid the existence of embedded protons. We also prepared a G/SiO2/500 μm-Si sample where Si is electrically grounded to earth  the doping occurs over a long time after the irradiation is turned off, (ii) the SiO2-layer in the substrate is indispensable for the effect to take place. On the basis of these findings, we proposed the electron-hole pair creation and the graphene-SiO2 interlayer Coulomb attraction as the underlying mechanism for the irradiation-induced strong hole doping. Also, from a detailed Ip-dependent study, we predicted that a much higher doping density and lower R would be achieved by longer irradiation. This could not performed in this work due to practical limitations of the beam facility. When compared with a conventional field effect gating or an ion-gel electronic double layer (EDL) gating, the current gating-free irradiation method does not require to apply bias voltage to dope carrier. One could perform the proton irradiation on a bare SiO2/Si substrate and transfer the graphene later on, which, owing to the slow post-irradiation doping mechanism, should yield the doping without any irradiation-driven defects at all in graphene. Also, by using a micro proton beam confined over a small area, one may create a local doping at a desired location, which might be useful in devising various graphene devices. Traditionally, high energy irradiation has been regarded as a tool of defect- or impurity-related physics and engineering. However, our work demonstrated that it can be used to control the electrical property of a solid as well- graphene in this case-which can be applied to other atomically thin solids such as transition metal dichalcogenides  and black phosphorene, offering new opportunity for fundamental and application study of the 2d materials.We demonstrated that graphene on SiO4)2S2O8 solution. After rinsing using DI water, graphene was transferred onto the different types of substrate, including SiO2/Si and Al2O3. The detailed transfer techniques are explained in ref. Large scale monolayer graphene (1 cm × 1 cm) was synthesized by the CVD method under low pressure and high temperature−3 torr) in the MC-50 cyclotron at KIRAMS . Beam current and spot diameter were set to 170 nA and 3.5 cm, respectively. The maximum irradiation time allowed by such instrumentation is limited to 90 min for safety reasons, which corresponds to a flux Ip of 1016 ions/cm2.A 5 MeV-Proton beam was used for the irradiation under vacuum conditions  and a 4.2 K-cooled bolometric detector. The Raman spectra were recorded with a WITec CRM200 Raman system using a Nd:YAG laser (532 nm) as excitation source in which the laser power was set to a value below 0.1 mW in order to avoid heating.The optical transmission in the Far-infrared frequency region was measured using a commercial Fourier Transform Infrared Spectrometer .


The aim of this study was to analyze the expression of PD-L1 and MHC-I in 19 human tumor cell lines and changes after interferon gamma (IFN-γ) treatment, in order to evaluate the potentiality of combining anti-PD-L1 antibody with other immunotherapies.Nineteen human tumor cell lines were cultured according with ATCC guidelines: 5 colon , 4 ovarian , 3 breast , 3 lung , 2 prostate (LnCap and PC-3), and 2 pancreatic (CFPAC-1 and ASPC-1). Cells were analyzed by flow-cytometry for PD-L1 (clone 29E.2A3) and MHC-I expression. The surface expression of PD-L1 was considered as low, medium, or high based on the percentage of positive cells . Cells were also analyzed for PD-L1 mRNA expression by RT-PCR. Experiments were performed with or without IFN-γ pre-treatment .The expression of PD-L1 was as follows. Low: 4/5 colon , 1/4 ovarian (OVCAR-3), 2/3 breast , and 1/2 pancreatic (ASPC-1). Medium: 1/5 colon (Caco-2), 2/4 ovarian , 2/3 lung , and 1/2 prostate (LnCap). High: 1/4 ovarian (ES-2), 1/3 lung (H441), and 1/2 prostate (PC-3), 1/3 breast (MDA-MB-231), and 1/2 pancreatic (ASPC-1). After IFN-γ pre-treatment, 14/19 cell lines showed a >50% increase of PD-L1 and 14/19 a >50% increase of MHC-I (either percentage positive or MFI). In 13/19 cell lines both markers increased. IFN-γ pre-treatment caused an increase >100% of PD-L1 mRNA expression in 14/19 cell lines. CFPAC-1 (pancreatic) showed an increase of surface PD-L1 without mRNA change; on the opposite, H1703 (lung) showed mRNA increase without changes in surface expression.Tumor cells express different percentage of PD-L1 and MHC-I in their surface. In most of the cells analyzed, both molecules are increased by exposure to IFN-γ. Based on these observations, immunotherapies aiming to increase IFN-γ in the tumor microenvironment, such as therapeutic vaccines or T cell adoptive transfer, can facilitate immune recognition of tumor cells by an increase of MHC-I on the surface of tumor cells. On the other hand, the increased PD-L1 expression in the tumor can be an ideal target for anti-PD-L1 antibody treatment.


