Spain’s financial crisis has been characterized by an increase in unemployment. This increase could have produced an increase in deaths of women due to intimate partner-related femicides (IPF). This study aims to determine whether the increase in unemployment among both sexes in different regions in Spain is related to an increase in the rates of IPF during the current financial crisis period.An ecological longitudinal study was carried out in Spain’s 17 regions. Two study periods were defined: pre-crisis period (2005–2007) and crisis period (2008–2013). IPF rates adjusted by age and unemployment rates for men and women were calculated. We fitted multilevel linear regression models in which observations at level 1 were nested within regions according to a repeated measurements design.Rates of unemployment have progressively increased in Spain, rising above 20 % from 2008 to 2013 in some regions. IPF rates decreased in some regions during crisis period with respect to pre-crisis period. The multilevel analysis does not support the existence of a significant relationship between the increase in unemployment in men and women and the decrease in IPF since 2008.The increase in unemployment in men and women in Spain does not appear to have an effect on IPF. The results of the multilevel analysis discard the hypothesis that the increase in the rates of unemployment in women and men are related to an increase in IPF rates.The decline in IPF since 2008 might be interpreted as the result of exposure to other factors such as the lower frequency of divorces in recent years or the medium term effects of the integral protection measures of the law on gender violence that began in 2005. The world economic crisis that broke out at the end of 2007 severely affected the banking sector. In Spain, banks stopped lending credit to companies and individuals, giving rise to dramatic destruction of jobs. For this reason rates of unemployment rose from 13.9 % in the year 2008 to 26.2 % in 2013 [It is known that mortality rates in countries facing financial crisis have changed historically with respect to periods of economic growth. Currently, death rates seem to be decreasing in several of these countries in crisis, but causes of mortality behave differently –6. When Our hypothesis in this article is that in the current financial crisis period in Spain, the massive destruction of employment for both men and women could favor or promote a change in homicides of women due to intimate partner violence, also known as intimate partner-related femicides (IPF). As has been observed in other studies, when comparing perpetrators of femicide with other abusive men, unemployment appears to be a significant demographic factor even after controlling for other relevant characteristics –19. As oThis study aims to explore how the increase in unemployment among both sexes in the different Spanish regions is related to rates of IPF during the current period of financial crisis. Prior studies showed differences in IPF among the different Spanish regions, which shows that it is important to take geographic distribution into account in the analysis of IPF in Spain –29.An ecological longitudinal study was carried out in 17 Spanish regions- Autonomous Communities- in the period 2005 to 2013. The autonomous cities of Ceuta and Melilla were excluded from the analysis because they are not comparable with the other regions studied, given that they are autonomous cities and not regions. Two periods were defined, considering 2008 as the year of the beginning of the economic crisis [The data on the number of IPF were collected from the Federation of Separated and Divorced Women, whose website has collected and published the cases of IPF for ages 15 and over since the year 1998 . OfficiaThe authors confirmed that 100 % of the deaths recorded by official government sources in Spain during the study period appear in the database of the Federation of Separated and Divorced Women. The Federation of Separated and Divorced Women was founded in 1973. Their activities included the defense of equal rights among men and women and recognition of women’s rights in order to eradicate discrimination in Spanish society. The Federation’s web page records all IPF related deaths in Spain. The authors reviewed this information and added cases to their own database. They double-checked later on that the number of annual deaths due to IPF recorded by the Federation equaled the number of deaths published by official sources such as the Ministry of Equality. The only