The outstanding charge transport properties of graphene enable numerous electronic applications of this remarkable material, many of which are expected to operate at ultrahigh speeds. In the regime of ultrafast, sub-picosecond electric fields, however, the very high conduction properties of graphene are not necessarily preserved, with the physical picture explaining this behaviour remaining unclear. Here we show that in graphene, the charge transport on an ultrafast timescale is determined by a simple thermodynamic balance maintained within the graphene electronic system acting as a thermalized electron gas. The energy of ultrafast electric fields applied to graphene is converted into the thermal energy of its entire charge carrier population, near-instantaneously raising the electronic temperature. The dynamic interplay between heating and cooling of the electron gas ultimately defines the ultrafast conductivity of graphene, which in a highly nonlinear manner depends on the dynamics and the strength of the applied electric fields. A linear energy–momentum relation of graphene results in a high direct-current electron mobility, but this is not necessarily true at terahertz frequencies. Here, the authors show that its ultrafast conductivity is dependent on a highly nonlinear interplay between heating and cooling of the electron gas. E and momentum p of an electron E=pvF , where vF≈c/300 is the Fermi velocity—the electron group velocity, and c is the speed of light in vacuum12356789Graphene stands out among other electronic materials for its unique band structure, which in the broad vicinity of the Dirac point (the point of lowest electronic energy) is characterized by a linear relation between the energy The very high direct-current conductivity in graphene11131315917202122In the following, we present a unifying view on electron transport in graphene in arbitrary ultrafast electric fields. We demonstrate that the conductivity of graphene in a very wide range of applied electric field strengths and frequencies can be described within a simple thermodynamic approach: by taking into account the statistically determined thermal balance maintained within the entire electron population of graphene interacting with the applied electric field, and without requiring detailed knowledge of the microscopic electron kinetics.ETHz propagates through the sample and induces a current j=σETHz in graphene, proportional to its conductivity σ, as shown in −1) and frequencies (0.4–1.2 THz)To study ultrafast charge transport in graphene, we use an all-optical, contact-free conductivity sampling method called nonlinear ultrafast THz spectroscopy2425EF=0.07±0.01 eV, corresponding to a sheet carrier density of Ns=(6.0±2.0) × 1011 cm−2, and the electron momentum scattering time at the Fermi level of τ(EF)=140±20 fs. All measurements in this work were performed at room temperature.Our single-layer graphene sample is grown by chemical vapour deposition (CVD) on copper and deposited on a silica substrate12−1 fields the conductivity almost vanishes. As explained below, this dramatic conductivity reduction is a direct result of the accumulation of thermal energy, absorbed from the applied ultrafast electric fields, within the highly correlated electronic system of graphene, which on the THz timescale behaves as a hot electron gas. This affects the conductivity of graphene, which is dependent on the electronic temperature.An overview of the experimental results is shown in 2122Tel, while the total kinetic energy of all electrons makes up the thermal energy of the entire gas—the electron heat. The energy distribution of the electrons in a thermalized gas is described by the Fermi–Dirac function μ is the chemical potential and kB is Boltzmann's constant. The process of thermalization, the equipartition of energy among all electrons in a gas, occurs via electron–electron interactions—elastic scattering processes in which the electrons exchange their energy and momentum. In graphene, this process is extremely efficientE(p)=pvF. Indeed, given the available phase space, any two electrons in graphene i and j, occupying any two states , can change their states, so that if one electron increases its energy and momentum by ΔE and Δp, respectively, the second one will decrease its energy and momentum by the exact same amounts ΔE and Δp, as illustrated in 303132333435Tel. This motivates our thermodynamic, rather than microscopic, approach to describe the THz conductivity in graphene: instead of following the microscopic electron kinetics, on the THz timescale we can consider the macroscopic statistical properties of the whole ensemble of electrons in graphene.A large ensemble of electrons, an electron gas, has a natural, statistically determined state—the thermalized state. In such a state, all electrons in the gas are equilibrated and are characterized by a common temperature EF∼100 meV the thermal broadening of the electron distribution can easily be of the same order as the Fermi energy itself, especially at elevated temperatures . That is, for the description of the electric conductivity in graphene one should consider not only the electrons at the Fermi level , but a broader electron distribution, as illustrated in σ(ω) using3437Examining the thermal distribution (Fermi–Dirac) function, we note that, unlike in metals which typically have a Fermi energy on the order of several electron volts, in doped graphene with a typical τ(E) is the electron momentum scattering time, which may also be energy dependent.Here −1. We first consider the case of the weakest applied THz field of 2.3 kV cm−1, where the amount of energy transferred to the electron system of graphene is negligible, and find that Tel=300 K, μ=EF=0.07 eV and τ(EF)=140 fs (see In 0 fs see .D(E)fFD(E), which now corresponds to a certain elevated electron temperature Tel, as illustrated in μ. The reason for this is that the incident THz field only induces intra-band transitions, and hence the carrier density must remain conserved at all times: μ remained constant, an increase in electron temperature would lead to an increase in carrier density. Therefore, to provide for the necessary carrier density conservation Ns=const, the increase in electron temperature must necessarily be accompanied by a corresponding decrease in chemical potential. This provides a qualitative explanation of the observed reduction of the intra-band THz conductivity for a hotter electron distribution in graphene, shown in 38Increasing the THz field strength leads to a significant reduction of the graphene conductivity, especially at higher frequencies. As applying stronger fields to graphene facilitates more energy transfer to its electronic system, this leads to a modification of the electron distribution Ns=const , and use the electronic temperature Tel as a free fit parameter. At this point, not only the temperature-dependent electron distribution (see τ(E) should be considered. To simplify our model, we consider two main electron momentum scattering regimes in graphene (see ref. τ(E): (i) long-range scattering on Coulomb impurities, and (ii) short-range scattering on disorder. The former is expected to dominate in CVD graphene, such as our sample, with the momentum scattering time being directly proportional to the electron energy τ(E)=γE (ref. τ(E)=β/E /EF and β=τ(EF) × EF.For a quantitative description of the measured THz conductivity spectra shown in tion see , but als=γE ref. . The latβ/E ref. . Here γ −1 are indeed best described using the scenario of long-range Coulomb scattering, as expected for CVD graphene. We find effective electron temperatures in the range 300–936 K. At the same time, the alternative scattering scenario does not describe our data very accurately. Nevertheless, qualitatively the result of both electron momentum scattering regimes in graphene is the same: they both lead to lower electronic conductivity with increasing frequency and strength of applied electric fields, as a result of adding heat to the electron gas in graphene. The validity of this thermodynamic model of conductivity, assuming the electron momentum scattering on Coulomb impurities, is further confirmed by using it on a different CVD graphene sample with a different set of material parameters, mainly a higher Fermi energy at 300 K  determined at Tel=300 K, the known electron cooling dynamics33ETHz(t) in the entire range of peak field strength .The time-domain calculation provides useful insights into the dynamical heat balance of the electron gas in graphene during the interaction with ultrafast electric fields. The temporal evolution of the instantaneous temperature −1, respectively. As solid lines in the same figure, we show the conductivity spectra calculated with the split-step time-domain method, corresponding to the same experimental conditions. Our modelling approach, not containing any free parameters and based on the simple time-dependent thermodynamic balance maintained in the electronic system of graphene, describes the entirety of our experimental observations very well  in Q, we calculate the average electron heating efficiency η=Q/(A × Fp)—the ratio of the time-averaged thermal energy retained in the electron gas of graphene  to the total absorbed THz energy , where Fp is the known fluence of the THz signal, see η≈15% for the whole range of peak THz fields (2.3–120 kV cm−1), with the rest of the deposited electric field energy being converted to phonons on the same timescale. We emphasize that these values are specific not only to the cooling dynamics of the graphene sample, but also to the actual shape of the THz waveform, defining the dynamics of the highly nonlinear interaction between the electric field and graphene  yields the value Q/Ns∼0.5 eV. Indeed, electrons with energies as high as 0.78 eV were observed by optical probing of the density of states of doped graphene, excited with ultrafast THz fields of ∼200 kV cm−1 strength in ref. 203945The high electron temperatures we find see  imply thIn conclusion, we have shown that the ultrafast electron transport in single-layer graphene can be understood within a simple picture of a time-dependent thermodynamic  balance maintained within the electronic population of graphene: The primary mechanism of interaction between the electric fields and graphene is the conductivity, directly converting the energy of the applied electric field into the energy of induced electric current. However, the extremely efficient electron–electron interaction in graphene30313233−1, operating at ∼THz frequencies910The timescale for electron gas thermalization in graphene, previously determined to be sub-50 fs refs , 33, 34,On the other hand, the high electron temperatures and relatively slow cooling dynamics in graphene are clearly advantageous for THz detectors1541725Etrans(t) (through the substrate with graphene), from the known, measured reference waveform Eref(t) (through the substrate without graphene). As the sample response and the field strength in the THz waveform are coupled to each other through the absorbed heat Q(t) of the carriers, we numerically solve the following system of nonlinear equations. The transmitted waveform is the convolution of the response function of the sample and the incident waveform:We propagate the incident THz pulse numerically through the sample, thus calculating the transmitted waveform T(t) is the inverse Fourier transform of the complex transmission function t(ω) (see Q(t). We calculate this from the energy balance of the experiment—the absorbed THz energy is equal to the difference between the energy of the incident THz waveform and that of the reflected and transmitted waveforms:where t(ω) see . Note thR(t) describes the dynamics of the heat dissipation from the carriers. We take these dynamics from the experimental study in ref. Etrans(t). Finally we obtain the nonlinear conductivity using where How to cite this article: Mics, Z. et al. Thermodynamic picture of ultrafast charge transport in graphene. Nat. Commun. 6:7655 doi: 10.1038/ncomms8655 (2015).Supplementary Figures 1-8, Supplementary Note 1, Supplementary Methods and Supplementary References


