A stable modulation depth up to −23.26 dB can be achieved. Due to the cooperative effect of the “bright-bright” elements, the amount of the gold strips in each unit cell determines the number of the stop-bands, providing a simple and flexible approach to develop multispectral devices. Further investigations illustrate that the location of the stop bands not only can be adjusted by varying the length of gold strips, but also can be dynamically controlled by tuning the Fermi energy level of graphene, and deep modulation is acquired through designing the carrier mobility. With the sensitivity as high as 2393 nm/RIU of the resonances to the varieties of surrounding medium, the structure is also enabled to be an index based sensor. The results will benefit the on plane or integrated micro-structure research with simple structure and flexible tunability, and can be applied in multi-band stop filters, sensors and other graphene-based multispectral devices.Dynamically tunable band stop filter based on metal-graphene metamaterials is proposed and numerically investigated at mid-infrared frequencies. The proposed filter is constructed by unit cells with simple gold strips on the stack of monolayer graphene and the substrate of BaF By controlling the geometry of microelements in the metamaterial structures, the electric permittivity and magnetic permeability can be arbitrarily designed. Recently, the combination of metamaterials and plasmonics reveals an analogous physical phenomena of EM induced transparency (EIT), which is termed as plasmon induced transparency5 (PIT). The unit cell of PIT based metamaterials usually consists of bright mode and dark mode resonators10. The bright mode or super-radiant mode can be excited directly by an external incident field, and has a large scattering cross section5. In contrast, the dark mode or sub-radiant mode cannot be directly excited, but it can be excited by the local field of the bright mode via near field coupling and exhibits a larger quality factor due to the weak radiation coupling11.Electromagnetic (EM) metamaterials, a kind of artificially structured EM materials, are usually composed of periodically arranged subwavelength microstructures or ‘meta-molecules’, and have attracted great attention for their unique EM properties that the natural materials cannot realize. Some of the most typical meta-molecules are the metal split ring resonators14 and the bright to dark mode coupling16, but usually come with an unsatisfactory modulation depth. Moreover, the optical properties of metamaterials based on metallic structures cannot be changed once the structure is fabricated. To improve above situation, some actively controlled elements, such as liquid crystals19, liquid metals21 and semiconductors14, are applied in these metamaterials devices. In recent years, graphene, a flat monolayer of carbon atoms packed into a dense 2D honeycomb crystal lattice, has become a very promising material for its unique electrical properties24, such as high electron mobility, flexible tunability, relatively low loss and tight field confinement. The most attractive one among these properties is that the conductivity of graphene can be dynamically tuned by changing the Fermi energy through chemical doping26 or electrostatic gating27. Therefore, the combination of graphene and metamaterials can provide an effective approach to improve the dynamically tunable resonant properties of the multispectral devices31.Metamaterials with multiple PIT windows have many promising applications in optical information processing systems, effective switchers and ultrasensitive sensors. Traditional PIT based multispectral devices are usually designed by two approaches: the bright to bright mode couplingIn this work, by introducing a monolayer of graphene into a simple metamaterial structure, dynamically tunable dual-band and multi-band stop filters are proposed and researched. With two gold strips acting as typical bright mode resonators, a dual-band stop filter with an ideal modulation depth as high as −23.26 dB is achieved. To reveal the cooperative effect of the bright mode resonators, the electric field and surface current distributions are further investigated. The number of the stop-band can be easily increased by adding the gold strips in each unit cell, hence a filter with multi-band stop is obtained. The dynamically tunable properties of the proposed filters are realized by adjusting the graphene Fermi energy, and the modulation depth can be designed through varying the carrier mobility. Furthermore, the proposed structure can also be used as an index based sensor for its ultra-high sensitivity to the surrounding medium. All these results demonstrated that the presented metal-graphene based metamaterials provide promising applications in multi-band stop filters, sensors and other graphene-based multispectral devices.2 substrate assuming the refractive index n = 1.46 are separated by a monolayer of graphene and a thin layer of SU-8 photoresist. As shown in Fig. w = 0.5 μm are symmetrically attached on the center of the unit cell and separated from each other by a small gap d = 0.2 μm. The lengths of the short and long gold strips are set as l1 and l2, respectively. The periods of the unit cell are 4.2 μm both on x and y directions. The top metallic pad serves as the electrodes along with the square metallic ring at the back of the dielectric layer to tune the Fermi energy level of the graphene by applying a gate voltage. The thickness of the gold strips and the SU-8 photoresist layer are 75 nm and 300 nm, respectively.The proposed dual-band stop filter is schematically depicted in Fig. 33:w, kB, e is the incident angular frequency, Boltzmann constant, reduced Planck constant and electron charge, respectively. T is the temperature in kelvin, which is fixed to 300 K in this study. The value of chemical potential μc is equal to that of the Fermi energy level Ef when kB < μc, which can be written as n is the carrier concentration and the Fermi velocity is 6 m/s. Hence the Fermi energy is determined by the carrier concentration, which is proportional to the gate voltage Vg on the graphene as n = εdε0Vg/eh, where εd and h is the permittivity and thickness of the insulated substrate material. The relaxation time μ is the carrier mobility. Recent literatures showed that the graphene carrier mobility could reach higher than 105 cm2V−1s−1 in the experiment34, and is set as 2050 cm2V−1s−1 corresponding to the relaxation time Ef = 0.1 eV.In the mid-infrared region, the complex conductivity of monolayer graphene, consisting of interband and intraband contributions, can be given by Kubo formulax-direction (p-polarized) is used as the normal incident light. Figure 5. As depicted by the dash curves in Fig. l = 2.8 μm (black) and 2.0 μm (red) show strong resonance dips at 34.6 THz and 43.3 THz, respectively, which are exactly corresponding to the two resonances of the proposed filter. So, different from the traditional PIT effect of near-field coupling between bright elements and black elements, both the short and long gold strips in the dual-band stop filter are act as bright elements, and the two deep transmission dips are actually resulted from the simple cooperative effect of the two bright elements.A plane wave polarized parallel to the x-component electric field Ex, y-component electric field Ey and the amplitude of electric field |E| with surface current density are calculated and depicted in Fig. In order to further demonstrate the cooperative effect of the gold strips in the proposed structure, distributions of l leads to a significant red shift of the resonance frequency, suggesting that the required stop band can be reached by designing the special length of the gold strips. Figure l2 increases from 2.4 μm to 2.8 μm, while the higher one remains unchanged for the fixed length of short gold strip. Similarly, the varieties of the short gold strip length l1 decreasing from 2.2 μm to 1.8 μm corresponds to a blue shift of the high-order stop band, while the location of the low-order stop band keeps as a constant because of the fixed length l2. The shift of the resonance frequency results a broader transmission band. All of the results are in good agreement with those in Fig. As we noted above, the length of gold strip plays a key role in the location of the transmission dip. To explore this relationship, the resonance frequencies of the single gold strip structure versus its length are displayed in Fig. A(f) = 1 − R(f) − T(f), where T(f) and R(f) is the transmission and reflection coefficient. As shown in Fig. Ef = 0.1 eV and 0.55 eV at the corresponding center frequencies of the low-order stop bands. As expected, stronger confined electric fields are observed around the long gold strip at the Fermi energy of 0.55 eV.As discussed above, the graphene conductivity is a function of the Fermi energy, which can be flexibly tuned by the applied gate voltage between two metallic pads. Figure τ ranging from 0.01 ps to 0.1 ps, corresponding to the values of the graphene carrier mobility μ changing from 1000 cm2V−1s−1 to 10000 cm2V−1s−1. It is evident that the increasing of graphene carrier mobility has no effect on the location of the resonance frequencies, but raises the peak of the transmission band contributing to the increment of the modulation depth, due to the reduction of the graphene absorption. Thus, by introducing the monolayer graphene into the filter, its stop bands can realize dynamical tunability through controlling the graphene Fermi energy, and then a relatively good modulation depth can be achieved by designing the carrier mobility of graphene.Though the structure couldn’t be dynamically adjusted by graphene carrier mobility in the experiment, the modulation depth can be further improved by choosing the monolayer graphene with a high value. When the Fermi energy of graphene is fixed as 0.1 eV, Fig. Note that the resonance frequencies of the proposed structure are sensitive to the refractive index of the surrounding medium. Figure l1 = 1.8 μm, l2 = 2.2 μm and l3 = 2.6 μm. Similarly, the four-band stop structure can be obtained simply by increase the number of gold strips to four as depicted in Fig. Finally, multispectral metamaterials are realized with the Fermi energy and relaxation time are fixed as 0.1 eV and 0.0629 ps, respectively. The diagram of the structure with triple-band stop are shown in Fig. In conclusion, dynamically tunable band stop filters based on metal-graphene metamaterials are investigated at mid-infrared frequencies. The calculated transmission spectrums exhibit broad stop bands, and the number of the stop-band can be increased simply by adding the gold strips in each unit cell. The center frequency of the stop band or the location of resonance not only can be adjusted by varying the length of gold strips, but also can be dynamically controlled through the graphene Fermi energy. The increment of graphene carrier mobility will enhance the amplitude of the transmission peak to realize low loss transmission in pass band. Furthermore, the proposed structure can also be used as an index based sensor for its high sensitivity to the surrounding medium. The results will benefit the on plane or integrated micro-structure research with simple structure and flexible tunability, can be applied in multi-band stop filters, sensors and other graphene-based multispectral devices.x and y directions is performed with the periodic boundary condition. A p-polarized plane wave is used as the incident light. The conductivity of the gold is described by the Drude model with the plasma frequency wp = 1.36 × 1016 rad/s and the scattering rate Γ = 3.33 × 1013 rad/s in this work. The monolayer graphene is modeled as a surface current in the boundary conditions, which is defined as the product of graphene conductivity and the electric field in the frequency domain. The surrounding medium are assume to be uniform with the refractive index nr = 1. The transmission coefficient T(f) and reflection coefficient R(f) are calculated from S-parameters in the simulated results, and given as T(f) = |S21|2, R(f) = |S11|2 respectively.The proposed structures are numerically calculated using finite element method (FEM). In the three-dimensional simulations, Floquet ports with perfectly matched layer (PML) absorbing boundary conditions are used in the z-direction, and the unit cell both in