difference between the two sources was that the information recorded by the Federation is disaggregated. Previous studies show similar percentages of overlap between the database of the Federation of Separated and Divorced Women and the National Observatory on Violence against Women of the Spanish government , 29, 31.Unemployment data were obtained from the Labor Force Survey (LFS) conducted by the National Institute of Statistics of Spain (INE). Unemployment data are collected each trimester through questioning a wide representative sample of the Spanish population about their employment situation during the past weeks. This is the most reliable data to measure unemployment in Spain. An unemployed person is defined as “16 years and over who, during the survey week, was without work, was available for work and actively seeking employment” [The dependent variable was the annual rate of IPF in the 17 Spanish regions adjusted by age. The independent variable considered was the annual unemployment rate (men and women) in the 17 Spanish regions during the study period. The denominator for calculating unemployment rates was the total labor force . AnotherFinally, for the multilevel regression study, a dummy variable was created taking the value of 1 in a period of crisis (2008–2013) and 0 otherwise. Thus it is possible to observe the probability of variation in IPF between the two periods.We calculated crude annual unemployment rates for men and women and age-adjusted rates of Intimate Partner-related Femicide for the years 2005 through 2013 . With respect to unemployment data, in period 1 the unemployment rate ranged from 3.79 to 10.20 % for men and from 7.2 to 20.1 % for women. In period 2 the unemployment rate for men ranged from 11.18 to 27.5 %, while for women rates ranged from 12.4 to 30.9 %. Finally, the percentages of foreign population observed in period 1 ranged from 2.11 to 14.11 %. For period 2, these percentages ranged from a minimum of 3.01 % to a maximum of 18.54 % . In neither of the two models was the percentage of immigrant population significant, nor was the period of crisis, in explaining IPF  . HegemonAdditionally, it must be taken into account that in Spain the number of divorces and separations decreased by 19.18 % between 2005 and 2012 . DivorceIt is also possible that there is an explanation related to the measures for integral protection against gender violence laid out by Organic Law 1/2004 . In the The low number of cases of IPF in years studied may have influenced our results. As has been mentioned in previous studies, there is no established date for the start of the financial crisis in Spain . HoweverIn Spain there has been an increase in male and female unemployment rates as a consequence of the economic crisis, there has also been a decrease in IPF rates. It does not seem that unemployment has influenced the observed tendency of IPF, which should therefore be attributed to exposure to other factors such as the decrease in divorce or the protective effects of the gender violence law in effect prior to the beginning of the crisis. However, the decrease in IPF could be related to an increase in invisible IPV cases. Therefore, it is important to continue this line of research with other studies that contemplate other IPV outcomes such as calls to help lines or filing formal police reports. It would also be interesting to analyze the situation in other countries where the government response to the financial crisis has been different.


It is believed that at low hydrogen flow, those remained oxygen or other oxidizing impurities make the graphene films p-doped and result in decreasing the carrier mobility.Hydrogen flow during low pressure chemical vapor deposition had significant effect not only on the physical properties but also on the electrical properties of graphene. Nucleation and grain growth of graphene increased at higher hydrogen flows. And, more oxygen-related functional groups like amorphous and oxidized carbon that probably contributed to defects or contamination of graphene remained on the graphene surface at low H Hydrogen plays a dual role during growth on copper (Cu) substrate [2, methane (CH4) used as a carbon precursor should chemisorb on the Cu surface to form active carbon species, that is, (CHx)s (x = 0 ~ 3), which subsequently react to form graphene. However, such dehydrogenation reactions are not thermodynamically favorable even on Cu surface [2 more readily dissociates on Cu and produces active H atoms [4, and lead to production of surface bound (CH3)s, (CH2)s, or (CH)s radicals. It also controls the grains' shape and dimension by etching away the weak carbon-carbon bonds [2 gas during growth steps affects the morphology of graphene film such as the sizes, shapes, and dimensions of graphene grain boundaries  [Since the first report of the special characteristics of graphene, a planar sheet of sp physics ,2 to the physics , photovo physics , or micr physics -9. Howev physics ,11, alth physics -14. One  physics -18. Espe physics . To obtaubstrate . Without surface . Molecul H atoms . These Hon bonds . So, cha zigzag) ,22.2 flow during the low pressure CVD also had significant effect on the electrical properties of graphene. We found that in our process conditions, H2 seemed to act more like as a nucleation/growth promoter and a defect suppressor  of CVD-grown graphene film in our process conditions.In this article, we observed that the H2 and argon (Ar) atmosphere at 950°C for 15 min. The Ar flow was set at 100 sccm during the annealing step for all the samples. After that, CH4 gas was forced to flow at 100 sccm during the growth step, and the H2 flow was systematically (10 to 500 sccm) varied for different samples. Then, the chamber was cooled down to room temperature without H2 and CH4 flows. Table 2/Si substrate and put into acetone for 10 h to dissolve PMMA. All the samples were thermally annealed after transferring to the SiO2/Si substrate under nitrogen gas protection for further characterization. The as-grown and transferred films were characterized by field emission scanning electron microscopy  to check the proper growth of the graphene film. Raman spectra were recorded using a Renishaw Raman microscope  with an Ar laser wavelength of 514 nm, and X-ray photoelectron spectroscopy  was used to confirm the quality of the film. The atomic force microscopy (AFM) was done to analyze the morphology of grown films, grain boundaries, and thickness using the commercial AFM n-Tracer . Electrical contacts were made by thermally evaporating Au/Cr with thickness of 30/5 nm for transport measurements.Graphene samples of various sizes (ranging from 1 × 1 to 3 × 3 cm) were grown on Cu foils  purchased from Sigma-Aldrich . The Cu foil substrate was ultrasonically degreased by being immersed in acetone, methanol, isopropyl alcohol (IPA) solution, and deionized (DI) water and then dried and baked for 5 min. After the foil was placed in a quartz chamber, the chamber was evacuated and heated up to 950°C very rapidly using halogen lamps. Annealing was performed under H2 flow of 10, 100, 200, 300, 400, and 500 sccm at four different growth durations of 1, 5, 10, and 15 min. Figure 2 flow rates of 10 (low H2 flow) and 400 sccm (high H2 flow). The corresponding growth schematics during each elapse time are shown in Figure A large number of graphene samples were prepared, corresponding to the H2 flow rate (10 sccm), no nucleation occurs for smaller growth durations, e.g., t < 5 min. The spontaneous nucleation and subsequent growth of graphene occurs only when a critical level of supersaturation is reached. At lower H2 flow rates and hence at low partial pressure, this critical level of supersaturation is achieved only at longer growth durations. As the H2 flow rate increases, this threshold of supersaturation is achieved at relatively shorter growth durations. Further, our results also suggest that as the H2 flow increases the density of nuclei increases , the nucleation and growth are reduced significantly, as compared in Figure 2 partial pressure, the graphene domain feature irregular shapes [As shown in Figure low rate 0 sccm, nlow rate 0 sccm, nlow rate 0 sccm, nlow rate 0 sccm, nr shapes . The dom2 flow (10 to 500 sccm) at a growth time of 15 min. As can be observed from Figure 2 flow increases from 10 to 500 sccm, the intensity of defect peak in the Raman spectra decreases and is finally unnoticeable for 500 sccm of H2, indicating that the quality of grown graphene increases with increasing H2 flow. The D peak is located at approximately 1,350 cm−1, attributed to the breathing mode of sp2 atoms, and originated by the existence of some defects such as edges, grain boundaries, functional groups, or structural disorders [−1, respectively. The G peak becomes sharp, and the D peak shows high intensity. While for the sample grown at high H2 flow (500 sccm), the peak positions of G and 2D are red shifted to around 1,585 and 2,683 cm−1, respectively, as can be observed in Figure 2 flow, as can been seen in Figure 2. The FWHM of 2D peak is 28 to 39 cm−1 for all the