Wrinkles as intrinsic topological feature have been expected to affect the electrical and mechanical properties of atomically thin graphene. Molecular dynamics simulations are adopted to investigate the wrinkling characteristics in hydrogenated graphene annulus under circular shearing at the inner edge. The amplitude of wrinkles induced by in-plane rotation around the inner edge is sensitive to hydrogenation, and increases quadratically with hydrogen coverage. The effect of hydrogenation on mechanical properties is investigated by calculating the torque capability of annular graphene with varying hydrogen coverage and inner radius. Hydrogenation-enhanced wrinkles cause the aggregation of carbon atoms towards the inner edge and contribute to the critical torque strength of annulus. Based on detailed stress distribution contours, a shear-to-tension conversion mechanism is proposed for the contribution of wrinkles on torque capacity. As a result, the graphane annulus anomalously has similar torque capacity to pristine graphene annulus. The competition between hydrogenation caused bond strength deterioration and wrinkling induced local stress state conversion leads to a U-shaped evolution of torque strength relative to the increase of hydrogen coverage from 0 to 100%. Such hydrogenation tailored topological and mechanical characteristics provides an innovative mean to develop novel graphene-based devices. Possessing exceptional chemical145891113151627Annular graphene as a popular structures in nanoelectronicsRo of the initial structure is defined as the outer boundary, and the atoms inside the inner radius Ri is the inner boundary . It is contrary to our expectations that the torque strength of graphane annulus composed of weak spsp2 bonds. In order to systematically reveal the effect of hydrogenation on torsion strength of graphene annulus, we further calculated the torque strength of graphene with given inner radius at varying hydrogen coverage from 0–100%. As shown in H < 50%, and reaches a minimum at H = 50%. When H > 50%, the torque strength anomalously increases with increasing hydrogen coverage. Each point of Ri = 1, 1.5, 3 nm. We also consider the effect of the size of outer radius Ro on the torque strength of annulus with fixed inner radius Ri, and the variation of torque strength is negligible for both graphene and graphane over a wide range of outer radius Ro.  before the onset of torsion failure. Each atom is colored according to the displacement in the out-of-plane direction, and the difference in color intensity reveals the difference in wrinkle amplitude. Annular graphane with Ri = 1 nm has the largest amplitude among these three situations while the graphane with Ri = 3 nm has the smallest wrinkle amplitude. Such anomalous trend of wrinkle amplitude tuned by hydrogen coverage provide innovative fabrication concept for flexible electronic devices and strain sensors.It is noticeable that the three curves depicted in Ri, and the linear fitting lines of graphene and graphane are parallel to each other. It can be concluded that hydrogenation enhanced flexibility increases the wrinkling amplitude and decreases the wrinkle number compared to the wrinkling characteristics of pristine graphene.Another important wrinkling characteristic is wrinkle number surrounding the inner edge. Here, we consider the wrinkle number of fully hydrogenated annular graphene with different inner radius. Ro = 10 nm and Ri = 1, 1.5, 3 nm at varying hydrogen coverage from 0–100% under fixed boundary conditions. The evolution of torque strengths versus hydrogen coverage still follows the U-shaped trend with a minimum value at H = 50%. We also consider a larger scale graphene annulus with Ri = 9.5 nm and Ri/Ro = 0.3, which has the same Ri/Ro, and the surface area of the larger annulus is one order of magnitude larger than the model of H < 100% because of the conversion of sp2 to sp3 bonding33H = 0%, 50% and 100%, as well as the corresponding distribution of radial normal stress and circular shear stress, respectively. The wrinkles are apparently enhanced by hydrogenation and the stress distribution is strongly affected by the wrinkle configurations, especially at the regions close to the inner edge where failure initiates. By comparing the stress contour of graphene annulus at H = 0% and 50%, the stress distribution of randomly hydrogenated graphene  force field failure . The torRi<R<Ro, we first generated models of fully hydrogen functionalized annular graphene (H-coverage=100%) by bonding hydrogen atoms on both side of the graphene sheet alternatively .


Programmed death-ligand 1 (PD-L1) has been recognized as a critical and promising target in therapies that direct immune escape of cancers. However, its association with aggressive clinicopathological features in solid tumors remains unclear. We investigated this question by synthesizing published articles.Electronic databases were searched for relevant studies. Outcomes of interest included age, gender, tumor size, tumor size, lymph node metastasis and tumor cell differentiation.A total of 61 studies involving 17 types of malignancies were included. The overall expression rate of PD-L1 was 44.5% . Patients with regional lymph node metastases , large size tumor  or poor differentiated tumors  were associated with higher PD-L1 expression rate. However, no significant association was observed between young and elder patients , or male and female patients . A numerically higher PD-L1 expression rate was detected in polyclonal antibodies (57.2%) than monoclonal antibodies (39.6%). In addition, the PD-L1 expression rate reported by studies from Asian areas (52.3%) was numerically higher than those from non-Asian areas, namely Caucasians (32.7%).This meta-analysis indicated that patients with larger tumors, regional lymph node metastases, or poor-differentiated tumors were associated with a higher PD-L1 expression rate; in addition the expression rate of PD-L1 in Asians might be higher than that of Caucasians. This information might be useful in screening candidates for relevant tests and treatments. Programmed cell death ligand 1 (PD-L1), also known as B7 homolog 1 (B7H1), plays a critical role in T cells co-inhibition and exhaustion via the binding with programmed death 1 (PD-1) receptor expressed on activated T cells. RecentlPD-L1 expression has been observed in various solid tumors, including carcinomas of the esophagus, gastrointestinal tract, pancreas, breast, lung, and kidney –8. InhibRecent clinical trials show that blockage of PD-1/PD-L1 pathway with anti-PD-1 or anti-PD-L1 antibodies can produce durable remission in several different malignancies including, but not limited to, melanoma, lung, renal and bladder cancer–14. AlthA number of recent studies have reported on the clinicopathologic features of PD-L1 positive cancer. However the results of these studies were not consistent and sample sizes were relatively modest. Since PD-L1/PD-1 is a common pathway that functions in a wide spectrum of cancers, we performed a meta-analysis by incorporating all the available evidence to evaluate the association between clincopathological features and PD-L1 expression, aiming to identify those with high PD-L1 expression that may benefit from anti-PD-L1 therapy.Our initial search yielded 1158 potential literature citations. Of these, 863 were excluded after scanning titles and abstracts, leaving 295 citations for full-text assessment. Of the 295, 61 studies met the inclusion criteria and were used for this meta-analysis. The remaining studies were excluded largely because of the following reasons: (I) reported data regarding clinicopathological features was not stratified by PD-L1 status, (II) the data was reported in an unsuitable format and therefore unable to be compared to the others studies, or (III) they included data that overlapped with other studies.  were PD-L1 expression positive, and 821 (42.4%) of 1936 younger patients were PD-L1 expression positive. No significant association was found between PD-L1 expression and age  Figure .Thirty-eight studies, including 6675 patients, were analyzed for the association between PD-L1 expression and gender. Of 4309 male patients, 2298 (53.3%) were PD-L1 expression positive, and 1404 (59.3%) of 2366 female patients were PD-L1 expression positive. No significant association was found between PD-L1 expression and gender  Figure .The association between PD-L1 expression and size of tumor was analyzed in Thirty-eight studies, including a total of 6273 patients. Of 3417 tumors of large size, 1953 (57.6%) were PD-L1 expression positive, and 1070 (37.5%) of 2856 small size tumors were PD-L1 expression positive. There was a significant association betweenPD-L1 expression and tumors of large size  Figure .Thirty-four studies, including 6300 patients, were analyzed for the association between PD-L1 expression and lymph node metastasis. Of 2530 lymph node-positive patients, 1354 (53.5%) tested positive for PD-L1 expression, and 1443 (38.3%) of 3770 lymph node-negative patients tested positive for PD-L1 expression. A significant association was found between PD-L1 expression and the presence of lymph node metastases  Figure .Twenty-three studies, including 4735 patients, were analyzed for the association of PD-L1 expression and differentiation of tumor cells. Of 1791 patients with poor tumor differentiation (grade III), 668 (37.3%) were PD-L1 expression positive, and 880 (29.9%) of 2944 patients with well differentiation (grade I/II) were PD-L1 expression positive. A significant association exists between PD-L1 expression and tumor cell differentiation  Figure .Further subgroup analysis was performed by stratifying the studies according to the patients' origins (Asian & Non-Asian), primary anti-PD-L1 antibodies , IHC evaluation for PD-L1 (percentage & H-score), cut-off values for percentage (5% & 10%) and H-score (≤ 50 & > 50). The conclusions drawn from the subgroup analyses are similar to that of the overall analysis  that promote cell proliferation, and induce PD-L1 expression as well . On the EGFR) mutation status has a similar predictive role in the efficacy of EGFR-targeting therapy for patients with non-small cell lung cancer (NSCLC) [With regard to the immune evasion of solid tumor mediated by PD-1/PD-L1 immune-checkpoint pathway, a series of specific inhibitors are currently under investigation and in clinical development such as nivolumab, a fully human IgG4 PD-1 immune checkpoint inhibitor antibody, pembrolizumab (formerly known as MK-3475 or lambrolizumab), a high affinity humanized IgG4 monoclonal antibody targeting PD-1, and MPDL3280A, an engineered IgG anti-PD-L1 antibody , 15, 28. (NSCLC) . As for  (NSCLC) .The higher expression rate of PD-L1 among patients of Asian origin, as shown in our results, may be associated with higher frequencies of virus-associated malignancies in Asian areas. PD-L1 over expression has been reported to be associated with viral infection and chronic inflammation . IntratuTo the best of our knowledge, this is the first study to comprehensively demonstrate the relationship between PD-L1 status and clinicopathological parameters of solid tumors. However, several limitations existed: (I) Different tumors may have different biologic behaviors, but we failed to conduct this particular subgroup analysis as data for some tumor types was insufficient; (II) Cut-off values distinguishing high or low levels of PD-L1 expression determined by IHC evaluation and the primary antibodies varied in different types of tumors, which might cause heterogeneity of the overall results; (III) Different studies adopt different thresholds for the size of large tumors, as well as the age for elder patients, which can also lead to the heterogeneity of the pooled result. (IV) As a result of the lack of sufficient data, we were unable to evaluate the relationship between clinicopathological parameters and PD-1/PD-L1 status on the tumor infiltrating lymphocytes (TILs) which is also associated with immune evasion of tumor and responsiveness of immunotherapy.Regardless of the above limitations, this comprehensive analysis demonstrates the relationship between PD-L1 expression and clinicopathological features of solid tumors. The results may lead to improvement in the outcome of anti-PD-1/PD-L1 therapy by guiding patients' stratification in a convenient way. Further large-scale clinical studies should be performed to demonstrate the association between PD-L1 expression and clinicopathological characteristics in the subgroups of different tumor types. Further effort is also warranted to investigate the relationship between PD-1/PD-L1 status on TILs.In summary, meta-analysis of the literature shows that PD-L1 over expression on the tumor cells correlates with poor prognostic features including large tumor size, lymph node metastasis and poor differentiation of solid tumors. The PD-L1 expression rate in Asians might be higher than that in Caucasians. This information might prove to be helpful in screening candidates for relevant treatments.We conducted a systematic search of PubMed, Embase, and Cochrane databases for articles published from inception to July 2015. The following keywords were used: “PD-L1” or “B7-H1” or “CD274” or “PD-1” or “CD279” or “programmed cell death 1” combined with “cancer” or “tumor” or “carcinoma” with limits “human”. An additional search through Google Scholar and a manual search through reference lists of relevant reviews were additionally performed. Three authors  independently carried out the search. As Chinese investigators, we restricted our searches to studies published in either English or Chinese.Eligible studies fulfilled the following inclusion criteria: (i) original articles; (ii) the expression level of PD-L1 is tested by immunohistochemistry (IHC) staining on tumor cell specimens; (iii) data of the binary clinicopathological factors stratified by PD-L1 status were available. Studies that failed to meet the inclusion criteria were excluded.http://www.cochrane.de). The data on lead author, publication year, tumor type, patients' characteristics, patient origin, primary antibody, and PD-L1 status, clinicopathological features stratified by PD-L1 status were extracted by two investigators (LZY and XYT) independently. For uniformed data analysis, T1 was considered as small size, and T2, T3, and T4 as large size for CRC, EC and UC. For the histological grade, grades I and II (well/moderate differentiation) were grouped together vs grade III (poor differentiation). We defined large tumors in other tumor types and elder patients according to the criteria of each individual study. Reviewers (LZY and XYT) used the Newcastle-Ottawa scale specific to cohort study to assess all included studies. Discrepancies were discussed by all investigators to reach consensus. All eligible studies were of high quality.The data collection and assessment of methodological quality followed the QUORUM and the Cochrane Collaboration guidelines  and Review Manager 5.2 . The relationship between PD-L1 expression rate and antibody types, patients' origin, and IHC evaluation methods were investigated using independent statistical t-test respectively. Comparisons of dichotomous measures were performed by pooled estimates of odds ratios (ORs), as well as their 95% CI. Subgroup analysis was conducted according to patients' origin, primary antibody  and IHC evaluation method  respectively. All CIs had two-sided probability coverage of 95%. A statistical test with P-value less than 0.05 was considered significant.An extensive search strategy was made to minimize the potential publication bias. Graphical funnel plots were generated to visually assess publication bias. The statistical method to detect funnel plot asymmetry was the Begg's test.