Hydrophilic graphene sheets were synthesized from a mixture of magnesium and gallic acid (GA) modified graphene oxide (GO) in a self-propagating high-temperature synthesis (SHS) process, and hydrophilic graphene sheets displayed the higher C/O ratio (16.36), outstanding conductivity (~88900 S/m) and excellent water-solubility. GO sheets were connected together by GA, and GA was captured to darn GO structure defects through the formation of hydrogen bonds and ester bonds. In SHS process, the most oxygen ions of GO reacted with magnesium to prevent the escape of carbon dioxide and carbon monoxide to from the structure defects associated with vacancies, and GA could take place the high-temperature carbonization, during which a large-area graphene sheets formed with a part of the structure defects being repaired. When only GO was reduced by magnesium in SHS process, and the reduced GO (rGO) exhibited the smaller sheets, the lower C/O ratio (15.26), the weaker conductivity (4200 S/m) and the poor water-solubility because rGO inevitably left behind carbon vacancies and topological defects. Therefore, the larger sheet, less edge defects and free structure defects associated with vacancies play a key role for graphene sheets good dispersion in water. When N2H4 was used as reducing agent, N can be doped in the as-prepared graphene. Although NaBH4 showed excellent ability in GO reduction, the hydrolysis property made it difficult to get a stable NaBH4 aqueous solution2 elimination from the carbon lattice to form the atomic vacancies or graphene fragment, which cause the structural deterioration of as-prepared grapheneKnown as GO rich in oxygen containing groups such as hydroxyl, carbonyl and epoxy group, and the chemical reduction of GO is the most efficient method for low-cost and large-scale production of graphene121314–4~10−3 m/s), generation of high temperatures , and rapid heating (103~104 K/s). The SHS technique is also characterized by process simplicity, short reaction time, easy-to-build equipment, low energy requirements, and the possibility of obtaining complex or metastable phases. GA has three hydroxyl and one carboxyl groups in its benzene ring which has the same oxygen-containing groups with GO. The network holes of GO can be darn with GA by hydrogen bonds and ester bonds, and GO sheets can also be connected together by gallic acid. In this paper, GO was modified by GA to form GOGA, and GOGA was further reduced by magnesium in SHS process under air atmosphere condition 4 (8.6)4 (12)GA and rGO, indicating that the delocalized π-conjugated structure was well restored in rGOGA and rGOGA was higher than that of rGO, but rGOGA displayed much better hydrophilic than rGO. Those results are far beyond our expectations. A hypothesis was proposed that graphene composition is not the main factor to influence the hydrophilic or hydrophobic property, but its structure characteristics must play a key role on its hydrophilic or hydrophobic property.In order to investigate the reasons for excellent water-solubility of rGOvey scan , and no GA, rGOGA and rGO. A series of absorption bands ranging from 1000 to 1750 cm−1 exhibited the presence of C=O bonds (at 1720 cm−1), O–H bending (at 1621 cm−1), O-H deformation (at 1406 cm−1), C-O groups in epoxy (at 1221 cm−1) and C-O groups in alkoxy (at 1057 cm−1)29−1, due to the presence of moisture intercalated within hydrophilic GO sheetsGA), the characteristic bands for oxygen containing groups did not change remarkably, but some new FT-IR absorptions of GOGA appeared in the range of 1490–1190 cm−1, which indicated that gallic acid molecules were adsorbed or darned on GOGA is shifted by 44 cm−1 from 1720 cm−1 to 1676 cm−1 compared to GO, corresponding to the formation of O=C-O-C groups, which demonstrates that a portion of gallic acid are combined with GO by esterification. After samples undergo magnesium self-propagating high temperature synthesis process, the characteristic bands for oxygen-containing groups of rGO and rGOGA almost completely disappeared, but the bands of aromatic carbon  at 1575 cm−1 and (Larger π-conjugated C=C structure) at 1560 cm−1 appeared. These observations suggested that GO and GOGA were well reduced, and the larger-area π-conjugated structure was established in rGOGA.GA, rGOGA and rGO were further characterized by Raman spectroscopy. As shown in −1) and G band (~1585 cm−1), which are E2g vibration mode in-plane and A1g breathing modeGA was shifted by 7 cm−1 from 1589 to 1582 cm−1, displaying a high reduction degree2 cluster regions is the inverse of the ratio of the D and the G band integrated intensities (ID/IG)3234D/IG ratio was 0.89, and the ID/IG ratio of GOGA increased to 0.91. However, ID/IG ratio of rGO was 0.97, rGOGA ID/IG ratio declined to 0.89. From those results, the following conclusions can be drawn: (1) the most oxygen ions of GO reacted with magnesium to form MgO, reducing the number of carbon atoms vacancies of the carbon lattice; (2) the average size of the sp2 domains of rGOGA was larger than rGO slightly; (3) gallic acid can repair the structure defects of graphene during magnesium thermal reaction. Apart from D band and G band, there are two Raman bands with weaker intensity called 2D and D + G located at 2680–3000 cm−1 as shown in insets of et al.GA by comparing these two bands. Compared with rGO (or GO), the enhancement of I2D/ID+G ratio of rGOGA can be clearly observed, which suggests that the full recovery and the larger-area π-conjugated structure for rGOGA.GO, GOGA and rGO. GA shows an obvious shift to higher 2θ angles , suggesting that rGOGA was well ordered with two-dimensional sheets and well removal of surface functional groupsGA, a weak shoulder peak at 26.5° of rGO was found, which revealed a small portion of the reduced graphene sheets agglomerated together or formed graphite structureXRD was used to characterize graphite, GO, rGOGA and rGO red shifted to 264 nm owing to the restoration of the conjugated structure, which indicated the removal of oxygen-containing functional groups and the disappearance of n→π* transition band41GA formed with a part of the structure defects being repaired during a SHS process.Samples were also characterized by UV-vis absorption, as shown in 373839GA. By comparison, GA was adsorbed on the surface and edge of GO by the interaction between GA molecule and defects of GO  as precursor. The merit of this method is that the process carried out in the air atmosphere without external heating source, and gallic acid can repair the structure defects of graphene during SHS process. The obtained graphene sheets could be stably dispersed in water for more than one month. The wrinkles, scrolls, irregular structure defect associated with vacancies and grain boundaries (rather than C/O atomic ratio) determine the hydrophilic property. While the less edge defects and free structure defects associated with vacancies play a key role for graphene sheets good dispersion in H2O. Moreover, the synthetic process can be utilized to produce large-scale graphene by an environmentally friendly way. Obviously, these advantages will facilitate its industrial applications.In conclusion, a novel and facile approach to the synthesize graphene sheets based on magnesium SHS process is reported using GO modified by gallic acid (GO3 (99.8%), KMnO4 (99.5%), H2SO4 (98%), HCl (35%), magnesium powder and magnesium ribbon et al. were purchased from Tianjin Kermel Chemical Reagent Co., Ltd.Natural graphite powder (80 mesh) was supplied by Tianjin Chemical Co., Ltd and used as received. NaNO−1. Raman spectra were obtained by using a micro-Raman system on  with an excitation energy of 2.41 eV (l = 514.5 nm). XRD patterns were measured on a Bruker XPS (Germany) using Cu Kα radiation. UV-vis detection was carried out by UV/Vis Spectrometer . TEM and HRTEM images were obtained from a JEM-2100 transmission electron microscope . Electrical conductivity of samples was measured on a ST2722 semiconductor powder resistivity tester . X-ray photoelectron spectroscopy (XPS) analysis was carried by .FT-IR was recorded from a TESOR 37  and operated by attenuated total reflectance (ATR) in the wavenumber range of 4000–500 cm3, and 125 ml H2SO4 were loaded in a 1000 ml round-bottom flask with magnetic stirring. After being cooled with ice-water bath at 0 °C for 1 h, 17.5 g KMnO4 was added into above reaction system. After reaction was kept at 0–5 °C for 1 h and then heated at 35 °C for 2 h, the mixture was diluted with 250 ml deionized water and reacted at 98 °C for 45 min. 200 ml deionized water and 50 ml H2O2 (30%) were added into above suspension under the condition of magnetic stirring. After centrifugation and dry, GO was obtained.GO was prepared by modified Hummers’ methodGA was prepared in three steps: (1) 1.5 g gallic acid was loaded into 500 ml GO (5 mg/ml) solution, and the mixture was dispersed by ultrasound for 40 min. (2) the mixed solution was allowed to stand at room temperature for 3 h. (3) The mixed solution was dried at 90 °C under air atmosphere for 24 h to obtain the mixture of gallic acid-modified GO (GOGA). As a control, different addition amount of gallic acid (1.0 g and 2.0 g) in equal mass of GO (were labeled as GOGA1 and GOGA2) were prepared with the same method.Exfoliation of GO was obtained by ultrasonication in water for 2 h. GOGA powder were mixed evenly. The mixture was put in a crucible, and mixture was covered with a thin layer of magnesium powder on the surface of mixture. Mixture was ignited by magnesium ribbon under air atmosphere condition. After the reaction is over, the mixed products were transferred to a beaker containing 250 ml of 3 M HCl. Products were transferred into the acid solution to remove MgO and residual Mg. The resulting rGOGA was collected with filtration, washed with deionized water until the filtrate turned out to be of neutral. Obtained rGOGA was dried at 60 °C under high vacuum conditions for 10 h. As a control, rGOGA1, rGOGA2 and rGO were prepared from GOGA1, GOGA2 and GO reduction by magnesium with the same methods.3.0 g Magnesium powder and 1.5 g GOThe contact angle of the samples was measured on DSA 100 (KRUSS) contact angle system at ambient temperature. Before the measurement, 20 mg power samples were dispersed in 500 ul ethanol with ultrasonic treatment 1 h. In the following, the suspension was dropped onto a glass slide to form a uniform film.Electrical conductivity of samples was measured on a ST2722 semiconductor powder resistivity tester . The powder sample was filled into a test slot, then applying a pressure about 18 MPa. The electrical resistivity tester would reflect the electrical resistivity of the sample directly. The electrical conductivity of samples was calculated through the following equations:How to cite this article: Cao, L. et al. Hydrophilic Graphene Preparation from Gallic Acid Modified Graphene Oxide in Magnesium Self-Propagating High Temperature Synthesis Process. Sci. Rep.6, 35184; doi: 10.1038/srep35184 (2016).