Azadirachta indica A. Juss) is one of the most versatile tropical evergreen tree species known in India since the Vedic period (1500 BC–600 BC). Neem tree is a rich source of limonoids, having a wide spectrum of activity against insect pests and microbial pathogens. Complex tetranortriterpenoids such as azadirachtin, salanin and nimbin are the major active principles isolated from neem seed. Absolutely nothing is known about the biochemical pathways of these metabolites in neem tree. To identify genes and pathways in neem, we sequenced neem genomes and transcriptomes using next generation sequencing technologies. Assembly of Illumina and 454 sequencing reads resulted in 267 Mb, which accounts for 70% of estimated size of neem genome. We predicted 44,495 genes in the neem genome, of which 32,278 genes were expressed in neem tissues. Neem genome consists about 32.5% (87 Mb) of repetitive DNA elements. Neem tree is phylogenetically related to citrus, Citrus sinensis. Comparative analysis anchored 62% (161 Mb) of assembled neem genomic contigs onto citrus chromomes. Ultrahigh performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was used to quantify azadirachtin, nimbin, and salanin from neem tissues. Weighted Correlation Network Analysis (WCGNA) of expressed genes and metabolites resulted in identification of possible candidate genes involved in azadirachtin biosynthesis pathway. This study provides genomic, transcriptomic and quantity of top three neem metabolites resource, which will accelerate basic research in neem to understand biochemical pathways.Neem ( Azadirachta indica is an evergreen tree, native to the Indian subcontinent. It belongs to Meliaceae family plants, which are the major source for diverse limonoids , Anuganalu, Hassan (Genotype 2)) and Tamil Nadu  for this study .Mature neem leaves were collected from neem trees for DNA isolation. CTAB method  was used for isolation of neem genomic DNA , with feNeem endosperm explants, of 4–5 mm length pieces were cultured on Murashige and Skoog solid medium (pH 5.6) containing 0.45 mg BAP, 0.80 mg NAA and 150 mg Casein hydrolysate. Cultures were maintained in growth chamber at 25 ± 2 °C with 55 ± 5% of relative humidity, for 16 h photoperiod and allowed for formation of callus tissue. After 2 weeks, mature callus was sub-cultured to fresh media every fourth or sixth weeks.http://454.com).Whole genome shotgun DNA library was prepared using Illumina TrueSeq DNA sample preparation kit (FC-121-2001). The paired-end (PE) (2 × 100 nts) sequencing was carried out using Illumina HiSeq 1000 at the Next Generation Genomics Facility at Centre for Cellular and Molecular Platforms (C-CAMP). We prepared whole genome shotgun 454 library for Genotype 1  using the rapid library preparation kit from Roche . The 454 sequencing was carried out using GS FLX+ chemistry as per Roche/454 manual instructions . The RNA-seq library was prepared using 1 µg of total RNA according to Illumina’s TruSeq RNA sample preparation kit (RS-122-2001).q) and percentage (p) value was assigned as 20 and 100, respectively . After quality filtering, we obtained a total 75–192 millions of paired-end reads and 61–71 millions of singleton reads (De novo assembly of neem genome was performed using Velvet program (Illumina PE reads were pre-processed using FASTX-Toolkit (v 0.0.13). The read quality score cut-off  assembled contigs, obtained from Velvet  and MIRAThe completeness of the assembled genome was checked by using eukaryotic core gene-mapping approach (CEGMA) . CEGMA uhttp://www.repeatmasker.org/RepeatModeler.html). Gene expression was quantified by mapping RNA-Seq reads . The program was run with RM-BLAST (NCBI) database as an input for the repeat modelling. We trained Repeat Modeler using genomes of other published plant species including P. trichocarpa (39.40%), R. communis (51.74%) and A. thaliana (15.29%) for pipeline validation (SRA1085705) from lidation . We down® III RT enzyme (200 u/µl) . The qPCR was performed on an Applied Biosystems, 7900HT Fast Real-Time PCR system machine. Real-time PCR was performed in a 384-wells optical reaction plate  using SYBR green PCR mastermix , which contains AmpliTaq Gold® DNA polymerase and ROX as a passive reference dye. Cycling conditions were 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s with 40 cycles. We compared the fold change in qPCR experiments for selected genes from azadirachtin biosynthesis with conserved eukaryotic (rice) elongation factor 1α (eEF-1α) gene (AK061464.1). All reactions were performed in triplicate with elongation factor primers and water as an internal control. The primer sequences of selected genes are listed in the Total RNA from leaf, callus and developing endosperm  was isolated using TRIzol reagent method and quantified using a Qubit Fluorometer. The cDNA synthesis was performed using total RNA (1 µg) with oligo(dT) random primers (50 µM) and SuperScripthttp://pgrc.ipk-gatersleben.de/misa/) with assembled neem genome sequences. The SSRs containing contigs were extracted for SSRs motif variability prediction. The previously published SSR marker regions (Identification of simple sequence repeats (SSRs) or microsatellite was done using MIcroSAtellite tool (MISA)  and Insertion Deletion (InDels) markers were identified by mapping Illumina short reads from Genotype 2 and Genotype 3 to reference Genotype 1 assembly using Bowtie2 . The aliA. thaliana, B. napus, C. papaya, C. sinensis, N. tabacum, P. dactylifera, P. trichocarpa, R. communis, S. bicolour and V. vinifera using Bowtie2  chromosomes using MUMMER program  was done using Proteinortho program . ComparaWe purchased neem standard metabolites such as azadirachtin , salanin  and nimbin . High purity