In addition, as a proof-of-concept, highly B-doped graphene nanoplatelets were used as an electrode of an electrochemical double-layer capacitor (EDLC) and showed an excellent specific capacitance value of 448 F/g in an aqueous electrolyte without additional conductive additives. We believe that B-doped graphene nanoplatelets can also be used in other applications such as electrocatalyst and nano-electronics because of their reliable and controllable electrical properties regardless of the outer environment.For the utilization of graphene in various energy storage and conversion applications, it must be synthesized in bulk with reliable and controllable electrical properties. Although nitrogen-doped graphene shows a high doping efficiency, its electrical properties can be easily affected by oxygen and water impurities from the environment. We here report that boron-doped graphene nanoplatelets with desirable electrical properties can be prepared by the simultaneous reduction and boron-doping of graphene oxide (GO) at a high annealing temperature. B-doped graphene nanoplatelets prepared at 1000 °C show a maximum boron concentration of 6.04 ± 1.44 at %, which is the highest value among B-doped graphenes prepared using various methods. With well-mixed GO and g-B Graphene is one of the most promising materials for nanoelectronic applications including transparent conductive electrodes, electrocatalysts, field effect transistors, chemical/biological sensing platforms and energy devices because of its large surface area, high chemical stability and mechanical or electrical robustness. For this reason, the demand for graphene has increased dramatically in recent years123sp2-bonded carbon networks with hetero atoms such as boron or nitrogen, which is called substitutional doping5678101112141516176101112One of the most promising routes to modulate the electrical properties of graphene is the chemical substitution of carbon atoms in On the other hand, the chemical treatment of rGO or GO has advantages for bulk synthesis and incorporation into a solution process, which is quite beneficial for the easy formation of structural electrodes for energy applications. However, the doped graphene nanoplatelets produced from rGO or GO have poor doping uniformity and low doping efficiency. Therefore, investigations into techniques for substitutional doping of graphene nanoplatelets suitable for bulk synthesis and uniform doping with high efficiency are necessary to improve the electrical properties of doped graphene nanoplatelets and expand their applications into energy-related materials.et al. reported that rGO can be doped with boron from boron trichloride (BCl3) gas by heat treatment, but only a low efficiency of 0.88 at % boron doping was obtainedet al. reported that B-doped graphene nanoplatelets could be synthesized by the reaction of GO with a borane-tetrahydrofuran adduct (BH3-THF) under reflux in aqueous solution. However, the reaction time was too long, and the boron concentration was quite low at 1.1 at %Most studies on substitutional doping of graphene nanoplatelets using GO or rGO as starting materials have focused on nitrogen-doped graphenes. N-doped graphene nanoplatelets have been extensively synthesized by the treatment of GO with nitrogen-containing chemicals such as ammonia and urea, and doping efficiencies of approximately 5–8.05 at % have been achieved192021232O3). The GO/B2O3 mixture was annealed at various temperatures to systematically control the efficiency of boron doping and the degree of reduction of GO. B-doped graphene nanoplatelets prepared at 1000 °C showed the maximum boron concentration of 6.04 ± 1.44 at % which is the highest value among B-doped graphenes produced by CVD, arc discharge or the substitutional doping of GO or rGO. In addition, B-doped graphene nanoplatelets show a uniform distribution of boron, indicating that this simple method is very useful for bulk synthesis of B-doped graphene nanoplatelets with uniform high-concentration boron doping. Finally, as a proof-of-concept, we have also demonstrated highly B-doped graphene nanoplatelets as an electrode in an electrochemical double-layer capacitor (EDLC), indicating their potential for use in energy storage applications. Highly B-doped graphene nanoplatelets prepared at 1000 °C showed excellent specific capacitance value of 448 F/g in an aqueous electrolyte, which is 3-folds higher than that of a thermally reduced GO electrode without boron (135 F/g). The improved specific capacitance of B-doped graphene nanoplatelets is due to their great enhancement in electrical conductivity and specific surface.In this study, we demonstrated that B-doped graphene nanoplatelets can be prepared by simple thermal annealing of GO nanoplatelets combined with boron oxide  was also prepared as a control.Graphene oxide (GO) obtained by the oxidation and exfoliation of graphite is the most promising candidate as a starting material for bulk synthesis of doped graphene nanoplatelets. 2O3 at 196.0 eV, demonstrating that boron atoms are linked with carbon atoms in the sp2 carbon network of rGO. The peaks at 190.8 and 192.6 eV arise from the B atoms of BC3 and BC2O, respectively3 at 190.8 eV becomes more predominant at 1000 °C. This demonstrates the formation of more B-C bonds in carbon network and the reduction of more oxygen groups with increasing temperature.BT-rGO and T-rGO samples annealed at various temperatures were quantitatively and qualitatively characterized by X-ray photoelectron spectroscopy measurements (XPS). 9272O3, as confirmed by X-ray diffraction (XRD) measurement (2O3 and β-B2O3) and a g-B2O3 glass-like phase of boron (III) oxide are known to exist, where the local structure of g-B2O3 is closest to the structure of α-B2O3312O3 shift toward lower 2Θ positions with increasing temperature. In particular, the interplanar spacings of g-B2O3 calculated from the first halo increases from 4.00 Å (2Θ = 22.2°) at 25 °C to 4.48 Å (2Θ = 19.8°) at 1000 °C. Furthermore, thermogravimetric analysis (TGA) of g-B2O3  is also slightly increased from 88.7 to 89.0 cm−1. This confirms the boron doping as reported in the literature9−1 due to the many defects derived from insufficient reduction and boron doping in the structure, as well as a higher intensity ratio of D and G band (ID/IG) of 0.93, compared to 0.82 for T-rGO. The in-plane crystallite sizes calculated using the equation reported in the literatureRaman spectroscopy is the most powerful and non-destructive technique for identifying the structure and quality of graphene39332O3. The atomic percentage of boron determined from the EELS mapping is 6.88%, which is approximately consistent with that from the XPS analysis.The boron atoms on the basal plane of BT-rGO are uniformly distributed, which is confirmed by the electron energy loss spectroscopy (EELS) mapping. Finally, we investigated the effect of boron doping on the electrical properties of BT-rGO and demonstrated, as a proof-of-concept, a highly B-doped rGO electrode in an electrochemical double-layer capacitor (EDLC) to confirm the potential of this material for use in energy storage applications. The current-voltage (I-V) curves in 9et al., even though no conductive additives such as carbon black were usedGraphene has many inherent advantages as an electrode of a supercapacitor, such as its high surface area, high electrical conductivity, light weight, and excellent flexibility362138262 adsorption measurements were performed to investigate the specific surface area of the BT-rGO electrode. As shown in The specific capacitance of a supercapacitor is mainly affected by the electrical conductivity and specific surface area of the electrode. As mentioned above, the great enhancement in the electrical conductivity of the BT-rGO electrode definitely contributes to a reduction in the resistive loss in the capacitor circuit, and thus using a doped electrode can improve the capacitance without the use of conductive additives. Furthermore, Brunauer-Emmett-Teller (BET) N2O3 as a dopant. At a high annealing temperature, the oxygen-containing groups of GO are removed, and defects thus obtained can act as active sites for the reaction between carbon and boron atoms released by thermal decomposition of g-B2O3. BT-rGO annealed at 1000 °C shows the maximum boron concentration of 6.04 ± 1.44 at %, which is the highest value among B-doped graphenes produced by CVD, arc discharge or the substitutional doping of GO or rGO. Boron atoms are uniformly doped into the graphene networks and the amount of boron can be easily controlled by changing the annealing temperature. Furthermore, this process is suitable for gram-scale production of B-doped graphene nanoplatelets using laboratory equipment. Therefore, B-doped graphene nanoplatelets could be utilized in various energy storage and conversion applications requiring three-dimensional nanostructures and facile synthesis. We also demonstrated, as a proof-of-concept, a highly-B-doped graphene electrode in an electrochemical double-layer capacitor (EDLC). B-doped graphene nanoplatelets have an improved electrical conductivity due to the increased charge carrier concentration derived from boron doping and defect-like small pores in the basal plane of the graphene sheet. This improved conductivity leads to a high specific capacitance of 448 F/g without the use of conductive additives such as carbon black. We believe that B-doped graphene nanoplatelets can be used in other applications such as biological/chemical sensors, electrocatalyst and nano-electronics because of their reliable and controllable electrical properties regardless of the outer environment, unlike N-doped graphene nanoplatelets.In summary, we herein demonstrated that boron-doped graphene nanoplatelets (BT-rGO) can be prepared by the simultaneous reduction and doping of GO using simple thermal annealing with g-B2SO4 and reagent-grade B2O3 were purchased from Sigma-Aldrich, and KMnO4 and 30% H2O2 aqueous solution were obtained from Yakuri. All chemicals were used without further purification. Graphene oxide (GO) was prepared via the modified Hummers method using expandable graphite flake as a starting material2O3, 20 mg) using an ultrasonicator for 1 h. Then the suspension was dried using a freeze dryer (Operon FDCF-12001) and subjected to thermal annealing at 300, 500, 700 or 1000 °C using a tubular furnace in an N2 atmosphere for 1 h. Finally, the obtained black powder was washed with hot distilled water and filtered, and then this procedure was repeated three times in order to remove the residual boron oxide. Thermally reduced GO without boron substitution (T-rGO) was prepared as a control. In brief, 40 mL of GO suspension was dried using a freeze dryer and subjected to thermal annealing as described for the preparation of BT-rGO. The obtained T-rGO black powder was also washed and filtered as described above.Natural grade, ~100 mesh, 99.9%  graphite powder was purchased from Asbury Graphite Mills, Inc. The 98% HTM, FEI) with an accelerating voltage of 300 kV and electron energy loss spectroscopy (EELS). X-ray photoelectron spectroscopy (XPS) measurements were carried out on a PHI 5000 VersaProbe (Ulvac-PHI) using monochromatic Al Kα radiation in order to quantitatively and qualitatively characterize BT-rGO and T-rGO. Raman spectroscopy  was also performed for characterization. The specific surface areas of T-rGO and BT-rGO were determined using the Brunauer–Emmett–Teller method  with liquid nitrogen. Electrical conductivity measurements were carried out using a four-probe system consisting of a four-point cylindrical probe head (JANDEL Engineering Ltd.), a direct current precision power source , and a nanovoltmeter . Cyclic voltammetry  was performed to characterize the electrochemical properties with a three-electrode system with an aqueous 6 M KOH. A Pt plate and Hg/HgO served as a counter and reference electrodes, respectively. To fabricate a working electrode, BT-rGO powder (or T-rGO) and polyvinylidene fluoride  were mixed in a mass ratio of 95:5 and dispersed in N-methylpyrrolidone (NMP) solvent using a paste mixer. The resulting paste was spread onto nickel foam (1 cm × 1 cm) and dried at 120 °C under vacuum. The prepared electrode was immersed in DI water as a non-solvent for 12 h to remove NMP solvent where exchange occurred between NMP and waterIdV)/(vmV), where I is the response current density (A/cm2), V is the potential, v is the potential scan rate (mV/s), and m is the mass of the electroactive materials in the electrodes (g). The mass (m) of the prepared electrode determined by a microbalance was approximately 1 mg/cm2.The surface morphology and thickness of GO and BT-rGO were characterized using an atomic force microscope  at room temperature in non-contact mode with 10 nm standard cantilevers . The structural and chemical composition of BT-rGO were characterized using a transmission electron microscope .Supplementary Figures 1-6