The combination treatment also produced synergistic increase in P-ERK1/2 level in spleen. Immunohistochemistry shown that SEP induced the PD-L1 expression in melanoma tissue possibly by promoting IFN-γ excretion, which led to the synergistic anti-tumor effects of aPD-L1 and SEP. Furthermore, in the purified T lymphocyte from the naive mice, the combination of SEP and αPD-L1 had more potent than SEP or αPD-L1 in promoting T lymphocyte proliferation and cytokines secretion including IL-2 and IFN-γ, at least partially by activating MEK/ERK pathway. Our study provides the scientific basis for a clinical trial that would involve combination of anti-PD-L1 mAb and SEP for sustained melanoma control.The increased PD-L1 induces poorer prognosis in melanoma. The treatment with PD-1/PD-L1 antibodies have a low response rate. The combination immunotherapies are the encouraging drug development strategy to receive maximal therapeutic benefit. In this study, we investigated the enhanced antitumor and immunomodulatory activity of combined SEP and αPD-L1 in B16-F10 melanoma-bearing mice. The results shown that combined SEP and αPD-L1 presented significant synergistic antitumor effects, increased the frequency of CD8 The average survival time was 6–12 months and a 5-year survival rate under 10% with traditional therapies2. Recently, due to the profound understanding of immunobiology for melanoma development, immunotherapies have become the standard treatment regimens for the patients with advanced melanoma2.Melanoma comprises only 5% of all skin cancers, but approximately 80% of all skin cancer-related deaths are caused by melanoma3. However, the coinhibitory receptors such as anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death 1 (PD-1) are able to down-regulate the immune system by preventing T cell over-activation, promoting self-tolerance and avoiding autoimmunity4. After expressed approximately 48 h after T cell activation, CTLA-4 binds to B7-1 in limiting T cell activation2. When the activated T cells enter tumor microenvironment, they become “tolerated”  by engagement of PD-1/B7-H1 (PD-L1) signaling pathway5. Therefore, these negative regulation mechanisms decrease T cell anti-tumor activity in cancer immunotherapy. So far, the immunotherapeutic drugs such as CTLA-4 antibody (ipilimumab) and PD-1 antibodies (pembrolizumab and nivolumab), have been approved for the treatment of the advanced melanoma by the US Food and Drug Administration (FDA)2. In addition, the PD-1 receptor ligand (PD-L1) antibodies (BMS-936559 and atezolizumab) have demonstrated promising activity for treating melanoma in preclinical mouse models and clinical trials6. Furthermore, concurrent CTLA-4 and PD-1/PD-L1 inhibition and combination with other immunotherapeutic strategies, have been a promising approach for the melanoma patients with the increased benefits7. In a randomized phase III trial in treating patients with metastatic melanoma, an increased response rate and improved progression-free survival were observed with the ipilimumab-nivolumab combination when compared with ipilimumab alone8.T lymphocytes play a critical role in cell-mediated immunity and cancer immunotherapy. The classic two-signal activation model includes both TCR signaling pathways and CD28/B7 costimulatory pathway9. Several immunoceuticals composed of polysaccharides have been used for treating cancers such as lentinan, schizophyllan and krestin9. These polysaccharides are be characterized by low toxicity and limited side effects10.Many polysaccharides, isolated from mushrooms, fungi, yeasts and plants, have attracted more attention recently due to their immunomodulatory and anti-cancer effects9. Strongylocentrotus nudus egg polysaccharide (SEP), a D-glucan containing an a-1,4-linked backbone and a-1,6-linked branches, was isolated and purified from Strongylocentrotus nudus eggs11. In our previous studies, SEP prevented the growth of both S180 and H22 hepatocellular carcinomas in vivo by enhancing splenocyte proliferation, CD4+ and CD8+ T cell numbers as well as cytotoxic T lymphocyte (CTL) activity, and increasing IL-2 and TNF-α secretion levels in the serum12. The studies also found that SEP regulates intracellular signaling pathway associated with splenocytes proliferation and cytokine expression12.Sea urchins belong to the echinoderm phylum. Sea urchin eggs are a kind of favorite seafood for their good taste and high nutrition in ChinaAccording to the above studies, SEP can activate T cells possibly by mediating signaling pathway in lymphatic tissues, while the PD-L1 antibodies can upragulate T-cell effector function by blocking PD-1/B7-H1 (PD-L1) signaling pathway in peripheral tissues including the tumor microenvironment. Therefore, we hypothesize that the combined treatment with SEP and anti-PD-L1 mAb produce an additive and even synergistic antitumor effect via immuneregulation in melanoma.in vivo tumor growth, T lymphocytes counts, CTL cytotoxicity and cytokines expression. We also investigated the potential effects of combination treatment on MEK/ERK signaling pathway in spleen. In addition, by using the purified T lymphocyte from the untreated mice, we studied the effects of MEK/ERK signaling pathway on the T lymphocyte proliferation and cytokines secretion induced by the combined SEP and αPD-L1 in vitro.In the present study, we investigated the enhanced antitumor and immunomodulatory activity of combined SEP and anti-PD-L1 mAb in B16-F10 melanoma-bearing mice via analyzing in vitro. As expected, neither SEP or αPD-L1 mAb alone, nor combination could inhibit B16-F10 cells growth compared with the control group   and tumor volumes  compared with the control group  and tumor volumes . Furthermore, we noticed that combination of SEP with αPD-L1 showed more potent than SEP or αPD-L1 on reducing tumor weights  and tumor volumes .We examined the oup Fig. . αPD-L1 P < 0.05 at every ratio, n = 3). Furthermore, combination of SEP and αPD-L1 resulted in the additive upregulation of cytotoxicity activity of splenocytes in B16-F10-bearing mice compared with SEP  or αPD-L1 .CTL plays an important role in inhibiting tumor cell. Therefore, we measured the cytotoxic activity of splenocytes against B16-F10 cells. The results showed that the cytotoxic activity of splenocytes at E/T ratios of 10:1, 20:1 and 40:1 were increased in three treatment groups in B16-F10-bearing mice compared with the control group . Furthermore, the combination of SEP and αPD-L1 led to significantly additive increase of CD3+, CD4+ or CD8+ T cells in spleen compared with SEP  or αPD-L1 .To investigate the effect of each treatment on the cellular immunity, the cell counts for CD3+, CD4+ or CD8+ T cells was also observed in three treatment groups compared with the control group   or αPD-L1 .Similarly, in tumor, the increased percentage of CD3P < 0.05 for each cytokine, respectively, n = 6). Furthermore, combination of SEP and αPD-L1 promoted significantly additive increase of IFN-γ and IL-2 levels compared with SEP  or αPD-L1  in spleen . Furthermore, combination of SEP and αPD-L1 significantly increase IFN-γ and IL-2 levels compared with SEP  or αPD-L1  in tumor . Furthermore, combination of SEP and αPD-L1 had more potent than SEP  or αPD-L1 . The results suggests that these facilitated the T cell proliferation and cytokines in B16-F10-bearing mice partially via the MEK/ERK signaling pathway.One of the key pathways in the cytokine expression involves the MEK/ERK signaling pathway in spleen. Therefore, we investigated the effect of the combination of SEP and αPD-L1 on the ERK1/2 phosphorylation. As shown in Fig. We used ICH methods to examine PD-L1 expression in melanoma cancer tissue. All immunostained tumor sections were examined by light microscopy, and images were photographed with a microscope camera  at a magnification of × 100. We found that the PD-L1 expression was significantly induced by SEP in melanoma cancer tissue compared with that in the control group Fig. . The αPDP < 0.05, respectively, n = 3). Furthermore, combination of SEP and αPD-L1 had more potent than SEP  or αPD-L1 . However, after the pretreatment with MEK inhibitor PD98059 for 30 min, the the increased ratios induced by SEP or/and αPD-L1 was partially decreased .To assess the role of MEK/ERK signaling pathway in T lymphocytes immunomodulation during SEP or/and αPD-L1 treatment, we used western blot analysis to detect the expression of ERK1/2 and p-ERK1/2 in T lymphocytes after 48 h exposure. Our results in Fig. P < 0.05, respectively, n = 6). Furthermore, combination of SEP and αPD-L1 had more potent than SEP  or αPD-L1 . However, after the pretreatment with MEK inhibitor PD98059 for 30 min, the T cell proliferation induced by SEP or/and αPD-L1 was partially inhibited .To investigate the effects of MEK/ERK signaling in T lymphocyte proliferation, CCK-8 kit was used. As shown in Fig. P < 0.05, respectively, n = 6). Furthermore, combination of SEP and αPD-L1 had more potent than SEP  or αPD-L1 . However, after the pretreatment with MEK inhibitor PD98059, the increased IL-2 and IFN-γ secretion in SEP or/and αPD-L1 groups was partially inhibited  secrete a series of cytokines, such as IL-2, IFN-γ and TNF-α, to enhance the production of CD8+ cytotoxic T cells9. The IL-2 can stimulate T cell proliferation and differentiation into mature CD4+ and CD8+ T cells to enhance the adaptive immunity16. The activated CD8+ T cells can concentrate to the tumor microenvironment and secrete some cytokines, including IFN-γ. The IFN-γ expression levels by CD8+ T cells are closely related with the specific cytotoxic activity for antitumor effects17. In our study, the ELISA results showed that combined SEP and αPD-L1 induced T cells to produce the additive IL-2 and IFN-γ increase in splenocytes and tumor from B16-F10 melanoma-bearing mice when compared with single treatment, which either promote proliferation and differentiation of immune cells to inhibit tumor growth or lead to cytotoxic effects to tumor cells18.CD419. Previous studies have demonstrated that many drugs can induce PD-L1 expression on tumor cells by increasing the IFN-γ excretion20. In our study, SEP can induce IFN-γ excretion in melanoma tumor tissue, which could inderectly stimulate the PD-L1 expression, which provide the rationale for combined cancer treatment of SEP and αPD-L1.We used ICH methods to examine PD-L1 expression in B16-F10 melanoma tumor tissue. The results shown that SEP significantly induced the PD-L1 expression in melanoma cancer tissue, which may partially responsible for the synergistic anti-tumor effects of aPD-L1 and SEP as tumor PD-L1 expression disabling the antitumor function of preexisting tumor antigen-specific T cells has been thought to be prerequisite for αPD-1/αPD-L1 mAb therapy to work21. However, blocking the PD-1/PD-L1 immune checkpoint pathway by anti-PD-1 or anti-PD-L1 antibodies, causes activation