MS grade solvents  were obtained from Merck Millipore .For metabolites analysis, we collected samples from various neem tissues  and dried at 37 °C for two days. The samples were ground into fine powder using Pestle and Mortar, and stored at −80 °C until use. There were no commercially internal standards available for neem metabolites quantification; therefore, we used estrone-d4  as an internal standard to construct the standard curve and also for absolute quantification. The calibration curves for the quantification of all three neem metabolites were linear over a 64-fold concentration range  and 73-fold concentration range (nimbin) with linear regression correlation coefficients ranging from 0.998 to 0.999 . TypicalNeem metabolites were extracted from the dried powder (2 mg) using 1 mL of methanol followed by 5 min sonication and centrifuged for 5 min , the supernatant was transferred to fresh micro-centrifuge tubes. About 5 µL of supernatant was spiked into 35 µL of methanol along with internal standard (10 µL estrone-d4 from 100 µg/mL). The analyses were done by injecting 10 µL of the sample into the UHPLC-MS/SRM system . The intense product ions were selected for the LC-MS/SRM analysis . We used following LC conditions: solvent system A-Water (10 mM Ammonium Acetate) containing 0.1% FA and B-Acetonitrile containing 0.1% FA, Flow-200 µL/min, Column- C-18 column , Gradient- 2% B at 0 min, 2% B at 3 min, 40% B at 10 min, 95% B at 15 min, 2% B at 15.1 min, 2% B at 15.1–20 min. MS conditions: spray voltage, 3,000 V; ion transfer capillary temperature, 270 °C; source temperature 100 °C; sheath gas 18, auxillary gas 5 (arbitrary units); collision gas, argon; S-lens Voltage was optimized for individual metabolites; scan time of 50 millisec/transition and ion polarity positive. In case of azadirachtin (15.6 pg to 1 ng), nimbin (3.4 pg to 0.25 ng) and salanin (7.8 pg to 0.5 ng) on column was used to construct the standard curve. The overall scheme for the quantification of neem metabolites is shown in the The quantified metabolites data points from different explants or tissues were used to compare the gene expression pattern using WGCNA package in R  for trace novo assembly resulted in 216 Mb (version 1.1) with 21X coverage using Velvet software. This analysis produced a total of 94,780 scaffolds where 90% of the genome was covered by scaffolds length longer than 1,000 bp. The N50 was about 22 Kb with 31.13% of GC content and the longest scaffold length was 241 Kb. In addition to Illumina data, we also generated 1.13 Gb  data using Roche 454 GS FLX + chemistry for the Genotype 1. The average read length was 410 nts and longest 454 read length was 1,596 nts. The 454 assembly was generated using MIRA program (We sequenced the neem genome using Illumina and 454 platforms. Illumina HiSeq paired-end (2 × 100 nts) sequencing yielded 13.86 Gb of high quality data for the neem Genotype 1 . D program . The draGene prediction was performed using two programs, Augustus and GenScan, which led to identification of 40,130 and 52,617 genes, respectively. In total, we identified 44,495 genes after merging and clustering of genes from these two programs. The genic region of neem genome was about 114 Mb (42.5%). The initial gene prediction was carried out without masking the repeat regions in the genome to avoid missing of simple sequence repeats in the coding regions and to predict proper exon/intron boundaries. To annotate the neem genes, annot8r program was used by incorporating functional proteins from GO, EC and KEGG and merged with BLAST results . There wde novo assembly of neem transcriptome using Trinity program. These transcripts were further used as supporting expression evidences for the genes that involved in metabolites biosynthesis pathways. The protein sequences for 44,495 genes were compared with proteome of 23 sequenced plant species. Of these, 23,125 genes (52%) were classified into 18,327 families  and monocot (rice) plants . 27,498 De novo repeat identification in neem genome was performed using RepeatModeler (SRA1085705) using SOAPdenovo2 program. During our re-analysis of neem genome assembly from Krishnan et al. dataset (SRA1085705), we found 22 to 35% of genome harbour repeats (SRA1085705 (tModeler . This antModeler . The lon repeats . In addiA1085705  as compaSimple sequence repeats (SSR), single-nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels) are the most abundant DNA markers in any plant genomes. Three sequenced neem genotypes were compared to assess genome-wide genetic diversity. This analysis identified 140807, 108020 and 95840 SSRs from neem Genotype 1, Genotype 2 and Genotype 3, respectively  and S14Bin-silico polymorphism in SSR loci. Nearly 100 nts from upstream and downstream regions of SSR motif from Genotype 1 (reference) were used for SSR polymorphism analysis. These analyses resulted in 2,199 and 2,120 polymorphic SSRs in the Genotype 2 and Genotype 3, respectively. The concordance SSR analyses showed 571 SSRs were polymorphic among three neem genomes  of the genes. We identified 130,668 (2.78%) and 159,375 (3.21%) SNPs, for exonic regions of coding genes from Genotype 2 and 3, respectively. There were 31,312 and 37,864 SNPs in the coding sequence which represents the synonymous amino acid changes in protein coding regions of Genotype 2 and 3, respectively. There were 84,194 and 102,663 SNPs that showed non-synonymous amino acid substitutions in the protein coding regions of Genotype 2 and 3, respectively. The presence of SNPs in start and stop codons were less as expected. The details of SNP annotation is summarized in We have quantified neem metabolites using high sensitive UHPLC-MS/SRM method, which can detect the picogram (pg) level of neem metabolites . The concentration of neem metabolites in mature seeds , nimbin  and salanin ) was higher than other neem tissues. The concentration of Azadirachtin was always higher in mature seeds, followed by developing endosperm, shoot, root, cambium, pulp, flower, bark and leaf. Nimbin concentration was higher in seed, followed by bark, cambium, endosperm, root, shoot, flower, pulp and leaf. Similarly for salanin, the trend was higher in seeds followed by endosperm, bark, cambium, root, shoot, leaf, pulp and flower. The quantification of neem metabolites from various tissues is shown in Genes involved in various steps from tirucallol to azadirachtin are not yet established. Therefore, we devised bioinformatics approach by clustering expressed genes along with amount of metabolites  in each tissue using Weighted Correlation Network Analysis (WGCNA) method. This WGCNA clustering analysis identified azadirachtin biosynthesis genes that are up-regulated in developing endosperm, and had a minimal expression in other tissues such as leaf, flower, fruit coat and pulp . From thWe filtered reads which map to chloroplast and mitochondrial genomes of other plant species. The assembly and annotation statistics for organelle genome are shown in The mitochondrial genome of neem was also assembled . N50 forGenome size and chromosomal architecture was not available for neem tree. Synteny analysis was performed to obtain conserved chromosomal blocks of neem genome and genes in a closely related plant species. Our analysis revealed that the neem genome is phylogentically related to citrus. Therefore, we used the citrus genome (nine citrus pseudomolecules) as a reference to order neem contigs. This comparative analysis anchored 24,902 neem contigs onto 9 citrus chromosomes . This anNeem is medicinally, agriculturally and environmentally important tropical tree in Indian subcontinent. Neem is well-known for its complex tetranortriterpenoids compounds such as azadirachtin, nimbin and salanin, which are the main constituents in insecticidal and pharmaceutical formulations . Unlike de novo assembled neem genome. Our prediction of genes  and TE-related genes (35%) are comparable with well annotated rice genes (http://rice.plantbiology.msu.edu)  of genome based on estimated genome neem size by msu.edu) . More thSRA1085705). This analysis predicted more than 20% of repeats and constitutes higher gaps with lots of Ns (up to 60%)  repeats in the neem genome in contrast to previous study . They ha to 60%) . Accordi to 60%) . Our assOther highlight of our study is that we sequenced three neem genotypes from varied environmental conditions, which assisted us to identify DNA molecular markers such as SSRs, SNPs and InDels. We obtained about 2.9 SNPs and 0.22 InDels per 1,000 nts on the reference neem genome (Genotype 1). SNP and InDel markers density distribution in neem genome are lower than citrus genome (3.6 SNPs and 0.6 InDels per Kb) . The lowCitrus sinensis) at the family level  a steroid triterpenoid, is a possible precursor for azadirachtin biosynthesis in neem . In the Our study added genomic, transcriptomic and metabolites data to support limonoids biosynthesis pathway genes in neem. To understand the neem metabolites bio-synthesis, we quantified major metabolites using a sensitive UHPLC-MS/SRM method. We identified the known secondary metabolite pathway genes including farnesyl diphosphate synthase (Ai02g10209), squalene synthase (Ai02g1634), geranyl diphosphate synthase (Ai02g31423), mevalonate kinase (Ai02g16520) and other unannotated genes upstream to squalene . Across Neem is an important tropical evergreen tree in India, used for centuries in agriculture and traditional medicine. In this study, we report the detailed analysis of neem genomes, transcriptomes and metabolites. We identified possible candidate genes involved in azadirachtin biosynthesis pathways. Genomic resources such as sequence of genomes, genes, transcripts, SSRs, SNPs and InDels from this study will have a profound application to study diversity, traits association, with biopesticidal properties and biochemical pathways in neem and other species of Melieacea family.10.7717/peerj.1066/supp-1Table S1Click here for additional data file.10.7717/peerj.1066/supp-2Table S2Click here for additional data file.10.7717/peerj.1066/supp-3Table S3Click here for additional data file.10.7717/peerj.1066/supp-4Table S4Click here for additional data file.10.7717/peerj.1066/supp-5Table S5Click here for additional data file.10.7717/peerj.1066/supp-6Table S6Click here for additional data file.10.7717/peerj.1066/supp-7Table S7Click here for additional data file.10.7717/peerj.1066/supp-8Table S8Click here for additional data file.10.7717/peerj.1066/supp-9Table S9Click here for additional data file.10.7717/peerj.1066/supp-10Table S10Click here for additional data file.10.7717/peerj.1066/supp-11Table S11Click here for additional data file.10.7717/peerj.1066/supp-12Table S12Click here for additional data file.10.7717/peerj.1066/supp-13Table S13Click here for additional data file.10.7717/peerj.1066/supp-14Table S14Click here for additional data file.10.7717/peerj.1066/supp-15Table S15Click here for additional data file.10.7717/peerj.1066/supp-16Table S16Click here for additional data file.10.7717/peerj.1066/supp-17Table S17Click here for additional data file.10.7717/peerj.1066/supp-18Table S18Click here for additional data file.10.7717/peerj.1066/supp-19Table S19Click here for additional data file.10.7717/peerj.1066/supp-20Table S20Click here for additional data file.10.7717/peerj.1066/supp-21Figure S1Click here for additional data file.10.7717/peerj.1066/supp-22Figure S2Click here for additional data file.