Hence, there is a major interest in in vivo imaging of this receptor. A PET tracer would allow non-invasive in vivo visualization and quantification of the MCHR1. The aim of the study was the ex vivo evaluation of the MCHR1 ligand [18F]FE@SNAP was injected directly into the jugular vein of awake naïve rats for ex vivo brain autoradiography, biodistribution and additional blood metabolite analysis. Blocking experiments were conducted using the unlabeled MCHR1 ligand SNAP-7941.[18F]FE@SNAP was observed in the lateral hypothalamus and the ventricular system. Both regions were significantly blocked by SNAP-7941. Biodistribution evinced the highest uptake in the kidneys, adrenals, lung and duodenum. Specific blocking with SNAP-7941 led to a significant tracer reduction in the heart and adrenals. In plasma samples, 47.73 ± 6.1 % of a hydrophilic radioactive metabolite was found 45 min after tracer injection.A high uptake of [18F]FE@SNAP uptake was significantly blocked in the lateral hypothalamus, there is strong evidence that [18F]FE@SNAP is a highly suitable agent for specific MCHR1 imaging in the central nervous system. Additionally, this finding is supported by the specific blocking in the ventricular system, where the MCHR1 is expressed in the ependymal cells. These findings suggest that [18F]FE@SNAP could serve as a useful imaging and therapy monitoring tool for MCHR1-related pathologies.Since [ The melanin-concentrating hormone receptor 1 (MCHR1) plays a key role in energy homeostasis and obesity . Further11C]SNAP-7941 was developed as the first PET tracer for the MCHR1 and was evaluated in a preclinical study [18F-fluoroethylated analogue [18F]FE@SNAP  and selectivity  towards the MCHR1 FE@SNAP for specific MCHR1 brain imaging in healthy rats. Therefore, [18F]FE@SNAP was administered IV for ex vivo brain autoradiography and additionally to study biodistribution and to search for potential circulating metabolites. It is noteworthy that IV application was performed through the jugular vein, allowing animals to be awake and conscious, hence excluding the well-known significant anaesthetic influence on imaging results FE@SNAP and [18F]altanserin were performed in a microfluidic device (Advion NanoTek®) followed by a purification in a conventional synthesizer (Nuclear Interface®) as described elsewhere [18F]FE@SNAP was formulated in physiological saline solution .Radiosyntheses of [lsewhere . [18F]FEn = 3) received vehicle (400 μL), and rats of the blocking group (n = 3) received SNAP-7941  via the jugular vein 30 min prior to tracer application. Via the jugular vein, 51.33 ± 26.2 MBq of [18F]FE@SNAP  was then administered to all rats. After 45 min, rats were sacrificed by IV ketamine injection and decapitated, and the brains were removed and immediately quick frozen in isopentane (−45 °C) for ex vivo autoradiography. Other organs including the eyes, tongue, muscle, epidermal white adipose tissue (WATep), heart, lung, stomach, pancreas, liver, duodenum, colon, spleen, kidneys, adrenals, testis, bladder and bone as well as blood and urine were removed, weighed and measured in a gamma counter . Radioactivity concentrations were normalized to dose and weight and expressed as percent injected dose per gram (%ID/g). To determine significant differences, a two-tailed t test with α = 0.95 was performed using the statistics add-on in Microsoft Excel® 2013. A value of P < 0.05 was considered as significant.Conscious and freely moving rats of the baseline and blocking groups were always examined simultaneously: rats of the baseline group  to separate cellular components. Sample cleanup was performed by vortexing plasma with the equivalent amount of acetonitrile and by subsequent centrifugation  to remove precipitated proteins. The obtained supernatant was applied to radio-thin-layer chromatography  and analysed via a Canberra-Packard Instant Imager.For analysis of potential circulating metabolites, blood samples from the baseline group were collected into heparinized tubes and immediately stored on ice before processing. The blood was centrifuged  were cut in a cryo-microtome  into 50-μm-thick slices and thaw-mounted onto superfrost slides . Samples were placed on Phosphor Imager plates  for an exposure period of 18 h and then analysed with a Cyclone Phosphor Imager . Data analysis was performed with OptiQuant® data processing software Version 5.0. Due to high-MCHR1 appearance, special emphasis was laid on the hypothalamus FE@SNAP  showed tracer uptake into the rat brains, with increased accumulation in the lateral hypothalamus and the ventricular system. In the blocking group (n = 3), whole-brain uptake was significantly higher than in baseline animals. Specific blocking with 15 mg/kg SNAP-7941 evinced a significant reduction of tracer uptake in the lateral hypothalamus and in the ventricular system  are shown in Fig. The results of the biodistribution (n = 3) evinced 51.50 ± 5.5 % of the parent compound and 47.73 ± 6.1 % of a hydrophilic radioactive metabolite (probably [18F]fluoroethanol) 45 min after tracer application.Analysis of metabolites in the blood (Preliminary small-animal PET measurements in a healthy rat under baseline conditions showed a high tracer uptake in the ventricular system Fig. .Fig. 5ExSpecific MCHR1 imaging is of high clinical interest for status monitoring in endocrine pathologies like obesity and diabetes. A PET tracer for MCHR1 comprises several advantages for clinicians and patients as the in vivo monitoring and following of the hormone receptor status and related pathologies. Moreover, it could support dose selection of MCHR1 antagonists in drug development .18F]FE@SNAP to specifically label MCHR1-rich regions like the lateral hypothalamus [18F]FE@SNAP was injected IV into healthy and conscious rats followed by ex vivo brain autoradiography, biodistribution and analysis of potential blood metabolites.The focus of this study was to investigate the potential of 1FFE@SNAP 18F]FE@SNAP was administered directly into the jugular vein of awake and conscious rats. IV application of [18F]FE@SNAP showed a high and specific uptake in the ventricular system—where the ependymal cells are recently known to express the MCHR1 FE@SNAP also in the adrenals, eyes, tongue, muscle, pancreas and colon which is in line with literature FE@SNAP in the lateral hypothalamus and in the ependymal cells of the third ventricle epithelium. Hence, as a consequential step, future small-animal PET experiments are feasible.The high resolution of ex vivo autoradiography using a Phosphor Imager allowed identification of specific uptake of [18F]FE@SNAP proved these characteristics, which provides strong evidence that it is a highly specific agent for MCHR1 imaging. Involvement of MCHR1 was reported in diabetes and obesity, and MCHR1 has also been related to asthmatic seizures, colitis, depression, anxiety and promotion of sleep. Against this background, [18F]FE@SNAP could serve as a useful tool for imaging and therapy monitoring for MCHR1-related pathologies.Since the MCHR1 is predominantly expressed in the lateral hypothalamus as well as in the ependymal cells of the third ventricle epithelium, a tracer for the MCHR1 should show specific uptake and be significantly blocked by an unlabeled ligand in these areas. [


A sample of 416 males from western and eastern Andalusia has been jointly analyzed for surnames and Y-chromosome haplogroups and haplotypes. The observed number of different surnames was 222 (353 when the second surname of the Spanish system of naming is considered). The great majority of recorded surnames have a Castilian-Leonese origin, while Catalan or Basque surnames have not been found. A few Arab-related surnames appear but none discernible of Sephardic-Jewish descent. Low correlation among surnames with different population frequencies and Y-chromosome markers, at different levels of genetic resolution, has been observed in Andalusia. This finding could be explained mainly by the very low rate of monophyletic surnames because of the historical process of surname ascription and the resulting high frequencies of the most common Spanish surnames. The introduction of surnames in Spain during the Middle Ages coincided with Reconquest of the territories under Islamic rule, and Muslims and Jews progressively adopted the present male line surname system. Sampled surnames and Y-chromosome lineages fit well a power-law distribution and observed isonymy is very close to that of the general population. Besides, our data and results show that the reliability of the isonymy method should be questioned because of the high rate of polyphyletic surnames, even in small geographic regions and autochthonous populations. Random isonymy would be consistently dependent of the most common surname frequencies in the population. To achieve a refined knowledge of the human population structure and its reconstruction is advisable to apply different but complementary knowledge bases, which are not only strictly biological. This multidisciplinary approach seeks to reconcile and interpret insights derived from historical events, population dynamics including demographic characteristics, cultural behaviours (mating patterns), and geography with expected strong effects upon extant gene pool.Surnames, considered key cultural markers, are being used to further investigate and enhance the genetic signals of population structure when analysing genetic data ,2. In moThe short evolutionary time of the generalized use of surnames in Europe, which started during Middle Ages, polyphyletism, illegitimacies, adoptions and changes of surnames from their original form (e.g. grammar spellings) all have weakened the strength of relationships between surnames and Y-C lineages. In the vast majority of the countries, the amount of recorded surnames are dozens of thousands, which