of T cells partially by increasing MEK/ERK pathway, promoting differentiation of effector and memory T cells, which suppresses cancer cell survival and enhances the antitumor responses of T cells21. Our previous study shown that SEP can activate MEK/ERK signaling pathway associated with T cells proliferation and cytokines excretion12. In the present study, the treatment with αPD-L1 or SEP in B16-F10 melanoma-bearing mice enhanced the ERK1/2 phosphorylation level in the spleen compared with the control group. However, the combination of SEP and αPD-L1 had synergistic effects in increasing ERK1/2 phosphorylation level in spleen, which allowed more effective T cells activation, proliferation and cytokines secretion such as IL-2 and IFN-γ, and further increase of antitumor effect.The actived PD-1/PD-L1 pathway inhibits the signaling pathways that lead to activation and expansion of T cells that recognize tumor antigens with the impaired generation of T effector and memory cells, which promote the survival of cancer cells and tumor tolerancein vitro from naive mice with/without MEK inhibitor PD98059 for 30 min, followed by 48 h of the coculture with the aPD-L1 and/or SEP with ConA. We found that the combination of SEP and αPD-L1 had more potent than SEP or αPD-L1 in the increase of P-ERK1/2 level. However, the pretreatment with PD98059 significantly reduced the increased P-ERK1/2 level induced by SEP or/and αPD-L1. Furthermore, SEP and αPD-L1 also significantly had more potent than SEP or αPD-L1 in promoting T cells proliferation and cytokines secretion including IFN-γ and IL-2. Similarly, the pretreatment with PD98059 significantly reduced T cells proliferation and cytokines secretion induced by SEP or/and αPD-L1. All these data indicated that the MEK/ERK signaling was at least partially responsible for T lymphocyte proliferation and cytokines secretion induced by SEP or/and αPD-L1.In order to further investigate the effects of MEK/ERK signaling in T lymphocyte proliferation and cytokines secretion, we pretreated splenocytes in vivo and in vitro in combination with αPD-L1 partially by activating the MEK/ERK signaling. Our study provides the scientific basis for a clinical trial that would involve combination of αPD-L1 and SEP for sustained tumor control in cancer patients.The antibodies targeting PD-1/PD-L1 pathways such as pembrolizumab and nivolumab has already been approved by FDA for treatment of melanoma. The αPD-L1 has demonstrated promising activity for treating melanoma in preclinical mouse models and clinical trials. It will be critical to increase the frequency of effector T cells and cytokines excretion in order to receive maximal therapeutic benefit. Our work suggests that administration of SEP may harness the therapeutic potential of effector T cells Strongylocentrotus nudus eggs as described previously11. Briefly, the crude, water-soluble polysaccharide from the eggs of sea urchins was separated and sequentially purified via Cellulose DE-52 and Sephacryl S-400 to yield the SEP for subsequent analysis. High performance liquid chromatography (HPLC) system with a TSK gel 4000 PWXL column and a Waters 2414 refractive index detector  was applied for analyzing the purity of SEP . The profile showed a single symmetrical and sharp peak with the purity of 98.0%. To remove endotoxin contamination, 1 ml of Affi-Prep Polymyxin Matrix was packed in a Bio-spin column; centrifuged at 200 × g for 2 min; and then 0.5 ml of either SEP (100 µg/ml), LPS (2 µg/ml) or a mixture of SEP (100 µg/ml) and LPS (2 µg/ml) was added. After an overnight incubation at 4 °C, the effluent was collected and centrifuged under the same condition. The endotoxin level in SEP was determined by using the E-TOXATE kit22.SEP was obtained from FITC-conjugated anti-CD3 mAb(#555274), FITC-conjugated anti-CD4 mAb (#553729) and PE-conjugated anti-CD8 mAb (#553033) were provided by BD Biosciences . Mice interferon gamma (IFN-γ) and interleukin-2 (IL-2) ELISA kits were purchased from R&D Systems, USA. The αPD-L1 antibody (clone 10 F.9G2) was bought form Bio-XCell (USA). ERK1/2 (SC-135900), P-ERK1/2 (Thr202) (SC-101760) and lipopolysaccharide (LPS) were from Santa Cruz Biotechnology . The anti-PD-L1 antibody  was from Cell Signaling Technology (USA) for immunohistochemistry (ICH). PD98059 (MEK inhibitor) was purchased from Calbiochem .® Number: CRL-6475™) was obtained from Cell Culture Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cells were maintained in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum in a humidified 5% CO2 atmosphere at 37 °C.Mouse melanoma cell line B16-F10  were used for 3 cells/well. After 24 h incubation, the cells were exposed to SEP and/or aPD-L1 at the designated concentrations and cultured at 37 °C in a humidified atmosphere for 48 h, 72 h and 96 h. 20 μL of MTT (5 mg/ml) was added to each well and incubated for additional 4 h at 37 °C. The medium was subsequently discarded, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a Molecular Devices Spectra Max M5 .To determine the cell viability of SEP and/or aPD-L1 on melanoma cell lines, cell survival was measured by MTT assay. Cells were plated in 96-well plate with a density of 5 × 105 cells mixed with matrigel into the dorsal area of male mice. On 2rd day, the mice bearing tumors were randomly divided into four groups (12/each group). Administration doses of SEP and aPD-L1 were designed according to the references14. Mice were intraperitoneally administrated with SEP at 16 mg/kg or aPD-L1 at 9 mg/kg with a volume of 0.1 ml/10 g for each drug. The mice in combination drug group were administrated with SEP at 16 mg/kg and aPD-L1 at 9 mg/kg. Control mice were given the same volume of saline. Figure 3) = 0.5 × length (mm) × width (mm) × width (mm). On day 16, all the mice were decapitated between 9:00 a.m. and 11:00 a.m. The tumors were obtained. And the inhibition rate (IR) of tumor growth was calculated by the following formula: IR (%) = [(A − B)/A] × 100, where A and B were the mean tumor weight in the control and treatment groups, respectively.B16-F10 tumors were established by injecting 1 × 1023. Briefly, B16-F10 cells were applied as target cells (T) and seeded in 96-well U-bottom microtiter plate at 1 × 104 cells/well in DMEM complete medium. The spleens were collected from treated mice in each group (n = 3) at the end of the experiment (day 16), and gently homogenized and passed through a 200-mesh sieve to obtain single-cell suspensions. After treatment with erythrocyte lysis buffer, the splenocytes were washed with phosphate-buffered saline (PBS) three times and resuspended in DMEM complete medium, which were used as effector cells (E). The ratios of effector cells to target cells were 10:1, 20:1, and 40:1. The plates were then continuously incubated for 20 h at 37 °C. 20 µl of MTT solution (5 mg/ml) was added to each well and the plates were incubated for another 4 h. To determine the percentage of the killed target cells, the following equation was used: CTL activity (%) = (ODT − (ODS − ODE))/ODT × 100%, where ODT, optical density value of target cells control, ODE, optical density value of effector cells control, and ODS, optical density value of samples.The cytotoxic activity of CTL was analyzed by MTT assay as previously described6 cells were used for antibody staining by using FITC-conjugated anti-CD3 mAb, FITC-conjugated anti-CD4 mAb, or PE-conjugated anti-CD8 mAb. The cell counts for the CD3+, CD4+ and CD8+ T lymphocytes were assessed via flow cytometry  and expressed as a percentage of the total number of lymphocytes.For flow cytometric analysis, spleen and tumor tissues were harvested at the end of the experiment (day 16). Single cell suspensions were prepared and a Ficoll-Hypaque purification step was carried out for the tumor-derived cell suspension. After the cells were washed twice with PBS and resuspended in 1% paraformaldehyde (PFA), about 2 × 10Mouse splenocytes in each group were obtained as described above. After incubation with LPS (4 μg/ml) for 48 h, the supernatants were collected for detecting IFN-γ and IL-2 levels using murine ELISA kits according to the manufacturer’s instructions. And IFN-γ and IL-2 levels in the tumor supernatants were measured by ELISA kits.The protein content of the spleen homogenates from the melanoma-bearing mice were measured by BCA method. The protein samples (20–30 μg per lane) were separated by SDS polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. These membranes were incubated with 1% bovine serum albumin in Tween-Tris-buffered saline and reacted overnight at 4 °C with the mouse or rabbits antibody (1:1000) against P-ERK1/2 and ERK1/2. After repeated washings, the immunoreactive bands were reacted with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody, then visualized by FluorChem™ Q chemiluminescent System . The O.D. value of reactive bands visible was determined densitometrically.2O for 10 min, and then counterstained with hematoxylin. After dehydration by using an ethanol series and xylene, each stained section was examined by light microscopy .The collected melanoma tumors were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin. Serial sections (4-μm) were prepared for ICH. After deparaffinization and antigen retrieval, the sections were preincubated in blocking serum, and then incubated overnight at 4 °C with the anti-PD-L1 antibody for ICH. Following incubation, the sections were washed three times in PBS and then incubated with biotin-conjugated secondary antibody for 2 h. Next, the sections were washed 3 times in PBS. Diaminobenzidine substrate was placed on each tissue section. After color development, the slides were immediately washed with H24. The T cells were cultured at 5 × 106–6 × 106 cells/ml in RPMI-1640 medium supplemented with 10% fetal calf serum. After the cells were pretreated with/without MEK inhibitor PD98059 (40 μM) for 30 min25, they were cultured with the aPD-L1 (10 nM) and/or SEP (50 μg/ml) with concanavalin A  for additional 48 h. Then the cell survival was determined by CCK-8 method, or the cell supernatants were collected, and IFN-γ and IL-2 levels were mesured by murine ELISA kits.T cells were purified from the spleens from naive C57BL/6NCrl mice, by using magnetic negative selection . The purity of T cells was more than 95% by as determined by flow cytometric analysisFor the effects on MEK/ERK signaling pathways, after the treatment with/wihtout PD98059 (40 μM) followed by aPD-L1 and/or SEP with ConA according to the above method, the T cells were collected and lysed in lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors . Western blot analysis was conducted for the detection of P-ERK1/2 and ERK1/2 expression accoriding to the above method.P < 0.05 was considered statistically significant.The statistical analyses were performed using one-way ANOVA, followed by LSD post hoc test in SPSS software (V 16.0).