Global demands on agricultural land are increasing due to population growth, dietary changes and the use of biofuels. Their effect on food security is to reduce humans’ ability to cope with the uncertainties of global climate change. In light of the 2008 food crisis, to secure reliable future access to sufficient agricultural land, many nations and corporations have begun purchasing large tracts of land in the global South, a phenomenon deemed “land grabbing” by popular media. Because land investors frequently export crops without providing adequate employment, this represents an effective income loss for local communities. We study 28 countries targeted by large-scale land acquisitions [comprising 87 % of reported cases and 27 million hectares (ha)] and estimate the effects of such investments on local communities’ incomes. We find that this phenomenon can potentially affect the incomes of ~12 million people globally with implications for food security, poverty levels and urbanization. While it is important to note that our study incorporates a number of assumptions and limitations, it provides a much needed initial quantification of the economic impacts of large-scale land acquisitions on rural livelihoods.The online version of this article (doi:10.1007/s11111-014-0215-2) contains supplementary material, which is available to authorized users. Population growth, dietary changes and increasing use of crop-based biofuel are placing ever greater demand on food production and its requisite resources  that this may result in increased urbanization and human migrations in order for rural communities to diversify their incomes , regardless of whether the land is under production because we assume that at this stage previous land users have already been excluded from accessing the acquired land. These criteria ensure that land rights have legally changed hands and that the ability of rural communities . Since data on average rural income were not available for the countries of interest, average income per capita was given as the gross national income (GNI) per capita in terms of purchasing power parity. These data were from the World Bank’s World Development Indicators database  against which future case studies can be measured. This is particularly noteworthy given the significant assumptions incorporated into our methodology  and, in turn, how they may influence our findings. Despite this, we find that where data are available our results agree quite well with case studies where rural communities were either displaced or their livelihoods were affected.While the loss of income and employment opportunities by rural communities is an important impact to consider, we also acknowledge that with each land deal comes a unique set of benefits to, impacts on and responses by the affected local communities  actually find their way to the populace just as the original agricultural income would. One way by which these changes in land tenure can potentially benefit and sustain the livelihoods of local communities is by providing employment opportunities with adequate income. While investing corporations regularly make estimates on new job creation, the actual number of jobs created is typically well below expectations, due to transitions to plantation style agriculture preferring mechanization and wage laborers , highlighting the fact that countries are differentially affected by and sensitive to consequences of large-scale land acquisitions. As per capita income can vary greatly between targeted countries  and infrastructural  means to seek employment through non-farm activities are often left intact, but the enduring livelihoods of households are threatened. Given the proximity of many land deals to urban areas Below is the link to the electronic supplementary material.


Sarcoidosis is a systemic disease of unknown etiology characterized histologically by the observation of non-caseating granulomas and several immunological abnormalities. Sarcoidosis is a multi-organ disorder which involves formation of granulomas in many tissues including the lungs (pulmonary) and others such as skin, bone, heart (extra pulmonary). Associations between human leukocyte antigens (HLA), the encoded cell surface receptor (HLA-DR) and sarcoidosis have been reported in several studies. Several HLA-DR alleles have been described as potential risk factors for sarcoidosis in distinct ethnic groups however evidence for a relationship between HLA-DR alleles and pulmonary and extra-pulmonary sarcoidosis (EPS) is still scarce. Although the etiology of the disease remains unclear, infectious and environmental factors have been postulated. Inflammatory cytokines and chemokines may play important roles in the pathogenesis of sarcoidosis and serum free light chain (FLC) numbers have been implicated in several immunologic disorders.The aim of the present study was to investigate HLA associations with serum cytokines and FLC in Iranian patients with pulmonary (n = 86) and EPS (n = 46).We found that among the 16 HLA DRB alleles only *7 and *12 were different in sarcoidosis patients. The levels of TNF-α and IL-8 in pulmonary sarcoidosis patients were higher than in EPS (P < 0.05) whereas the levels of FLC subunits in EPS were higher than in pulmonary sarcoidosis.This data may suggests a link between HLA-DRB *12 and sarcoidosis in Iranian population. Sarcoidosis MIM 181000) is a systemic granulomatous disease that was originally described over 100 years ago 81000 is . While mThe inflammatory response in sarcoidosis is characterized by the accumulation of CD4 T cells at the sites of disease activity . These TA genetic susceptibility to sarcoidosis has been supported by studies demonstrating familial clustering . First-dThe clinical presentation of sarcoidosis depends on epidemiological factors such as age, gender and race; the duration of the disease and the anatomical sites involved -18. AlthThere is evidence that patients with pulmonary plus EPS have greater severity of symptoms, restrictions in daily activities and quality of life and impairment of health status comparing to the patients with isolated pulmonary sarcoidosis . HoweverCytokines have been implicated in the pathogenesis of sarcoidosis for many years. In