have represented in turn several orders of magnitude greater than the observed number of specific Y-C haplogroups, and even haplotypes, with the usual number of screened SNPs or STRs, respectively. We will check these points, among others, by analyzing the distributions of surnames and Y-chromosome lineages in a large sample of Andalusian males.García (~3%) while broad groups of Y-chromosomes among western Europeans carry the haplogroup R1b1a2-M269 (60–80%) . Genomic DNA was extracted from total blood (≈5 ml) (taken on EDTA tubes) using standard proteinase-K digestion followed by phenol-chloroform extraction and ethanol precipitation. DNA samples were firstly genotyped for 17 Y-STRs (Short Tandem Repeats or microsatellites)  included in AmpFlSTR Yfiler Kit (Life Technologies). Granada sample (n = 249) was reduced for technical reasons to 223 individuals when typing the array of microsatellites. Alleles of microsatellites were designated based on the number of variable repeats included  are that it uses the less informative part of the data. This means it is strongly biased having a mean quadratic error from six to eight times higher than the maximum likelihood estimator  was 0.6250±0.0281. As expected, the weight of singleton surnames was higher (h = 0.6990±0.0412) than that calculated for repeated surnames (h = 0.5787±0.0368).Haplogroup diversity, h = 0) and the majority (24/27) were associated to R1b1a2-M269 haplogroup. Cases in which the same surname occurs two or more times within the same haplogroup accounts for 184 out of 257 of which 155 correspond to the abovementioned marker, with peak frequencies in western Europe (60–70%). In the study sample the frequency of R1b1a2-M269 was 62%. Furthermore, a significant relationships between frequency distributions of R1b1a2-M269 and surname occurrences was observed . When using Fisher's exact test, the P-value was very similar (0.016). The level of significance can be explained by large deviations between observed and expected counts. The categories of cells 4 and 2 (see M & M) with standardized residuals lower than 2.00 were those having a minor contribution to the chi-square (χ2) value. Similar statistical analyses for other haplogroups are unfeasible because their low number of cases in the population (≤ 5).A number of 27 out of 63 repeated surnames (42.86%) shared identical haplogroups  with R1b1a2-M269 being the most common haplogroup in all cases. The presence of other Y-chromosomal haplogroups: E-V13, E-M81, E-M34, J1-M267, J2-M172, I1-M253, I2-P215 and G2a-P15 would be interpreted as a consequence of the genetic history linked to the Iberian Peninsula, especially, to Andalusia, with a long, prevailing contacts with the Mediterranean world. The findings would be showing indeed that a remarkable number of descendants of distant migrants established in the Andalusia territory adopted the most common Spanish surnames.Haplotypes based on multiple Y-specific microsatellites represent more sensitive indicators of recent coancestry than haplogroups. They are highly variable and have much lower average population frequencies than do haplogroups, so the chance of sharing is less likely .H was particularly high  being the mean number of mutational steps (number of loci differences) between haplotypes (7 locus Y-STRs) 3.86±1.96 (3.75±1.89 when only examining the group of repeated surnames).From the 390 DNA male samples, the number of different haplotypes observed was 184 (7 locus Y-STRs) and 354 (17 locus Y-STRs). As expected, the extent of global haplotype diversity, i). A great number of haplotypes exhibited the condition of being singletons: 124/390  and 323/390 , ii). There are only a small group of 39 Y-chromosomes  that bearing different surnames share all the same haplotype: DYS19(14)-DYS389I(13)-DYS389II(16)-DYS390(24)-DYS391(11)-DYS392(13)-DYS393(13). When examining haplotypes across 17 loci the modal haplotype (n = 5) was: DYS19(14)-DYS389I(13)-DYS389II(16)-DYS390(24)-DYS391(11)-DYS392(13)-DYS393(13)-DYS438(12)-DYS439(12)-DYS437(15)-DYS448(19)-DYS456(15)-DYS458(15)-DYS635(24)-Y GATA H4(12)-DYS385 (11–15) and, iii) A low proportion of sharing haplotypes within surnames has been observed .It has been pointed out that unrelated men sharing surnames were significantly more likely to share haplotypes than are men carrying different surnames ,23. TablIn summary, our study sample shows few concordances between Y-C haplotypes and surnames. False paternities and mutational pressure can cause part of the discrepancies. Mutational events (genetic or linguistic) seem to have a low effect here due to the few generations passed from the introduction of surnames to the present and the rather rigid and perdurable orthography and phonetics of the Spanish language. False paternities would not be relevant here, because the most common haplotypes are mostly composed of different surnames and birthplaces of individuals carrying those haplotypes are different, too. Obviously, false paternity events tend to occur in the same locality or in its proximity.http://www.ifmj.org). The former list comprises 1620 Arab surnames and the latter one 5220 Sephardic surnames. Eleven different Spanish surnames with Arab origin were detected (5 first surnames and 11 second ones) all of them singletons in each group. Of the 5 first surnames, two are linked to the Y-chromosome R-M269 haplogroup whereas the rest were associated to E-M81, E-V13 and J2b2-M241 lineages. Thus, some relationships between specific NRY haplogroups and Spanish-Arab surnames with origin in northern Africa and eastern Mediterranean are observed in our Andalusian sample. By contrast, many of those Sephardic surnames are high or moderately frequent in Spain and coincident with those registered in our sample. Since Sephardic surnames are scarcely distinctive, in this case the provided information about the geographic origin on Y-C lineages related to Sephardic-Jewish surnames in our sample is consequently poor.First sampled surnames have been compared with available lists of Spanish surnames of Arab and Jewish origins ; toponymic ; occupational , and of appearance  would not be expected to show higher relationships with specific Y-C lineages than others. Some exceptional cases could be found out in extremely rare surnames in the population.The observed findings would be mainly explained because of the historical process of surname ascription or adoption, the high level of polyphyletism, and discrepancies between surnames and Y-chromosome lineage distributions in human populations. Main types of surnames such as patronymic . The three Y-C harbored the J2a1-M172 haplogroup with two of them sharing indeed 17 loci Y-STRs haplotype. These results suggest that the sampled “vc” males would be descendants of a member of that Muslim garrison, revealing then a clarifying case of Arab ancestry.The observed spectrum of surnames in the study Andalusia sample is supported by history. Castilian-Leonese men mainly carried out the Reconquest of southern Spain from the Muslim rulers, and many of those people resettled there. Leon and Castile regions had a high demographic size in the Middle Ages and played a dominant role during the process of the Reconquest of Spain. Thus, it can explain that the great majority of our sampled surnames have a Castilian-Leonese etymology whereas Galician and Portuguese surnames are occasional, despite that Huelva province is adjoining to Portugal. Only few surnames tracing to an Arab etymology have been detected but it was not the case for Catalan, Basque or even discernible Jewish surnames. Successive historical human movements into Andalusia have not significantly changed this scenario. A curious signal of preferences shown by Berber and Jewish people, by embracing particular Christian surnames, can be exemplified by “mo” (dark-haired man) and “rb”  both reaching moderate frequencies in Spain. The former registers six occurrences in the sample and all of them are linked to R-M269 lineage; the latter surname “rb” presents three occurrences and each Y-chromosome carries different haplogroups, being again the R-M269 one of them. It seems striking that a Mediterranean population as the Spanish is, mostly characterized by people with hair dark or dark-brown, the “mo” surname is less genetically diverse than those carrying “rb” surname. However, when the great majority of individuals within a population is well-defined by a certain phenotypic physical feature only people belonging to the extremes of the trait distribution can be successfully distinguished, and these outliers might be more genetically homogeneous and supposedly denoted with the same surname. On the contrary, the portion of the population which does not carry the common trait would be composed by people carrying the complementary trait in several degrees and forming then a more heterogeneous genetic group.Other examples do reference to “appearance” surnames, such as “k (surnames) in a sample of size n in equilibrium, for large n, is asymptotically θ ln n [k = θ ln(1+n/θ) [Larger, but economically feasible sample sizes than that analyzed here would not substantially increase the scope and reliability provided by the present study, due to the great disparity between the amounts of donors (sample size) and population surnames. The average number of alleles y θ ln n   ). Using a priori feasible though it would not provide probably a significant difference due to the very high value of the chi-square limit. The number of different surnames fluctuates considerably due to the birth and death process  governing usually singletons and quasi-singletons, and this number is difficult to define [By comparing observed surname distributions in the sample against the population where the sample was extracted, it is possible to infer whether that sample is biased or not. This comparison must be a routine for the study of human populations. Surname distributions in populations of most countries are known from national population censuses or from other data sources such as telephone and/or poll lists. Although neutrality tests cannot be performed on these huge data sets—hundreds of thousands- of surnames   almost surely national surname distributions would not fit well the IAM, due to similar causes than those acting on our study sample. However, a chi-square test between sampled and those all recorded population surnames is o define . Some stAs far as we know, it is the first time that a joint study of surnames and Y-chromosome markers in a representative population sample has been performed, at least in Spain. Other surveys ,2,22 useIn summary, it should be important to notice that the reliability of the isonymy method should be questioned for the high rate of polyphyletic surnames, even in small geographical regions and autochthonous populations. When isonymy is used for evaluating human inbreeding, estimated values are consistently dependent of the most common surname frequencies in the population. The low correlation observed in Andalusia between surnames with different population frequencies and Y-chromosome lineages, at different levels of genetic resolution, would be mainly explained by the very low rate of monophyletic surnames, because of the historical process of surname ascription. Both conclusions might be confidently extended to other countries in the world.S1 Table(DOCX)Click here for additional data file.