P<0.001) but not with gender, primary site, or lymph node status. The PD-L1 positivity rate and signal intensity are directly correlated (P<0.001) with a grade increase from G1 to G3. In particular in G3 cases, we observed a dichotomy between the morphology (WD- and PD-NENs) and Ki67. Moreover, our study demonstrated a significant association with the grade and PD-L1 expression levels in immune-infiltrating cells (P<0.001). In particular, G3 tumors are characterized by strong PD-L1 expression in both the tumor and infiltrating immune cells (P<0.001), reflecting an unfavorable environment for T-cell-mediated tumor aggression. These findings suggest that NENs might acquire resistance to immune surveillance by upregulating PD-L1 and inhibiting peritumoral and intratumoral infiltrating lymphocytes. Here we demonstrate that PD-L1 is currently the best-known biomarker for G3 NENs, becoming the new gold standard for G3 NEN discrimination. Furthermore, pharmacological approaches using anti-PD-1 antibodies may become the logical choice for the treatment of G3 cases with a poor prognosis.Neuroendocrine neoplasms (NENs) are rare, heterogeneous and ubiquitous tumors commonly localized in the gastrointestinal tract, lung, and pancreas. The clinical behavior of NEN is highly unpredictable; in fact, low-grade cases can unexpectedly be associated with metastases. Currently, the 2010 WHO NEN classification employs histological differentiation and the proliferation index for grading tumors but fails to provide reliable prognostic and therapeutic indications. Therefore, there is an urgent need for a better characterization of G2/G3 NENs. Similar to several other tumors, NENs possess immune-escape mechanisms, but very little has yet been done to characterize this crucial aspect. There are no available data describing PD-L1 expression in these tumors. Here we provide, for the first time, evidence of PD-L1 tissue expression in gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). PD-L1 expression was significantly associated with a high-grade WHO classification (G3) ( In 2000 and 2010, the WHO provided two different classifications of NENs based on the histopathological pattern, tumor grade, and stage intended to improve the therapeutic and prognostic evaluation.6 Most gastrointestinal NENs are well differentiated (WD) and characterized by a slow growth rate (hence the old term carcinoid). Gastrointestinal neuroendocrine carcinomas are highly aggressive malignancies and are mainly treated with a platinum-based chemotherapy.7 However, at certain locations, such as the esophagus or colon, poorly differentiated NENs are more frequent than their WD counterparts.8 The tumor morphology and Ki67 immunostaining have been defined as prognostic factors that can subdivide gastrointestinal NENs into two or even three subgroups with significantly different survival rates.9 However, the two current prognostic evaluation systems fail to guide the most efficient therapeutic strategy on the basis of the histological differentiation of the tumoral cell and their proliferative index. In particular, the 2010 WHO NEN classification identified three groups: NENs G1, NENs G2, and G3, also defined as neuroendocrine carcinoma (NEC).11 In general, the WD NENs (G1/G2) are much more common (70%) than the poorly differentiated NENs.12 However, some WD cases exhibit a high level of proliferation and, on the contrary, some poorly differentiated cases show a low mitotic activity. Besides, the defined range  appears very broad, including many heterogeneous clinical, morphological and prognostic conditions.13 A few recent papers14 indicated that despite the assessment of the proliferation fraction, some tumors, identified as NECs, do not show the expected poorly differentiated morphology. These reports indicate that these patients do not respond to cisplatin-based chemotherapy, the gold standard therapeutic approach to high-grade PD-NECs,14 and have a better prognosis. In contrast, WD-NENs, despite mostly featuring a low/intermediate differentiation grade, may contain regions with an increased proliferation rate that places them, in accordance with the WHO, in the WHO G3 category.15 Specifically, G3 NENs with a WD morphology are commonly Ki67 positive  and respond poorly to cisplatin-based treatment. In contrast, patients with a poorly differentiated NEC, but Ki67 expression of >60%, respond to cisplatin-based chemotherapy.18 In addition, PD-NEC may also include a combined component of a conventional carcinoma (MANEC),19 such as a squamous cell carcinoma or adenocarcinoma, but they do not typically contain a lower-grade WD-NEN.21 Therefore, the availability of biomarkers that could better characterize G3 GEP NENs could have crucial therapeutic implications, indicating the most efficient pharmacological strategy. Immunotherapy, which allows immune cells to attack cancer cells, results in long-term survival in patients with several different tumors, such as melanoma, non-small cell lung cancer, and renal cell carcinoma.22 Cancer immunotherapy has undergone major breakthroughs during the past decades, impressive results being reported by several studies.24 One key target of cancer immunotherapies has been the PD-1–PD-L1 pathway.26 Programmed death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274), is expressed on many cancer cells. PD-L1 binding to PD-1 results in a pattern of inhibitory signals, which inhibits CD8+ T cells’ proliferation, thus blocking the antitumor immune response and hence favoring tumor growth and metastasis.27 PD-1 has been detected in tumor-infiltrating lymphocytes (TILs) present in the tumor microenvironment, where PD-L1 aberrant expression is associated with a poor prognosis in several human tumors.29 Taube et al.30 demonstrated that the expression of PD-L1 in tumor cells and tumor-infiltrating immune cells was highly correlated with PD-1 expression in infiltrating lymphocytes. They also demonstrated that PD-L1-positive tumors are those that respond better to Nivolumab administration. Nivolumab is a monoclonal anti-PD-1 antibody that can block the aforementioned inhibitory signals that prevent activated T cells from attacking the cancer. Anti-PD-1 administration may improve the prognosis of patients characterized by both tumor cells’ PD-L1 expression and TILs, as shown by multiple studies.32 PD-L1 expression on tumor cells and immune-infiltrating cells, such as tumor-infiltrating macrophages, appears to be a crucial predictor of the antitumor T cells’ response. Importantly, in most clinical trials, the response rates of various tumor types to PD-L1/PD1 targeting therapies were correlated with the immune-histochemical expression of PD-L1, suggesting that PD-L1 expression could be a predictive biomarker of the tumor sensitivity to immunotherapy.33 Immunological approaches against NENs have not yet been explored, but in the present manuscript, we provide data that may better support the possible benefit deriving from the revolution in immunotherapy as regards the clinical evaluation of NENs and the identification of the most effective pharmacological strategy. PD-L1 tissue expression has not been addressed in gastroenteropancreatic NENs (GEP-NENs). Here we provide evidence of PD-L1 tissue expression in different NENs. PD-L1 expression, together with Ki67, offers a new and efficient benchmark for a broad range of grading of GEP-NENs. Therefore, results of the present study indicate that PD-L1 tissue expression may be the best GEP/NEN morphological characterization and grading marker. Furthermore, Nivolumab administration may become the pharmacological choice for Ki67>60% PD-NENs, identified in this study as the tumors with the highest PD-L1 expression.Neuroendocrine neoplasms (NENs) are commonly localized in the gastrointestinal tract, lung, and pancreas.The patients characteristics of the 57 cases of NETs are summarized in P=0.345; P<0.001; P<0.001) from G1 to G3 (P<0.001). Furthermore, we highlighted significant differences between the PD-L1 score and the Ki67 index and, in particular, observed a remarkable difference between individual Ki67 mean levels and the PD-L1 score  while 16 cases were positive (28%). The relationship between PD-L1 expression and gender was not significant P=0.345; . ImportaG1 to G3 . In addiG1 to G3  from 0 tG1 to G3 . PD-L1 tP<0.001; .P<0.001; PD-L1 expression was also detected in peritumoral and intratumoral immune-infiltrating cells. Immune-infiltrating cells were most frequently located at the interface between tumor cells and stroma, with similar morphological features to the lymphoepithelial lesions frequently present in MALT lymphomas. In our study, we highlighted a variable PD-L1 expression by grade in immune-infiltrating cells, as shown in 37 The PD-L1-positive rate was ∼30% in melanoma cases and 25–36% in non-small cell lung cancer; in NENs, we found 28% cases.39 Some studies explored, by immunohistochemistry (IHC), PD-L1 expression in different tumors and demonstrated that PD-L1 membranous expression on tumor cells and/or infiltrating immune cells is correlated to a better chance of response to anti PD-1 drugs.41 However, the prognostic role of PD-L1 remains controversial, mainly due to tumor heterogeneity, variability in the assays, location of intratumoral expression, and cutoff values for positive versus negative expression. Therefore, in the present study we investigated the impact of PD-L1 tissue expression on the grading of 57 GEP/NEN patients, associating PD-L1 expression with the morphological characterization. The characteristics of indolent or aggressive GEP-NEN were determined by tumor grade, differentiation, and proliferation status.42 The current WHO 2010 grading system was designed to provide clinicians with an effective tool for patients stratification and clinical management. Years later, this WHO classification fails to provide an efficient cutoff between G1, G2, and G3 likely because existing biomarkers discriminate only partially between different grades. Currently, the Ki67 expression level is associated with different prognoses and can only partially indicate the most efficient pharmacological strategy. Similarly, high-grade G3 NECs are defined by morphology and Ki67 expression .15 The broad interval indicated by Ki67 expression for G3 disease (21–100%) may include a variety of different neoplasms, with potentially different responses to therapies.43 Overall, the morphology-based classification of different categories is still challenging, especially for G3 cases. Our results indicate, for the first time, that PD-L1 tissue expression is present in 100% of G3 NENs, those cases in which the Ki67 expression is an unreliable benchmark for treatment choice and intratumoral grade heterogeneity assessment. The terminology of ‘neuroendocrine carcinoma’ for G3 neoplasms implies that they are histologically poorly differentiated, but some morphologically WD-NETs also have proliferation rates  that meet the threshold for G3 NECs. A recent landmark study showed that G3 NECs with a Ki67 index of <55% do not respond to cisplatinum-based chemotherapy, unlike G3 NECs with a Ki67 index >55%.45 This supports the concept that the current WHO G3 category is heterogeneous and that tumors at the lower end of the G3 range are, in fact, WD-NETs with an elevated proliferation rate . Membranous PD-L1 expression by tumor cells and immune infiltrates varied significantly by grade and was significantly associated with the high-grade WHO classification (G3) (P=0.001) but not with gender, primary site, or number of metastatic sites. Our findings highlight that the expression of PD-L1 is present in the proliferative, aggressive G3 type of GEP-NET and, only in G3, shows a moderate-to-strong signal. In particular, poorly differentiated NENs (NEN G3 and NEC) have a constant positive PD-L1 membrane expression that increases with the tumor aggressiveness. Consistently with previous studies based on different tumors,47 PD-L1 expression is correlated with a more aggressive subset of NENs identified using morphological data and routine staining. In accordance with this possibility, PD-L1 expression has been reported as the most important predictor of the responsiveness of various cancers to PD-L1 or PD-1 blocking antibodies.48 Moreover, our study demonstrated a significant association with grading and PD-L1 expression levels in infiltrating immune cells (P=0.001). PD-L1 expression on tumor cells and tumor-associated immune cells was also positively correlated with the progression of human malignant tumors.49 PD-1 is expressed by activated T cells50 and its engagement with PD-L1 inhibits TCR-mediated activation of IL-2 production and T-cell proliferation. These findings suggest that NENs might