particular, the role of IL-8 and TNF-α have been frequently studied -25. GranHLA molecules (low resolution molecular typing) it has not been possible to reach a consensus on the importance of HLA molecules in sarcoidosis [Serological and molecular analyses of HLA molecules have also been investigated in sarcoidosis. Unfortunately, because of the limitations of study design and techniques used to identify coidosis . CurrentImmunoglobulin free light chains (FLC) can exert various biological functions: enzymatic activity, binding to intracellular and extracellular proteins and cellular interactions . DiffereSarcoidosis patients (n =86) were recruited from Sarcoidosis clinic center from Massih Daneshvari Hospital between January 2012 and August 2014, Tehran-Iran. All patients met the diagnostic criteria for sarcoidosis established by the American Thoracic Society consensus panel . The incSpecific phenotypes of sarcoidosis were defined according to the ACCESS group . A summa95 controls were recruited as described previously  by randoHeparinized blood was collected from each case and control at the time of the interview and was sent by overnight courier to the DNA core laboratory for DNA isolation and purification. High molecular weight DNA was isolated from non-coagulated blood by detergent lysis and organic extraction. Purified DNA samples were diluted to a standard concentration (1 μg/ml) in 10 mMTris, 5 mM EDTA buffer and being frozen at −70°C until analysed. DNA integrity and concentrations were monitored using agarose gel electrophoresis and ethidium bromide staining.Determination of HLA-DR B1 subtypes was performed using single specific primer-polymerase chain reaction (SSP-PCR) methods .The PCR products were loaded onto a 0.5% agarose gel and the expression of various alleles was visualized by Gel DOC . HLA-DR subtypes were calculated according to the manufacturer’s instructions. Only primers that completely matched the target sequences result in amplified products under the controlled PCR conditions and the presence of an amplified DNA fragment is a positive indication of the existence of an allele-specific sequence in the genomic DNA.IL-8 concentrations were quantified by ELISA  according to the manufacturer’s instructions. Serum levels of TNF-α, IL-6 and IL-1β were also measured by ELISA kits  according to the manufacturer’s instructions.et al. as described previously [Total lambda (λ) and kappa (κ) FLC concentrations were determined in all sera using an ELISA adapted from Abe eviously ,44. In bt-test using GraphPad Prism (version 2.01). Results were considered statistically significant when p < 0.05.All conditions were performed in triplicate, and all experiments were repeated up to five times. Results are presented as mean ± S.E.M. Data were compared using the unpaired 2-tailed, student’s HLA-DRB1 *7 allele was significantly different between pulmonary (17.7%) and EPS subjects   and EPS patients   Table . In addi5) Table  and thisThe serum levels of IL-8 were significantly increased in both pulmonary and EPS patients compared with control subjects (Figure Serum TNF-α levels in pulmonary sarcoidosis patients were significantly increased compared with control subjects, but no increase was observed in patients with EPS (Figure There was a significant increase in serum κ (Figure In this study we investigated whether HLA-DRB * sub-typing could distinguish between sarcoidosis patients with pulmonary or extra-pulmonary involvement. We found that among the 16 HLA DRB alleles only *7 and *12 were different between sarcoidosis patients and controls with *12 being identified as a risk factor for sarcoidosis . This stAmong the alleles tested, only the expression of the *07 and *12 were significantly altered in sarcoidosis with the frequency of *07 being decreased whereas that of *12 being increased compared with control subjects. It has previously been reported that the presence of the HLA-DR B *7 allele is related to a good disease prognosis ,47. IndeVariations in HLA-DRs associations have also been reported in sarcoidosis patients across different ethnic groups. For example, although the frequency of HLA-DRw52 was significantly greater in Japanese sarcoidosis patients compared to control groups there was no significant association with HLA-B antigens . In addiWe also report enhanced levels of serum IL-8 in both pulmonary sarcoidosis and EPS patients. To our knowledge there has only been one previous report showing elevated IL-8 levels in sarcoidosis . Car andIn addition, serum levels of TNF-α were increased in patients with pulmonary sarcoidosis but not in those with EPS. TNF-α is a pro-inflammatory cytokine previously shown to play a critical role in the pathogenesis of Th1 responses and to be elevated in sarcoidosis patients . TNF-α pHowever, the failure to demonstrate increased serum levels of IL-1β and IL-6 in sarcoidosis requires further investigation. This may result from the involvement of distinct cell types present in blood and BAL since these cytokines and chemokines are all stimulated by the same stimuli involving NF-κB activation . This maWe also report elevated levels of both κ and λ FLC in the serum of EPS patients but not patients with pulmonary sarcoidosis. Sarcoidosis is known as a T cell mediated disease but B-cells may play a role in its pathogenesis particularly in chronic disease ,65. It iIn conclusion, we report for the first time that HLA DRB *12 may be indicative of sarcoidosis and that there is a differential HLA DRB allele usage between patients with EPS and pulmonary sarcoidosis. There were also differences in serum inflammatory markers between these two groups of sarcoidosis patients with patients with pulmonary sarcoidosis having greater levels of TNF-α and IL-8 than EPS and control subjects. Conversely, the expression of κ and λ FLCs was greater in EPS patients compared with pulmonary sarcoidosis patients. Overall, we report a link between HLA-DRB sub-types and inflammation in Iranian patients with sarcoidosis. We suggest that these novel markers should be readily incorporated into current laboratory analysis as they are relatively cheap and rapid, taking less than 48 h, and provide additional parameters for the differentiation of subtypes of sarcoidosis.