Trametes versicolor (TvL) on functionalized graphene oxide (fGO). The catalytic properties of the fGO-TvL nanoassemblies were found to depend on the number of the graphene oxide-enzyme layers present in the nanostructure. The fGO-TvL nanoassemblies exhibit an enhanced thermal stability at 60 °C, as demonstrated by a 4.7-fold higher activity as compared to the free enzyme. The multi-layer graphene oxide-enzyme nanoassemblies can efficiently catalyze the oxidation of anthracene, as well as the decolorization of an industrial dye, pinacyanol chloride. These materials retained almost completely their decolorization activity after five reaction cycles, proving their potential as efficient nano- biocatalysts for various applications.Multi-layer graphene oxide-enzyme nanoassemblies were prepared through the multi-point covalent immobilization of laccase from  Graphene, graphene oxide and their derivatives have attracted great interest in biotechnology and biomedicine applications such as in gene and drug delivery, bioimaging, as well as in the construction of biosensors and biomedical devices ,2,3, sinin vitro cascade reactions  bands can be used to evaluate the quality of graphene-based materials [D/IG intensity ratio was found to be 1.03 in the case of GO, while the ratios for both fGO-TvL multilayers nanoassemblies is equal to 1.07 indicating that the immobilization of the enzyme on the graphene nanosheets leaves the structure of GO unaffected.Raman spectra of the functionalized GO and fGO-TvL multilayer nanoassemblies (fGO-TvL-fGO-TvL-fGO-TvL and fGO-TvL-fGO-TvL-fGO-TvL-fGO) compared with the pristine GO and TvL are shown in xidation . The D paterials . The ID/The catalytic activity of the fGO-TvL nanoassemblies was determined using the oxidation of ABTS at room temperature as model reaction. The catalytic activity of TvL was decreased from 13.6 U/mg of free enzyme to 0.55 U/mg for fGO-TvL. The reduction of the enzyme activity upon immobilization observed here is in accordance with that reported for several enzymes and could be due either to partial denaturation of enzymes during the immobilization procedure or to mass transfer effects which can lead to a reduced catalytic efficiency compared to soluble enzymes ,43.As it can be seen from The thermal and operational stability of immobilized enzymes grafted onto various nanosupports is of great importance for technological applications of nanobiocatalytic systems, including biosensing and nanobiocatalysis ,45. In tPolycyclic aromatic hydrocarbons (PAHs) are highly toxic industrial pollutants, resulting from processes such as coal gasification, coking, and wood preservation. Due to their toxic effects PAHs pose a serious health risk to all forms of life, including humans, and some PAHs are known to be highly mutagenic and carcinogenic, thus their degradation is of high importance .In order to evaluate the ability of fGO-TvL nanoassemblies to catalyze the oxidation of PAHs, anthracene was used as model substrate and its biodegradation by fGO-TvL nanoassemblies in the presence of the mediator HBT (1 mM) was studied. The mediator HBT acts as a sort of electron shuttle between the enzyme and the substrate. Once oxidized by laccase, the mediator diffuses away from the enzymatic pocket and in turn oxidizes other molecules, extending the range of substrates susceptible to the enzymatic action and increasing the catalytic activity of the enzyme ,51.The degradation of anthracene by different fGO-TvL nanoassemblies is presented in Synthetic dyes originate mainly from textile and plastic industries and are resistant to biodegradation, resulting in serious health hazards to animals and humans. Laccases have been found to be able to catalyze the oxidation of dyes and have potential applications in detection  and biorTo investigate the ability of fGO-TvL to catalyze the degradation of dyes, we chose as model substrate pinacyanol chloride, a symmetric trimethinecyanine dye widely applied as an optical sensitizer in photographic processes . Figure If fGO-TvL nanoassemblies are to be employed for sustainable industrial applications, waste minimisation by reuse is an important issue. Reuse was therefore investigated with the oxidation of pinacyanol chloride as model reaction. As it can be seen from Trametes versicolor on functionalized graphene oxide and their use in different biocatalytic applications. The catalytic activity of the fGO-TvL nanoassemblies, as well as their ability to catalyze the degradation of anthracene and pinacyanol chloride, increased with the number of nanomaterial and enzyme layers and was higher when the external layer of the multi-layer nanoassemblies was formed by the enzyme. Moreover, the graphene oxide-enzyme nanoassemblies exhibited higher thermal stability than the free enzyme and reactivity was maintained at higher temperatures when the number of fGO-TvL layers increased. The high catalytic activity against various substrates together with the enhanced thermal and operational stability, make these structures promising nanobiocatalytic systems for use in bioanalytical devices, biofuel cells and biotransformations.In the present study, we demonstrated the development of novel graphene oxide-enzyme assemblies through the multi-point covalent immobilization of laccase from


Arabidopsis CRINKLY4 (ACR4) is a receptor-like kinase (RLK) involved in the global development of the plant. The Arabidopsis genome encodes four homologs of ACR4 that contain sequence similarity and analogous architectural elements to ACR4, termed Arabidopsis CRINKLY4 Related (AtCRRs) proteins. Additionally, a signaling module has been previously proposed including a postulated peptide ligand, CLE40, the ACR4 RLK, and the WOX5 transcription factor that engage in a possible feedback mechanism controlling stem cell differentiation. However, little biochemical evidence is available to ascertain the molecular aspects of receptor heterodimerization and the role of phosphorylation in these interactions. Therefore, we have undertaken an investigation of the in vitro interactions between the intracellular domains (ICD) of ACR4, the CRRs and WOX5. We demonstrate that interaction can occur between ACR4 and all four CRRs in the unphosphorylated state. However, phosphorylation dependency is observed for the interaction between ACR4 and CRR3. Furthermore, sequence analysis of the ACR4 gene family has revealed a conserved ‘KDSAF’ motif that may be involved in protein-protein interactions among the receptor family. We demonstrate that peptides harboring this conserved motif in CRR3 and CRK1are able to bind to the ACR4 kinase domain. Our investigations also indicate that the ACR4 ICD can interact with and phosphorylate the transcription factor WOX5. Mammalian systems have acquired signal transduction mechanisms via the use of receptor tyrosine kinases (RTK) to coordinate cellular processes such as proliferation, migration, differentiation, and cell-cycle control . RTKs arSimilar to RTKs in architecture, plant receptor-like kinases (RLKs) contain an extracellular ligand binding domain, a transmembrane helix, and an intracellular serine/threonine kinase domain and are involved in various plant signaling processes –9. It isArabidopsis CRINKLY4 (ACR4) is a RLK involved in the proper growth and development of the plant  ATP . Proteins were incubated at room temperature for 1 h. Reactions were then terminated with the addition of 4X SDS sample buffer and boiled. Proteins were separated by 12% SDS-PAGE and radioactive bands were analyzed by phosphorimaging.Active sACR4 protein was incubated with the inactive sCRRm proteins. For each reaction 1 μg of sACR4 was incubated with ~1 μg of sCRRm in 20 μl of Kinase Buffer  in Kinase Buffer as described above. Following incubation, mWOX5 protein was pulled-down using amylose resin. The phosphorylation reactions were brought up to 500 μl with Wash Buffer . 25 μl bed volume of amylose resin was added to each reaction and incubated an additional 1 h on a rotator at room temperature. Reactions were spun down to pellet the resin and the supernatant was removed. The resin was washed five times with 500 μl of Wash Buffer to remove unbound protein. After washing, the supernatant was removed and 100 μl of Laemmli buffer was added to the resin. The resin/protein mixture was boiled at 95°C for 5 min then centrifuged to pellet resin. Proteins from each reaction were separated by 10% SDS-PAGE and analyzed by phosphorimaging.A modified These were performed as described previously 26). Briefly, the target proteins  were blotted on to a nitrocellulose membrane after separation by 12% SDS-PAGE. After staining with a 0.1% Ponceau S solution, bands were then cut from the blot and destained. After washing and blocking steps, individual blotted bands were then added to microfuge tubes containing 1 ml of the appropriate peptide  at a final concentration of 100 μM in Tris-HCl buffer at pH 8.0. Blots were incubated on a rotating platform for 2 h at room temperature, followed by two washes with TBS at 5 min intervals. Peptides were also incubated with SUMO protein as a control. Peptide binding was detected by incubation with streptavidin conjugated Alkaline Phosphatase (AP) (Pierce) and AP Substrate kit (Bio-Rad) as described previously  ATP. We clearly demonstrate that WOX5 is phosphorylated by sACR4, in vitro (lanes 2–4). Since sACR4 and mWOX5 have similar migration patterns on an SDS-PAGE gel, we wanted to ensure the signal in the autoradiogram was due to WOX5 phosphorylation and not sACR4 autophosphorylation. Therefore, after incubation, the phosphorylated mWOX5 protein was pulled-down by amylose resin and extensively washed to remove autophosphorylated sACR4. Indeed, the appearance of the radioactive band was due to mWOX5 phosphorylation, as seen by the absence of sACR4 when incubated alone (lane 1). A separate control reaction performed under the same conditions with sACR4 and MBP confirmed that the phosphorylation was specific towards WOX5 and not the MBP solubility tag (lanes 5–7).The root tip –22. In aroot tip –43. Thused alone , lane 1.y and b series ions. For example, the CID tandem mass spectrum of the phosphopeptide encompassing the junction between the C-terminal end of the solubility tag and the N-terminal sequence of WOX5, GSAGAALG(pS)FSVK, is shown in 2+ ion (m/z of 567.3) carrying a +2 charge state. This is characteristic of a neutral loss of phosphoric acid which is indicative of a phosphorylated peptide. Exact assignment of the phosphorylation at Ser2 (in WOX5 amino acid sequence) was verified by the presence of site-discriminating ions including the phosphorylated b9  and y5  ions and the unphosphorylated b8  and y4  ion. The absence of the phosphorylated b series of ions corresponding to b2-b8 and y series ions y3-y4, further confirmed that phosphorylation occurs at Ser2. A total of four phosphorylation sites were identified within the WOX5 sequence  tandem mass spectrometry. The data dependent analysis of tryptic peptides and a search against the mWOX5 protein sequence  yielded sequence . Table 1In higher animals and plants, directed cell proliferation, specification, and differentiation during growth and development are guided by a variety of external stimuli that are perceived and interpreted by a multitude of cell surface receptors primarily mediated through RTKs in animals and by RLKs in plants. The accepted paradigm of signal transduction involves ligand perception/binding to the extracellular domain, activation of the intracellular kinase domain and subsequent activation of downstream signaling pathways through reversible phosphorylation events. Subsequently, downstream signaling components influence transcriptional activity in the nucleus allowing appropriate cellular responses to specific signaling cues.Arabidopsis through homo- and heteromeric complexes with ACR4. This study not only documented the overlapping expression patterns of the two RLKs in the distal root meristem using reporter lines but unequivocally demonstrated their association with the plasmodesmata. Significantly, it was demonstrated through a split-luciferase assay and FRET-FLIM analysis that direct molecular interactions between ACR4 and CLV1 occurred at the plasmamembrane and was mediated through the transmembrane domains.In recent years, however, it has become apparent that the concept of a single receptor-single ligand interaction is perhaps too simplistic. Thus, structural, biophysical, biochemical and functional studies of several RTKs such as the Epidermal Growth Factor Receptor (EGFR) family –12, 44, Arabidopsis plant demonstrate all family members are expressed