acquire resistance to immune surveillance by upregulating PD-L1 and inhibiting peritumoral and intratumoral infiltrating lymphocytes. Thus this hypothesis provides a strong rationale for PD-1/PD-L-targeted immunotherapy for NENs. Recent studies52 have demonstrated that PD-L1 has a critical role in regulatory T-cell (Treg) development and functional maintenance. Increases in FOXP3+Treg infiltration and PD-L1 expression have been revealed in gastric cancer tissues, colorectal carcinoma, and breast cancer. High PD-L1 expression and increased FOXP3+Treg infiltrates were both associated with high histological grade, and tumors with concomitant high expression levels of the two markers had the worst prognosis. Recently, it has been reported that PDL-1 blockade improves antitumor immunity and offers a promising cancer immunotherapy approach. The blockade of PD-L1 in non-small cell lung cancer might be one strategy to pursue for future immunotherapy.53 Several clinical studies have suggested that TILs have a critical role and a prognostic significance in certain human tumors. Recently, PD-L1 has also been found to be expressed in a broad range of cancers. The observation in our study that PDL-1 is overexpressed in NENs (G2–G3) indicates that tumor-related PD-L1 may be linked to a malignant potential and contribute to tumor progression by providing a protective mechanism against immune surveillance. The significant association between PD-L1 expression and grading confirms that tumor-related PD-L1 expression is indeed relevant to tumor development. In our study, we observed high levels of PD-L1 expression on the infiltrating immune cells of PD-NENs. Thus it is possible that the tumoral milieu promotes macrophages PD-L1 expression that, in turn, inhibits the function of TILs, favoring tumor progression. We recognize that 57 NENs cases, although rare tumors, are a small cohort, but the incidence of PD-L1-positive patients with grade G3 warrants a larger future study. PD-L1 expression could become the new gold standard for G3 NENs discrimination. To the best of our knowledge, this is the first report evaluating the characteristics and grading of PD-L1 tissue expression in patients with GEP-NENs, paving the way for a new and more efficient WHO classification. In fact, it is expected that a new WHO classification, to be published in 2017, should provide a new NEC category G4 (Ki67>55%), as recently proposed by Fazio et al.54 This subdivision, correlated with a timely diagnosis, may ensure the implementation of appropriate treatment and have a substantial impact on prognosis. Furthermore, based on these observations, pharmacological approaches using anti-PD-1 antibodies may become the logical choice for the treatment of G3 cases, currently burdened by a poor prognosis. Immunotherapy might be a valid alternative or support to other therapies in NENs. Moreover, PD-L1 might be a useful prognostic biomarker in GEP-NENs. At the moment, it seems that high-grade tumors might be the targets. In this context, it is important to carefully delineate those tumors that might better respond to this type of treatment.The expression of PD-L1 is significantly associated with the histopathological grade and poor clinical outcomes in various malignant tumors, such as gastric cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer, and bladder cancer.32 or endoscopic resection25 from January 2006 to December 2016 were reviewed at the National Institute of Gastroenterology Research Hospital ‘Saverio de Bellis’. The following clinicopathological characteristics were collected for all patients: age, gender, primary site, tumor grade (according to the 2010 WHO classification), and metastasis , and a representative paraffin block from each specimen was chosen for IHC analysis. Tumor size was measured either grossly (obtained from pathology reports) or microscopically; no case measured >5 cm. The criteria for further classification and grading were based on both the morphological features of the tumor and the proliferation rate. On H&E and PAS mucin-stained sections, the cytological characteristics of cells, growth patterns, presence of ulcerations, level of infiltration in the organ, and infiltration of mucosa were evaluated. In addition, we evaluated perineural infiltration, vascular permeation, presence of necrosis, intratumoral and peritumoral lymphocyte infiltration, and lymph node metastases . Intratu55 In the present study, all PD-L1 staining results were scored as both the approximate percentage of positive tumor cells only in the membranous site and the intensity of overall staining. In particular, the expression of PD-L1 was scored: 0: (no staining) negative; 1: weak expression, but weaker than the placenta membrane, staining in <10% of tumor cells; 2: moderate expression in ⩾10% of tumor cells; and 3: strong more than placenta membrane staining in ⩾10% of the tumor cells. For the data assessment, our cases were considered to be positive for PD-L1 expression only if they had scores of 2+ or 3+  were evaluated on the NENs, using an automated autostainer . The Real Envision DAB Substrate Kit (DAKO) was used according to the manufacturer’s instructions. Human placenta was included as positive control. The staining pattern of both antibodies was as expected: cellular membranous/submembranous and cytoplasmic with occasional dots, corresponding to the PD-1/PD-L1 interaction sites. The E1L3N antibody clone was preferred for further evaluations primarily because of the better signal-to-noise results. Ki67 IHC was carried out again on all cases on paraffin-embedded sections using an Ab anti-Ki67 diluted 1:100  following the manufacturer’s instructions. All cases were revised and re-evaluated and then assigned a precise proliferation index number that encompassed the amplitude limit of the WHO range. All IHC stained sections were initially evaluated and scored by two pathologists; discrepancies in the interpretation of scoring were resolved by consensus. PD-L1 expression was evaluated both on tumor cells and inflammatory cells as intratumoral and peritumoral in location. PD-L1 expression can be observed as a cytoplasmic or membranous immunoreactivity. The proportion of PD-L1-positive cells was estimated as the percentage of total tumor cells; tumor cells typically showed membranous staining with a variable cytoplasmic staining component. In literature, tumors with ⩾1% of tumor cells stained either in membrane or cytoplasm were considered positive for PD-L1 regardless of the intensity of staining.2+ or 3+ . Moreovet-test or Fisher’s exact test and χ2 test. All P-values were determined by two-sided tests and P-values<0.05 were considered significant. Statistical analyses were performed with StataCorp. 2007 Stata Statistical Software: release 10 .Correlations among the PD-L1 expression score of tumor cells, immune-infiltrating cells, and grading were statistically analyzed by


However, the effective work function values of metal gate in graphene FET are significantly deviated from their vacuum work function, which is similar to the Fermi level pinning effect observed in silicon MOSFETs with high-k dielectric. The degree of deviation represented by a pinning factor was much worse with HfO2 (pinning factor (S) = 0.19) than with Al2O3 (S = 0.69). We propose that the significant pinning-like behaviors induced by HfO2 are correlated with the oxygen exchange reactions occurred at the interface of graphene and HfO2.High-k materials such as Al Especially, graphene field-effect transistors (GFETs) have been extensively studied as a promising alternative for post-silicon metal–oxide–semiconductor field-effect transistors (MOSFETs)6. Recently, graphene barristors and related variations have achieved very high on/off ratios of up to 1078, even without any bandgap. Such progress is motivating further research on gate stacks that are compatible with graphene. Since graphene barristors rely on the sliding of the graphene Fermi level (EF) at the interface with the semiconductor, it is desirable to minimize the Fermi level pinning in the metal gate/high-k dielectric stack on a graphene channel.Graphene has received considerable attention because of its extraordinary electrical and physical properties, such as its high carrier mobility, excellent thermal stability, and top-down processing compatibilityin situ ozone treatments, functionalization of the graphene, or addition of a metal seed layer on the graphene to aid nucleation11. While the physical scaling of the gate dielectric on graphene has substantially progressed owing to these efforts, the electrical performance—such as quality and reliability—of the gate dielectric has not been thoroughly investigated. Alternatively, hBN has been extensively studied as a thin gate dielectric material for graphene15. The possibility of direct growth on a graphene due to the small lattice mismatch is a strong merit, but high leakage current with a dielectric constant lower than SiO2 (~3–4) indicates that the process is less matured for gate dielectric applications.So far, the physical scaling of gate dielectric has been the primary focus of study in the gate stack research for graphene because it is difficult to form a thin gate dielectric on graphene using atomic layer deposition (ALD) process because graphene does not have enough dangling bonds to initiate the chemical reactions for ALD. Thus, various processes have been explored, including surface modification by via interface dipole formation and Fermi level pinning18. Furthermore, the redistribution of the oxygen into the substrate strongly influences the effective work function of the metal gate. Thus, gate stacks on GaAs substrates are less influenced by Fermi level pinning than those on silicon, because the oxygen diffusivity in GaAs is lower than that in silicon20. These findings were critically important for the development of nanoscale silicon MOSFETs, because controlling the effective work function of the metal gate is crucial in controlling the threshold voltage of MOSFETs. Likewise, a proper understanding of the effective work function of the metal electrodes in GFETs will be very important to properly control the Dirac voltage (VDirac) of the graphene layers used in various graphene-based devices21.On the other hand, the metal gate might be the least studied component in GFET structure. In silicon MOSFETs, the redistribution of oxygen vacancies at the metal gate/high-k dielectric interface causes a work function roll-off phenomenon; this occurs 2 and Al2O3, and various metal gates covering a wide range of vacuum work functions from 4.28 to 5.65 eV. We found that the effective work function values of metal gates on the high-k dielectric and graphene stack are significantly deviated from the vacuum work function, which is similar to the Fermi level pinning effect of a metal gate/high-k dielectric stack in silicon MOSFETs. Fermi level pinning factors representing the degree of deviation were extracted from the slope of Dirac voltage and vacuum work function of top gate metal. The pinning factors were ~0.1914 for HfO2 and ~0.6912 for Al2O3, indicating that the Al2O3 gate dielectric is more suitable for controlling the Dirac voltage of GFETs. Finally, the origin of the Fermi level pinning effect in GFETs was studied using a discharge current analysis (DCA) method.In this work, the interactions between the gate metal, gate dielectric, and graphene in the graphene FETs have been investigated using two different dielectrics, HfO22.To compare the influences of different metal gates and high-k dielectrics on multiple GFETs, the baseline GFETs used in this work should have relatively small scattering in the device characteristics. Especially, the device should show symmetric I-V characteristics to accurately assess the impact of Fermi level pinning using the distribution of Dirac points. We applied various addition annealing and passivation processes to achieve symmetric I-V characteristics with minimal hysteresis and residual charges as described below2/highly p-doped silicon substrate (resistance < 0.005 Ω) was sonicated sequentially in acetone, methanol, and deionized (DI) water. The substrate was then cleaned using the SC1 process . Monolayer graphene was grown on a Cu foil (≥99.8%) using thermal chemical vapor deposition (CVD), and then transferred to the SiO2 substrate. First, the graphene was coated with poly(methyl methacrylate) (PMMA) at 500 rpm for 5 s and 3000 rpm for 50 s, and then baked at 80 °C for 5 min. Then, the Cu foil was etched at 50 °C using a Cu etchant. The PMMA/graphene film was cleaned with diluted HCl (HCl and H2O in a ratio of 5:13) then rinsed with DI water. A final DI water rinse was performed for 5 min, and then the PMMA/graphene