Perceiving social information such as the cooperativeness of another person is an important part of human interaction. But can people perceive the cooperativeness of others even without any visual or auditory information? In a novel experimental setup, we connected two people with a rope and made them accomplish a point-collecting task together while they could not see or hear each other. We observed a consistently emerging turn-taking behavior in the interactions and installed a confederate in a subsequent experiment who either minimized or maximized this behavior. Participants experienced this only through the haptic force-feedback of the rope and made evaluations about the confederate after each interaction. We found that perception of cooperativeness was significantly affected only by the manipulation of this turn-taking behavior. Gender- and size-related judgments also significantly differed. Our results suggest that people can perceive social information such as the cooperativeness of other people even in situations where possibilities for communication are minimal. Perceiving social information of an interaction partner is crucial for human interaction . EspeciaHow do people perceive the cooperativeness of others if possibilities to exchange visual or auditory information are limited during real-time interactions? Theories of human cooperation would predict that humans have clear beliefs and expectations about the behavior of others, so that individuals complying with these expectations would be seen as more cooperative . Humans n = 8), we observed a consistently emerging reciprocal “turn-taking behavior.” Although multiple coordination patterns were possible, it seemed as if participants were automatically expecting their partners to collect their points alternately and take turns. We wanted to test whether this turn-taking behavior was associated with perception of cooperativeness and installed a confederate who either maximized or minimized this behavior. Furthermore, do participants make person-related inferences about their interaction partner even when possibilities for communication are minimalistic?In the present study, we connected two participants with a rope, and they had to accomplish a joint task together. The task consisted of collecting points on a laptop computer which was located on each side and could only be reached if both interaction partners coordinated their movements . Importaf = 12, m = 12) who interacted with the same confederate in two sessions while they believed that they interacted with different partners in the two sessions. The confederate maximized “turn-taking”  in one and minimized “turn-taking”  in the other session. The order of the sessions was balanced. After each session, participants rated the cooperativeness of the interaction partner on a Likert scale (1–7) and gave estimates about person-related attributes such as the gender and size of their interaction partners.We tested 24 participants (t(23) = −7.11, p < .001) depending on whether the interaction partner was engaging in “turn-taking behavior”  or not  = −3.53, p < .01). The perceived body size of interaction partners was also significantly different dependent on the partner’s behavior (t(24) = 1.29, p < .001).Moreover, participants made different person-related inferences about the gender and size of the confederate . CooperaIn summary, our results suggest that people collect social information and make inferences about their interaction partner even when possibilities for communication are minimalistic. It is noteworthy that a reciprocal “turn-taking behavior” automatically emerged in the interactions and manipulating this behavior influenced the perception of cooperativeness as well as person-related inferences of the partner. Furthermore, our experimental paradigm of directly connecting two individuals and only allowing haptic force-feedback could serve as a useful method to further study how joint motor coordination is coupled to the perception of social information.