within all tissues examined, except for CRR2 and CRK1 which are not expressed in the siliques [in silico gene expression data sets to compare the mRNA levels of members of the ACR4 family in vegetative tissue and roots. Thus, analysis of the microarray data for various tissues/cell types in Arabidopsis at the Arabidopsis “electronic fluorescent pictograph” (eFP) browser (http://bar.utoronto.ca) [https://cornet.psb.ugent.be/) [in vivo towards defined biological pathways.Among the five members of the ACR4 receptor family, limited genetic and cell biology analyses have suggested these receptors may act in the same genetic pathway through functional redundancy based upon gene duplication and/or through activation of signaling cascades via receptor heterodimerization , 17, 20.siliques . In the onto.ca) , very clent.be/) , 53 specin vitro interactions between the ICDs of ACR4 and the CRRs to elucidate the molecular aspects of protein-protein interactions among the kinase domains of this receptor family. Ideally, crystal structure determination of complexes between ACR4 and the CRRs would facilitate an atomistic level understanding of these interactions. In the absence of structural information, we utilized hydrodynamic analyses, peptide binding studies and pull-down assays to demonstrate protein-protein interactions among members of the ACR4 family. Gel filtration studies and pull-downs show that, in the unphosphorylated state, the ICD of ACR4 can self-associate (lane 1) and also bind to all four CRR ICDs (right panels) although it is possible that they may have the capacity to form heteromers amongst themselves. Overall, given the ACR4-CLV1 interaction, it will be particularly interesting to see if the paradigm of heteromeric interactions holds for the ACR4 homologs as well. Such focused studies on the molecular basis of protein-protein interactions using in vitro biophysical/biochemical systems can inform on formulating new hypothesis-driven biological investigations.In this study we have analyzed the ssociate , lane 1 CRR ICDs , thus inArabidopsis genome are inactive, atypical RLKs, and it is likely that activation of differential signaling pathways can occur through heterodimerization among this RLK subfamily [We have also demonstrated that the ICD of ACR4 is capable of binding to the kinase-dead CRR1 and CRR2 ICDs regardless of the phosphorylation status . This obubfamily , 62–63. in vitro kinase assays demonstrate that active ACR4 kinase domain can phosphorylate the ICDs of all the CRR homologs, therefore suggesting that ACR4 signaling in vivo could potentially occur through heterodimerization with CRR family members. Similar mechanisms have been determined in plant RLKs in which cytoplasmic domains in heterodimeric receptor complexes can undergo trans phosphorylation [Arabidopsis that affect developmental functions in the plant [Our rylation , 64–65. rylation , 12. Altrylation –22, it dhe plant ,67. HoweBoth intramolecular and intermolecular protein-protein interactions are driven by contacts between specific residues on the interfacial faces of the participating domains. In contrast to stable complexes, biological signal transduction is regulated in large part by transiently formed states in which the proteins may switch between bound and unbound forms through modification-dependent conformational changes. While it is not possible to accurately define the state of the ACR4/AtCRR interactions based on current data, nevertheless our peptide-binding and HD exchange experiments indicate the importance of the N-lobe region of ACR4 kinase domain in heteromeric interactions. Specifically, our results suggest that the KDSAF motif in CRR3/CRK1 is important for contacts to the LLSLL region in ACR4 ICD  and is fin vitro. At this time, the relevance of these phosphorylation sites to the in vivo function of WOX5 is not known. Complementary in vivo experiments knocking out the potential phosphorylation sites in the WOX5 protein may drive phenotypic effects in transformed wox5 mutant plants aiding in the characterization of the functional significance of these phosphorylated residues. However, although WOX5 is deemed to be a key player in root stem cell regulation, it would be presumptuous to ignore the parallel inputs derived from other transcription factors [It appears that ACR4 is involved in at least two distinct signaling pathways with overlapping themes, stem cell differentiation in the root tip and differentiation of the epidermis in vegetative and reproductive tissue. As a mediator of stem cell differentiation in the root tip (20–22), ACR4 is described to perceive the extracellular CLE40 peptide ligand which promotes stem cell differentiation. Presumed activation of ACR4 then influences the expression domain of the WOX5 transcription factor, a protein that promotes stem cell proliferation. Our experiments have demonstrated that the ACR4 kinase can interact with and phosphorylate WOX5  factors  or poten factors .


Escherichia coli O157:H7 (EcO157) shed in cattle manure can survive for extended periods of time and intervention strategies to control this pathogen at the source are critical as produce crops are often grown in proximity to animal raising operations. This study evaluated whether neem (Azadirachta indica), known for its antimicrobial and insecticidal properties, can be used to amend manure to control EcO157. The influence of neem materials  on the survival of an apple juice outbreak strain of EcO157 in dairy manure was monitored. Neem leaf and bark supplements eliminated the pathogen in less than 10 d with a D-value (days for 90% elimination) of 1.3 d. In contrast, nearly 4 log CFU EcO157/g remained after 10 d in neem-free manure control. The ethyl acetate extractable fraction of neem leaves was inhibitory to the growth of EcO157 in LB broth. Azadirachtin, a neem product with insect antifeedant properties, failed to inhibit EcO157. Application of inexpensive neem supplements to control pathogens in manure and possibly in produce fields may be an option for controlling the transfer of foodborne pathogens from farm to fork. Escherichia coli O157:H7 (EcO157) [Concentrated animal feeding operations generate large amounts of manure waste , thus raEcO157) ,4. EcO157 ,4. EcO(EcO157) . PathogeAzardirachta indica) is a traditional and naturally available medicinal plant in India, South Africa, and Southeast Asia [Streptococcus mutans, S. salivarius, S. mitis, and S. sanguis [Listeria monocytogenes while ethanolic extracts showed higher inhibition for Staphylococcus aureus [Salmonella typhi, Shigella dysenteriae, E. coli, and Vibrio cholerae [Aeromonas hydrophila, Pseudomonas fluorescens, and E. coli) [Campylobacter jejuni [E. coli, but not against EcO157 according to an isolated study [Neem  . Extractr jejuni . Extractr jejuni . Thus, ned study . We evaluated the survival and fate of an outbreak related strain of EcO157 in manure supplemented with neem materials . Manure was from a medium-sized dairy in the central valley of California. The extractable fractions of neem leaves were also evaluated for their influence on the growth of EcO157 in 300-µL microcosms of nutrient medium. Microcosms were designed to determine the nature of extracts responsible for pathogen inhibition by neem.g for 10 min. The aqueous fraction was concentrated to 40 mL by rotary flash evaporation at 40 °C. The residual leaf paste was extracted by mixing for 1 h with 250 mL of 1:1 ethanol-ethyl acetate on a gyratory shaker. The organic extract was concentrated by flash evaporation and reconstituted in 25 mL ethanol. This extract from hereon will be called “ethyl acetate extract”. Five milliliters of the ethanolic extract were evaporated to dryness and reconstituted in 50 mL ethyl acetate and extracted with 100 mL of 1% sodium bicarbonate to remove the acidic components. The bicarbonate-washed ethyl acetate fraction was concentrated to dryness, reconstituted in 5 mL ethanol, and used in assays to determine the influence of neem extracts on EcO157. Dilutions of both “ethyl acetate extracts” were made in ethanol.Neem oil, powdered leaf, and bark were obtained from Neem Tree Farms . An aqueous extract of neem was prepared by extracting 50 g leaf powder with 200 mL RO pure deionized water. Extraction was carried out by shaking for 1 h on a gyratory shaker and the aqueous supernatant was separated by centrifugation at 10,000× 8 CFU and thoroughly mixed prior to incubation at 37 °C for 10 days. Overnight growth of MM123 in LB broth supplemented with 50 µg/mL kanamycin (to select for GFP) was centrifuged and resuspended in PBS prior to inoculations. GFP-labeled EcO157 cells were monitored at various intervals from 100 mg manure samples. One hundred-microliter portions of 10-fold serial dilutions of manure in PBS were plated on LB agar supplemented with 100 µg/mL rifampicin, 50 µg/mL nalidixic acid, and 50 µg/mL kanamycin and incubated overnight at 37 °C. The fluorescent colonies of MM123 were counted on a UV Transilluminator . Days for one log reduction  of the pathogen in manure were calculated from linear regressions of log cell number decline over time. Dairy manure used in this study was collected from a medium-sized dairy in Oakdale, CA, USA ,22. SurvGrowth of EcO157 strain MM149 in half-strength LB broth supplemented with aqueous or organic extracts of neem was monitored in a Bioscreen C microbial growth curve analysis system . MM149 was previously isolated from dairy manure and was chosen for its superior survival characteristics in dairy wastewaters ,22. WellMixing neem leaf at a concentration of 5% in dairy manure resulted in a 3 log reduction in numbers of MM123 within one day of incubation  and bothThe inhibitory effects of neem leaf and bark encouraged us to determine the extractable component of neem responsible for the inhibition of the pathogen. Since both leaf and bark behaved similarly in decreasing the populations of EcO157, aqueous and ethyl acetate extracts of leaves  were evaOptical densities measured at various sampling intervals were corrected for zero time values ranging from 0.05 to 0.6 for different treatments. The highest optical density at zero time was obtained with the full strength ethyl acetate extract. Some precipitation was noticed in this treatment but not at lower concentrations or with water or bicarbonate-washed ethyl acetate extracts. Clumping was not observed in any treatments. EcO157 shed in feces of ruminants can survive for extended periods of time ,26 and aE. coli [C. jejuni, a foodborne pathogen associated with contaminated meat and poultry, in a broth model meat system [E. coli were also inhibited in this model system, but it is not certain if the data can be extrapolated to pathogenic EcO157 or other shiga-toxigenic E. coli. Nonetheless, EcO157 in dairy manure was controlled by neem supplements.Neem leaf or bark eliminated the apple juice outbreak strain MM123 from dairy manure  in less E. coli , the oilt system . GenericL. monocytogenes and S. aureus [E. coli [E. coli [Since neem supplements controlled EcO157 in manure, we explored the nature of the active ingredient. The solvent extractable organic fraction from neem leaves is effective in totally inhibiting the growth of EcO157 in LB broth. The removal of acidic organic components with bicarbonate also resulted in some inhibition of the pathogen. Thus, it appears that the inhibitory activity of neem leaves is inherent to the organic fraction extracted by ethyl acetate containing both basic and acidic components. In contrast, chloroform and ethanol extracts of neem were previously found to be not inhibitory to EcO157 but inhibited the growth of two other foodborne pathogens, . aureus . Even th. aureus  extracte[E. coli . However[E. coli . Thus, rFoodborne pathogens in manure can be controlled with inexpensive treatments such as composting and/or supplementation of neem materials. Furthermore, pathogen transfer from point sources such as dairies and feedlots can be minimized by maintaining a safe setback distance for produce cultivation . ProduceFoodborne pathogen contamination from “ready to eat” produce is difficult to remove. Treating with neem supplements could be an inexpensive way to prevent pre-harvest contamination via manure from nearby animal raising operations. Supplementation of neem leaf and bark to manure resulted in elimination of pathogenic EcO157 in less than 10 days. The active principle for inhibition of neem leaves is localized in the organic extractable fraction. Neem supplementation to manure piles on dairies and feedlots, and also to produce fields, could be a novel strategy for on-site pathogen control.