film was transferred to the substrate and dried overnight.The 90 nm SiO−6 Torr). The Raman spectrum of the transferred graphene fits well with a Lorentzian function. As shown in Fig. To remove the PMMA layer, the samples were baked at 150 °C for 10 min and then submerged in acetone heated to 85 °C for 3 hr. Finally, thermal annealing was performed at 300 °C for 1 hr in a vacuum  evaporator, followed by the patterning of the channel and contact regions by photolithography and an Au etchant. Then, the graphene was etched by oxygen plasma according to the Au hard mask, which was performed at 50 W in a 200 mTorr environment for 2 min. The final graphene channel width and length was 30 µm and 9 µm, respectively. Then, the source/drain electrodes (100 nm Au) were formed by photolithography and a metal wet etch process.2O3 or HfO2 was deposited on the graphene channel as a top-gate dielectric using ALD. For HfO2, a Hf seed layer (2 nm) was deposited using the e-beam evaporator because of the nonuniform nucleation that arises without the seed layer11. Before the ALD process, the Hf seed layer was oxidized using 10 s H2O/30 s N2 purge steps (10 cycles) at 100 °C. Then, HfO2 was deposited over 400 ALD cycles using tetrakis(ethylmethylamino)hafnium /N2 purge (10 s)/H2O (0.2 s)/N2 purge (10 s) steps at 250 °C. In the case of Al2O3, the trimethylaluminum /N2 purge (20 s)/H2O (0.2 s)/N2 purge (20 s) cycle was repeated 300 times at 130 °C. Both dielectrics were densified at 300 °C for 1 hr under vacuum (~10−6 Torr). The thicknesses of the Al2O3 and HfO2 layers measured with a spectroscopic ellipsometer were both ~30 nm. Finally, four different top-gate metals—Pt, Au, Ti, and Al—were deposited and pattered using a lift-off process. The top-gate metals were chosen so their work functions covered the range from 4.28 to 5.65 eV. Figure −6 Torr) at 300 °C for 1 hr before the electrical tests were performed.AlI–V measurement unit (Keithley 4225-RPM)26. A pulsed I–V measurement method was used to minimize the influence of slow charge trapping effects. The pulse width and rise/fall time was 1 ms and 100 µs, respectively, which were the optimized test conditions for the top-gated GFETs used in this work. The gate voltage was increased in one direction starting from near the Dirac voltage to minimize hysteresis. Since the dielectric constant of Al2O3 is 6 and that of HfO2 is 20.6, the gate voltages were biased from −10 V to 10 V for the Al2O3 device and from −2.9 V to 2.9 V for the HfO2 device to generate matching maximum electric fields of around ±5.1 MV/cm. The mobility, hysteresis-induced charge trap density, and Dirac voltage was extracted from the electron-doped branch of the pulsed Id–Vg curve. The charge trap density of the top-gated GFETs at the Fermi level of graphene was extracted using the DCA method25. For the DCA measurements, square pulses were applied to the top-gate, ranging from 10 kHz to 1 MHz for Al2O3 and from 10 kHz to 500 kHz for HfO2. The frequency ranges were adjusted considering the influence of the different time constants of the charge traps in each dielectric. The capacitance–voltage curves of the top-gated GFETs were also measured using an impedance analyzer (Agilent 4294 A) to extract the EF value of graphene using the quantum capacitance. A detailed description of the DCA setup and analysis is given in Ref.25.The device characteristics were measured using a Keithley 4200 Parameter Analyzer with a pulsed Id – Vg) curves of the top-gated GFETs having different top-gate metals are shown in Fig. 2, which is much smaller than that expected based on the range of vacuum work function values. In contrast, the range of Al2O3 Dirac voltages was 1.10 V. This behavior is somewhat similar to the Fermi level pinning effects observed in silicon MOSFETs which limits the swing range of the threshold voltage so that it is much smaller than the range of the vacuum work function of metal gates. However, since the Fermi level pinning in silicon MOSFETs is related to trap states at the dielectric/silicon interface, whether we can attribute this observation to Fermi level pinning remains uncertain.The representative pulsed drain current–gate voltage  reactions—can be measured separately according to the response time; redox components primarily appear at higher frequencies23. Moreover, the quantities of these two types of charge generation mechanism can be extracted using the DCA method.To investigate this interesting phenomenon, the differences in the graphene and high-k dielectric interfacial reactions were investigated using the DCA method2O3 gave a charge density that was ten times lower than that of HfO2. This result indicates that there are significantly more oxygen-related charge generation events at the HfO2/graphene interface. This is a reasonable result considering that oxygen ions diffuse much faster in HfO2 than in Al2O333. Oxygen vacancies in dielectrics can create an interfacial dipole layer through electron transfer that affects the Fermi level pinning35. Hence, the high density of charge traps in the top-gated GFETs containing HfO2 corresponds with the small pinning factor extracted from the relationship between the Dirac voltage and the work function of the top-gate metal.Figure 2 can affect the Fermi level pinning. Figure I–V curve shift via Fermi level pinning is more pronounced at negative biases, i.e., lower work functions. This result implies that the electron doping of the graphene is weaker than expected for the lower work function metal gates, i.e. there is a mechanism prohibiting the electron doping of the graphene, especially in the case of HfO2, as graphically shown in Fig. − charging at the graphene surface and thus prevent electron doping. Figure 2, in contrast to Al2O3, providing corroborating evidence for our model. Thus, we can conclude that the strong Fermi level pinning observed for the GFETs fabricated with the HfO2 gate dielectric is due to the inhibition of electron doping, which occurs via counter-charge generation at the dielectric/graphene interface due to the interfacial oxygen accumulation. This mechanism is quite different from the original Fermi level pinning mechanism because the chemically induced charges are limiting the movement of Fermi level of graphene rather than the defect states.Especially, oxygen accumulated at the bottom interface of HfODct,redox) in the top-gated GFETs with HfO2 were 0.1914 and ~1014 cm−2 eV−1, respectively. These values show that HfO2 devices experience a more significant pinning effect than Al2O3 devices, which have a pinning factor of 0.6912 and Dct,redox = ~1013 cm−2 eV−1. These results indicate that the pinning effect is caused by redox reactions at the interface between the high-k dielectric and graphene. These results indicate that the scaling of gate dielectrics using higher-k materials will be more difficult for GFETs and will require stringent control over the interface and its passivation.In this research, the dependence of the Fermi level pinning effect on the dielectric was investigated using pulsed current–voltage and DCA methods. From the analysis of our measurements, the pinning factor and charge trap density induced by the redox component (Figure S1


Replicating prior work, L2 speaking rates were significantly slower than L1 speaking rates both across-groups , and across L1 and L2 within bilinguals. Critically, within the bilingual group, L1 speaking rate significantly predicted L2 speaking rate, suggesting that a significant portion of inter-talker variation in L2 speech is derived from inter-talker variation in L1 speech, and that individual variability in L2 spoken language production may be best understood within the context of individual variability in L1 spoken language production.Second-language (L2) speech is consistently slower than first-language (L1) speech, and L1 speaking rate varies within- and across-talkers depending on many individual, situational, linguistic, and sociolinguistic factors. It is asked whether speaking rate is also determined by a language-independent talker-specific trait such that, across a group of bilinguals, L1 speaking rate significantly predicts L2 speaking rate. Two measurements of speaking rate were automatically extracted from recordings of read and spontaneous speech by English monolinguals ( I.Cross-language and cross-talker variability are salient features of human speech production. Cross-language variability at the phonetic level is evident in the observation that similar speech sounds in different languages  often vary in their articulatory and acoustic details, giving rise to language-specific or dialect-specific articulatory settings . SimilarWithin the language/dialect-specific phonetic setting for a given language or dialect, acoustic-phonetic variation across individual talkers can exceed the extent expected based solely on physical variation across individuals. For example, within Standard American English, individual talkers differ in their characteristic voice onset times (VOTs) for voiceless stops. That is, controlling for speaking rate, some talkers produce longer VOTs than other talkers, and these talker differences are consistent across various places of articulation  or a consequence of more central, rather than peripheral, control processes in language and speech production. For example, working memory, IQ, and, lexical retrieval speed, amongst other cognitive and/or linguistic characteristics, may influence both first-language (L1) and second-language (L2) speech production. It is also possible that individual personality traits may bear some relation to patterns of speech production , as well as about who is saying it . For example, using sine-wave replicas of English speech in which the spectro-temporal dynamics of the signal  are preserved but the vocal source characteristics  are removed, The integration of linguistic (contrastive phonetic) and indexical  information suggests that some degree of idiolectal variability is likely constrained by language-specific structural features . In the case of bilingual talkers, for whom two linguistic sound systems must coexist, this may mean that the need to maintain a greater number of phonetic distinctions along any given acoustic dimension might further constrain the range of idiolectal variability. (Note that this account assumes integrated L1-L2 phonetic systems with constant co-activation of both languages.) Moreover, acoustic-phonetic variation along global acoustic-phonetic dimensions that are shared across a bilingual's L1 and L2 may be independently controlled in the two languages. For example, within a given language, speaking rate (in terms of syllables per second) may be substantially constrained by dialectal affiliation  of linguistically diverse bilinguals . We chose to focus primarily on timing in terms of speaking rate  because it can be automatically and consistently measured across languages. Moreover, as a global feature that sets a temporal frame (or tempo) for an utterance rather than conveys phonemic contrasts, speaking rate is exactly the type of acoustic feature that could be subject to both language/dialect- and talker-specific control. It is also more likely to be independent of the anatomical and physiological constraints of a particular vocal tract than other global features such as fundamental frequency or the long-term average speech spectrum. This set of conditions thus allows for the possibility of a dissociation between average L1 and L2 speaking rates in absolute terms (with L2 speaking rate invariably being slower than L1 speaking rate), but an association of relative L1 and L2 speaking rates across a group of bilingual talkers . The dissociation of average rates in L1 and L2 would establish that the parameter in question (speaking rate) is not an automatic consequence of the talker's vocal anatomy and physiology, but is instead a learned and/or centrally  controlled property. This then provides the necessary condition for investigating the relationship between relative L1 and L2 speaking rates as an indicator of the relationship between language- and talker-specificity in bilinguals.The present study seeks evidence for language-independent, talker-specific characteristics by examining a global, talker-controlled feature across both languages of a group of bilingual talkers. Specifically, we examined speech-timing patterns in a corpus that includes comparable recordings in each of the two languages of a group  than in a task that emphasizes speech production without language generation at the conceptual level (paragraph reading).II.A.Recordings for this study were all taken from a corpus of digital speech recordings that includes both read speech and spontaneous speech by bilingual