We have studied the finite-size effect on the dynamic behavior of graphene resonators and their applications in atomic mass detection using a continuum elastic model such as modified plate theory. In particular, we developed a model based on von Karman plate theory with including the edge stress, which arises from the imbalance between the coordination numbers of bulk atoms and edge atoms of graphene. It is shown that as the size of a graphene resonator decreases, the edge stress depending on the edge structure of a graphene resonator plays a critical role on both its dynamic and sensing performances. We found that the resonance behavior of graphene can be tuned not only through edge stress but also through nonlinear vibration, and that the detection sensitivity of a graphene resonator can be controlled by using the edge stress. Our study sheds light on the important role of the finite-size effect in the effective design of graphene resonators for their mass sensing applications. L), i.e., . The high-frequency dynamic range of nanomechanical resonators has enabled them to be used for development of lab-on-a-chip mass spectrometry [1 − cos(2πy/L)], which satisfies the essential boundary conditions [When a graphene resonator is actuated by an ac voltage, the electrostatic force nditions .xm, ym), the inertia becomesFor the case in which atoms are adsorbed onto a graphene resonator, we need to update the coefficient μ, while the other parameters of the Duffing equation are not affected by atomic adsorption, since the atomic adsorption only affects the inertia term dictated by the coefficient μ. When atoms are adsorbed onto a specific graphene site, the coordinates of which are given by  is the Dirac delta function. For the case in which atoms are adsorbed uniformly onto the entire area of a graphene sheet, the inertia term can be written aswhere Δm + Δm) − ω(m), where ω(m + Δm) is the resonant frequency of a graphene resonator onto which atoms are adsorbed, and ω(m) is the resonant frequency of a bare graphene resonator. Here, we note that since the elastic modulus of adsorbed atoms is much smaller than that of the graphene resonator, the frequency shift of the graphene resonator is mostly attributed to the mass of adsorbed atoms [Here, Δρ is the mass per unit length for adsorbed atoms. It is straightforward to compute the frequency shift (Δω) of a graphene resonator due to atomic adsorption such as Δω = ω of graphene and its resonant frequency (ω0) is given as [In order to validate our model, we computed the effective elastic modulus of a graphene sheet as a function of its size based on the harmonic oscillation of graphene. Specifically, we consider the vibration of a graphene sheet actuated by a force of graphene . The relgiven as ,45Eeff, h, L, ρ, and ν are the effective elastic modulus, thickness, length, mass density, and Poisson’s ratio of graphene, respectively, and ξ is a constant eigenvalue depending on the boundary conditions. Eeff – Ebulk)/Ebulk, where Ebulk is the elastic modulus of an infinitely long graphene sheet, as a function of the size of graphene sheets and their various edge structures. This dimensionless quantity shows how the edge stresses affect the elastic modulus of graphene. When the size of the graphene sheet with edge structures of AC-ZZ or ACH-ZZ57 decreases, the elastic modulus increases. This is attributed to the positive elastic moduli (Yxx and Yyy) of the edges. On the other hand, when the size of an AC56-ZZH graphene sheet decreases, its elastic modulus does as well, because of the negative elastic moduli of the edge. In addition, it is found that the edge eleasic modulus plays a crucial role in the elastic properties of graphene sheets when their size is L < 25 nm . It is shown in eff) of graphene can be asymptotically given byNow, we investigate the role that the edge stresses play in the harmonic oscillation behavior of a graphene resonator. Here, we define the frequency shift caused by edge stress as Δω0 is the bending rigidity of infinitely long graphene . As a consequence, the frequency change of graphene due to edge stress is represented in the form ofwhere κThe scaling law of the frequency shift due to edge stress elucidates that the frequency shift due to edge stress increases with decreasing the size of graphene. This theoretical prediction is consistent with our simulation results , which sTo quantitatively understand how the edge stress affects the dynamic behavior of a graphene resonator, we consider the resonant frequencies of two types of graphene resonators, the edge structures of which are given as AC-ZZ and AC56-ZZH. We measured the frequency differences between these two graphene resonators as a function of their length scales. We have studied the effect of edge stress on the nonlinear vibration of a graphene resonator. Nonlinear deformation can easily occur because the transverse deflection of a graphene sheet during its vibration is typically much larger than the thickness of the sheet. In addition, as described in –2426, th24p0 > 1 fN, at which the nonlinear vibration of graphene can be observed [p0 when the size of the graphene becomes L > 20 nm. The frequency shift of a small-scale graphene sheet (L < 20 nm) due to edge stress exhibits no nonlinear behavior. Moreover, we found that as the actuation force p0 increases, the frequency shift of a large-scale graphene resonator (L > 20 nm) due to edge stress is decreasing as the actuation force increases. This indicates that nonlinear vibration can reduce the effect of edge stress on the frequency dynamics of a graphene resonator. These observations indicate that the dependence of the frequency dynamics of graphene resonators on the edge stress is determined by two physical parameters such as the graphene size, L, and actuation force, p0. In particular, in case of L > 20 nm and p0 > 1 fN , the edge stress does not have any effect on the frequency dynamics of the graphene resonator.We consider the vibration of a graphene sheet actuated by a force of observed . It occuThis effect of nonlinear vibration on the edge stress-dependent frequency dynamics of a graphene resonator can be elucidated with using our modified plate theory . The nonp0, which is attributed to the edge elastic modulus that can annihilate the nonlinear effect. This theoretical prediction is consistent with the simulation results  is unable to enhance the detection sensitivity. The improvement of the detection sensitivity through nonlinear vibration is only possible when the length of the resonator is larger than 20 nm.In this work, we have studied the finite size effect on the dynamic behavior and the sensing performance of a graphene resonator with using a modified plate theory, which includes the effect of edge stress based on an energetic model. It is found that the edge stress plays an important role in the dynamic behavior and sensing performance of graphene resonators, whose length scale is below 20 nm. Moreover, it is shown that nonlinear vibration does not improve the detection sensitivity of a small-scale graphene resonator , whereas the edge stress is a key parameter that determines the dynamic behavior and sensing performance of the small-scale graphene resonators. Contrarily, for a large graphene resonator , the nonlinear vibration has a critical impact on the dynamic behavior and sensing performance, while these two properties are almost independent of the effect of edge stress. It should be noted that this work based on the modified plate theory is restricted to studying only the resonant frequencies of graphene sheets, while this current work may not be applicable for understanding the Q-factor. Our model does not include the intrinsic (flaw) factors, which affect the Q-factor of a graphene resonator. In order to understand the effect of edge stress on the Q-factor of a graphene resonator , the the