Understanding the modification of the graphene’s electronic structure upon doping is crucial for enlarging its potential applications. We present a study of nitrogen-doped graphene samples on SiC(000 However, engineering its electronic band structure in a controlled way is a crucial step towards graphene-based electronics. Inspired by semiconductor physics, the addition of electron donor or acceptor atoms to graphene has been proposed as a means to controllably shift the electronic bands of graphene. In this context, some experimental works have been performed using nitrogen12345678910121334526710n-type doping of the graphene sheet. Contrary to what has been reported in CVD-grown samples for simple subsitutionsIn a previous studyne/nN , acquired on the same samples, on which individual nitrogen dopants are visible. The main cone on each spectrum comes from graphene areas whose LEED pattern is rotated by 30° with respect to the LEED pattern of the SiC substrate. The ARPES spectra display several cones for each sample. This is because the sample consists in continuous graphene sheets with rotational boundaries and the beamspot, albeit small (~50 × 50μm2), integrates several orientations of the graphene lattice both laterally and in the stacked layers. The presence of Dirac cones in the ARPES spectra indicates that the insertion of low concentrations of nitrogen atoms in the graphene carbon lattice does not markedly modify the band structure of graphene. However, one can notice an increased width of the cones on the ARPES spectra at high concentration (171820vF is not affected by the doping: we measured vF for the highly doped sample is greater). The main effect expected from nitrogen-doping of graphene is an electronic doping i.e. an increase of the difference EF and ED are the Fermi and the Dirac energies, respectively. From the EDCs of the spectra, we found for this energy difference 0, 80, 190, 240 and 490 meV ±10% for T = 0, 7.5, 15, 30 and 90 min respectively . These doping levels are proportional to the exposure time meaning that our doping technique allows good control of the doping level of the graphene samples (see We have investigated the effect of nitrogen-doping on the band dispersion of graphene by ARPES experiments. In ntration  suggestintration . The rolectively . In ordec = 0.08%), although the effects on the band structure for this concentration are clear , two contributions can be observed: the graphitic N 1s peak is found at a binding energy of ~401.2 eV (approximately 1 eV higher than that of graphitic N atoms in the samples exposed during 15 and 30 min) and another peak lies at ~398.3 eV. The ratio between the two peaks is 10–15%, similar to the ratio between graphitic nitrogen and unidentified defects observed with STM34242526For the highly-doped sample . As already mentioned, significant variations of binding energies of the graphitic N 1s level  a small peak centered at 282.6 eV corresponding to carbon atoms belonging to the SiC substrate and (ii) an intense asymmetric peak at 284.4 eV associated with the sp2 carbon atoms of the graphene sheets. Despite the increasing shift of the Fermi level with respect to the Dirac energy  to affect preferentially the top layers while XPS probes all the graphene layers. We however note the following. The C 1s spectra  of the fi) the great uncertainty in XPS due to the small amount of nitrogen and (ii) the fact that we expect our doping method to dope preferentially the topmost graphene layer (measured with STM) whereas XPS also probes the buried layers.Finally, we note that the evaluation of the nitrogen concentration with XPS  agrees only semi-quantitatively with the evaluation from STM imaging. We believe the reasons for that are   that is then used to build the image shown in μ. Interestingly, this spatial variation, that ranges on the whole image between 100 meV and 340 meV with an average of 250 meV, is comparable to the dispersion of the values of μ measured at different locations on the sample   and the black curve is an average over tunneling spectra acquired at four different locations (at each location ten spectra were acquired) with four different tips, on the same sample, both on the same horizontal scale (the sample bias for the STS and the binding energy for the EDC). As mentioned above, the spatial inhomogeneities of the tunneling spectra are the smallest for this sample, making the comparison with ARPES data ideal.We now compare the ARPES and STS spectra. The averaged values of the Dirac point position found in STS together with the values found in ARPES are displayed on EF) proportional to the DOS of the doped graphene (which is linear in energy close to the Dirac point). The EDC looks indeed very much like a DOS of a doped graphene sample with μ = 80 ± 10 meV whereas the local minimum of the STS spectrum is found at ~−150 ± 15 meV. This difference is, within the experimental error, equal to the energy of the phonon created in the inelastic tunneling process and shifting the STS curve by this energy towards the Fermi level  and π states below the Dirac point is not modified in the presence of an attractive potential, which is the case when replacing a carbon atom by a nitrogen one. The variation of the number of electrons is therefore strictly equal to the total electronic population in the conduction band (above ED taken here as the origin of energies): π electrons per atom instead of one for carbon, this proves that ne/nN should be equal to unity:A complete treatment of this problematic is behind the scope of this article and will be published elsewhere, we only outline here the method and the results of interest to us. Let δρN(E) is the variation of the density of states induced by a single nitrogen atom.where δρ(E) is now equal to nNδρN(E), and the number of electrons ne is equal to nN = cN, where N is the number of atoms and c the nitrogen concentration, and we obtain:To go beyond, we extend this result to the case of a finite but small concentration of nitrogen atoms. Assuming negligible interactions between the impurities, vF is the Fermi velocity. In the present tight-binding formalism, the rigid band term is equal to W is the width of the band related to the transfer integral t, N we finally find:The term cAμ/W where A is a constant depending on the potential of the impurity. From this equation we can determine μ as a function of c,In this equation, the second term is not negligible. Close to the Dirac point, it can be approximated by c → 0, A, i.e. the impurity potential does not depend on the concentration. This is certainly not true: there are indications that the position of the nitrogen resonance tends to the Dirac point when c → 0decreases when c → 0 as observed  projected on lattice sites n(E) are calculated using the recursion methodV0) and its first neighbors (V1) which is sufficient to account for the local densities of states. They have been determined so as to fit at best ab initio calculations corresponding to c ~ 1% and are equal to V0/t = −1.47, V1/t = −0.9, respectivelyE = 0. To obtain the local so-called Mulliken charges we integrate the densities of states up to the Fermi level which, at infinite dilution, remains at the Dirac point. One can object that our calculations are not self-consistent but screening effects in graphene with a Fermi level at the Dirac point are still not very well understoodV0/t = −2.08, V1/t = −1.34. We assume that these potentials are appropriate when c = 0.15%. As a consequence, the excess π charge on the N atom and on the three first neighbours are much larger T = 7.5, 15, 30 and 90 min). The ARPES experiments were carried out at 30 K with a photon energy of 36 eV at the Cassiopée beamline of the Soleil synchrotron equipped with a Scienta R4000 electron energy analyzer; the overall energy resolution was set to 10 meV while the momentum resolution was 0.01 Å−1. The tunneling experiments were performed with a UHV Low-Temperature STM (4.2 K) from Omicron GmbH using electrochemically etched tungsten tips. dI/dV spectra were acquired with a lock-in detector at 710 Hz and a modulation amplitude of 24 mV. The STM images have been handled with WSxMα line. The emission angle was 0°. Between the different experimental setups , the samples were transported in the atmosphere and outgassed in UHV at ~800 °C prior to manipulation (except for the XPS measurements prior to which no outgassing was possible). It has been checked by STM that this exposure to air and subsequent annealing have no effect on the samples.The samples were grown by the confinement control sublimation (CCS) method464748How to cite this article: Joucken, F. et al. Charge transfer and electronic doping in nitrogen-doped graphene. Sci. Rep.5, 14564; doi: 10.1038/srep14564 (2015).