and monolingual English speakers. A key feature of this corpus is that it includes recordings in both the L1 and the L2 (English) of each bilingual talker. All talkers in the corpus were recorded producing a common set of materials that was selected to cover both read and spontaneous speech, and to be comparable across languages. The full set of recordings in each language consisted of six distinct subsets, three of which were sentence lists, one of which was a paragraph-length reading passage, and two of which were designed to elicit spontaneous speech. The sentence recordings are intended primarily for use as stimuli in speech perception experiments and were not analyzed for the present study. The paragraph is the widely translated fable, “The North Wind and the Sun,” as available for all languages included in the To date, this corpus  includes recordings from 119 bilingual talkers from 21 different L1 backgrounds, plus 27 monolingual English talkers who provided English recordings only. Additional and current information about the continuously updated and expanded corpus can be found online.In the present study, we analyzed the recordings of the paragraph-length reading passage (NWS), the picture story narratives (ST), and the open response questions (Q&A) by 86 bilinguals in both of their languages and by 27 monolingual English talkers (English only).B.n = 27) in the corpus.The bilingual talkers included in this study came from ten different L1 backgrounds . While English proficiency of the bilingual talkers varied, all talkers had achieved a level of English language proficiency that was sufficient to gain entry into a graduate program at Northwestern. Nevertheless, most of these bilingual talkers were enrolled, either by choice or by requirement, in intensive English language instruction as offered by the Northwestern University English Language Programs. Standardized English test scores  and their speech was recorded direct to disk onto an Intel Core 2 Duo iMac . All talkers completed a language background questionnaire before beginning the recording of the sentences, paragraph, and spontaneous speech in English. The bilingual talkers returned the following day for a second recording session during which they recorded the sentences, paragraph, and spontaneous speech recordings in their native language (the L1 recordings). All scripted materials were presented in the standard orthography of the language. Each session took ∼1–1.5 h.C.The primary measure of interest for the current study was speaking rate, which we measured in both the L1 and L2 speech samples from the read speech (NWS paragraph) and spontaneous speech recordings . We obtained two measures of speaking rate, speech rate and articulation rate, from each of the three speech samples in each language, both of which were based directly on the number of (acoustic) syllables  per second. The two speaking rate measures differed only with respect to the inclusion (speech rate) or exclusion (articulation rate) of silent pauses.From each speech sample we obtained the number of syllables using an automatic syllable detection algorithm implemented as a Praat script . This scAdditionally, we obtained the average number of syllables per utterance where utterance was defined as a stretch of speech surrounded by pauses of at least 100 milliseconds. This measure of average utterance length  was included as a predictor of speech rate and articulation rate in the statistical analyses because prior work  and within the data for each task ]. The specific dependent variable entered into each statistical model was a set of such z-transformed acoustic measurements. It is important to note that, while an untransformed speech rate measure is necessarily always slower than the matched untransformed articulation rate measure (they have the same numerator but different denominators), a z-transformed speech rate measure can be either larger, equal to, or smaller in magnitude than its z-transformed articulation rate counterpart due to the fact that the duration of pauses varies substantially within stretches of speech. Therefore, a given talker's average articulation rate measure (pauses excluded) may be close to the group-wise average articulation rate measure, but this same talker's average speech rate measure (pauses included) may be quite far (in either direction) from the group-wise average speech rate measure. This would indicate that the talker in question exhibits quite typical articulation rate but rather atypical pausing behavior relative to the group.The speech rate and articulation rate data were analyzed with linear mixed effects regression models using the lme4 . Five hyFor all analyses, the fit of the base model  was compared to the fit of additional models that included additional predictors . Models were compared by means of the anova function from the lmerTest package .H1. L1 speaking rate varies across languagesH2a. L1 English speaking rate by monolinguals is faster than L2 English speaking rate by bilingualsH2b. Within bilingual talkers, L1 speaking rate is faster than L2 speaking rateH3a. Within bilingual talkers, L1 speaking rate predicts L2 speaking rateH3b. Within bilingual talkers, L1 speaking rate predicts L2 speaking rate, when controlled for proficiency levelsThe specific hypotheses we tested are as follows:z-transformed as described above) for one of the three tasks .The overall structure of the data set for these analyses consisted of multiple entries for each talker in each language  with each entry representing a speaking rate measure . For this analysis, the fixed effect factors in the best fitting, maximal model were L1 , and a second within bilingual talkers . In the first regression model , the fixed effect factors of the best fitting, maximal model were language status , task , measure , and utterance length. All possible two- and three-way interactions among language status, task, and measure were also included as fixed effect factors. Language status, task, and measure were contrast coded; utterance length was centered. The random intercept was talker, with task and measure as random slopes. The multicollinearity of this model was low, with a condition number of 3.77. In the second regression model , the fixed effect factors of the best fitting, maximal model were language status , task , measure , L1 . In this multivariate analysis, the dependent variables were L2 speech rate and L2 articulation rate. The dataset consisted of multiple entries for each talker in each language with each entry representing a speaking rate measure  to test for the influence of L2 (English) proficiency on L2 rate, and to see if the effect of L1 rate on L2 rate remains when variation in L2 proficiency is controlled (H3b). In this model, the fixed effect factors were L1 rate, task , measure , L1  and articulation rate (excludes silent pauses) could be performed. The analysis of speaking rate differences across the various L1s showed that all of the main effects  reached significance except for talker age. (See z-transformed) was significantly faster than the speech rate measure (z-transformed). Finally, as expected based on prior work  is beyond the scope of the present study as it would require a detailed analysis of the morpho-phonological and syntactic features of the various languages. There was also a significant L1 by task interaction, involving either a relatively small read-spontaneous difference (Russian), a relatively great read-spontaneous difference (Korean and Turkish), or a different-from-typical difference within the two types of spontaneous speech (Russian and Hebrew). The task by measure interaction was also significant, as was the three-way interaction between L1, task, and measure, due to the fact that two languages (Mandarin and Hindi) showed a smaller read-spontaneous difference in speech rate than in articulation rate, whereas two other languages (Korean and Russian) showed a greater read-spontaneous difference in speech rate than in articulation rate. Overall, while this analysis across L1s showed some notable patterns, there was no clear, interpretable, and systematic trend that could be taken as a reliable indicator of a strong language-specific effect on speaking rate , measure (articulation rate faster than speech rate), and utterance length (faster rates for longer utterances).  for whom proficiency (Versant) scores were available], the critical relation between L1 and L2 speaking rates remained.  than in a task that emphasizes speech production without language generation at the conceptual level (reading). However, in the present study, the relationship between L1 and L2 speaking rates within bilinguals was similarly evident in both read and spontaneous speech . Nevertheless, in the analysis of L1 and L2 speaking rate across the various L1s and the analysis that directly compared L1 and L2 speaking rates, the data showed that speaking rate was generally modulated by task such that read speech was produced with a faster speaking rate than spontaneous speech in both L1 and L2 speech production. Moreover, within spontaneous speech, the task with picture prompts elicited speech with a faster rate than the free-form, question-and-answer task. Thus, speaking rate in both L1 and L2 decreased with increasing task complexity. Furthermore, the data showed an interaction between language status (as L1 or L2) and task, such that the L1 vs L2 speaking rate difference was smaller in the simpler task (reading) than in the less constrained spontaneous speech tasks whether picture-guided or question-prompted. Thus, while task differences did not appear to amplify or diminish talker-specificity in speaking rate, the difficulties of L2 speaking seem to accumulate across levels of processing such that L2 speech is particularly slow in complex tasks that require language generation at the semantic and syntactic levels compared to read speech where the talker does not need to generate well-formed phrases and sentences in addition to having to produce intelligible speech  languages of that individual talker. How, then, might this aspect of talker-specificity serve as a language-independent idiolectal marker? One possibility is that listeners develop expectations about the range of speaking rates to expect for L1 and L2 speech. Then, when an individual bilingual talker is encountered, and once the L1 or L2 status has been determined on the basis of the wide range of acoustic-phonetic deviations that typically characterize L1 vs L2 speech in a given language , listeners could adjust to that individual's speaking rate within the appropriate range. It is then possible that this L1- or L2-specific adjustment could form the basis for generalization to the other of this particular bilingual's language. If a listener has experience with a relatively fast talker in L1, then this listener may expect that this talker will also be a relatively fast talker in L2. This experience-based expectation about speaking rate may then combine with the other talker-specific factors  that facilitate talker identification and recognition of that talker's speech in either the L1 or the L2.It is also possible that talker-specific, rate-based expectations that listeners develop on the basis of exposure to one language  may extend to other rate-dependent acoustic-phonetic features that can quite easily generalize across languages. For example, even for an L1-L2 pair with different duration-based phonological contrasts , experience with a given talker's realization of the L1 contrast in relation to the L1 norms could potentially provide the basis for expected durations in this talker's realization of an analogous L2 contrast. Moreover, since fast and slow speech are associated with general hypo- and hyper-articulation, respectively, it may be possible for listeners to develop quite extensive, language-independent, talker-specific, rate-based expectations for L1 (or L2) speech on the basis of prior experience with L2 (or L1) speech. Such expectations could then facilitate recognition of this talker's speech across both languages even if prior exposure has been to only one of the talker's languages.In conclusion, our analyses of speaking rate in L1 and L2 within bilingual talkers has suggested that a significant portion of variation in L2 speech is derived from talker-specific variation in L1 speech. Notwithstanding the strong influence of the relationship between first- and second-language structure, and the similarly strong influence of experience- and training-related second-language proficiency, individual variability in L2 spoken language production is probably best understood within the context of individual variability in L1 spoken language production.