samples, as can be seen in Figure ID/IG ratio decreases from 0.42 to 0.08, whereas the I2D/IG ratio increases from 1.0 to 1.7 with increase of H2 flow from 10 to 400 sccm as can be presented in Figure 2 in Cu is 2 × 10−4 cm2s−1. H2 or atomic H can easily diffuse into the catalyst and compete with CH4 for chemisorption; H2 can also contribute to the fast removal of residual oxygen and contaminants from the surface [2 effects seem to be beneficial in improving the qualities of graphene in our process conditions.To check the crystallinity of graphene, we used Raman spectroscopy on all the prepared samples corresponding to Hisorders . As for  surface . These H−1, while G peaks appeared between 1,597 to 1,606, 1,592 to 1,601, and 1,580 to 1,589 cm−1 for samples at 10, 200, and 500 sccm of H2 flow, respectively  and carbonyl C = O and carboxylic COOH (288.3 to 288.7 eV) groups [2 hybridization is attributed to the carbon lattice, while sp3 is a result of oxygen-related functional groups like amorphous and oxidized carbon that probably contributed to defects or contamination [2 flow (500 sccm) shows higher sp2/sp3 peak ratio than that grown at 10 sccm in Figure 2 is observed at 284.6 eV with FWHM of 0.69 eV, while sp3 peaks at hydroxyl C-OH and carboxylic COOH are at 285.3 and 288.1 eV, respectively. As for the sample grown at low H2 flow (10 sccm), the sp2/sp3 ratio decreases significantly, and sp2 peak is broadened, which reveals that graphene is oxidized and amorphized at the low H2 atmosphere. It is also thought that residual oxygen or oxidizing impurities [2 atmosphere.C1s XPS spectra display an asymmetric shape typical of graphitic sp) groups ,30. The mination . The sampurities , which a2. Panels in Figure 2 each for a growth time of 15 min. As can be observed from the topography maps  under different H2 flows. In principle, the charge neutrality point (CNP) should be at Vg = 0 if the graphene is undoped [ne = Cg(Vg − VDirac), where Cg is the gate capacitance for a SiO/Si substrate [μ = (enρ)− 1.To further confirm the quality of the graphene films, charge transport measurements were performed. Figure  undoped ,34. The ubstrate . In orde2 flow rate. For the sample grown under 10 sccm of H2 flow, the CNP was at +31 V, but for the sample under 500 sccm H2 flow, the CNP was shifted to +10 V. Our results show that at low H2 flow all the film exhibited p-typed behavior [2 flow, the synthesized graphene becomes closer to a pristine graphene, and its mobility increases. Figure 2 flow. The changes in charge carrier density of graphene layers are related with changes in Fermi level of graphene layers. Thus, different H2 flow significantly modulates the Fermi level of graphene layers. The defects of graphene, which can be originated from the inevitable occurrence of residual oxidizing impurities in the chamber's atmosphere and gas feedstock, are suppressed under high H2 flow rate.In the experiments, more than six devices were made. There is a trend in which the mobility of the graphene increases and the CNP shifts toward zero with increasing Hbehavior . With th2 flow and is in good agreement with that reported for a monolayer graphene (97.1%), indicating all monolayer graphene films as confirmed from the AFM topography [The optical transmittance spectra as depicted in Figure pography . Our res2 flow conditions. It is believed that at low hydrogen flow, those remained oxygen or other oxidizing impurities make the graphene films p-doped and result in decreasing the carrier mobility. In our process conditions, H2 seemed to act more as a nucleation/growth promoter and a defect suppressor  of CVD-grown graphene film.In conclusion, we observed that hydrogen flow during the low pressure chemical vapor deposition had significant effect not only on the physical properties but also on the electrical properties of graphene. Nucleation and grain growth of graphene increased at higher hydrogen flows. And, more oxygen-related functional groups like amorphous and oxidized carbon that probably contributed to defects or contamination of graphene remained on the graphene surface at low HThe authors declare that they have no competing interests.SH and JP performed the experiments. MI, MA, JS, and JE analyzed the data. SH and JJ wrote the paper. All authors read and approved the final manuscript.


With much improved h-BN-tunneling-junction stability, we are able to probe all possible phonon modes of h-BN and graphite/graphene at Γ and K high symmetry points by inelastic tunneling spectroscopy. Additionally, we observe that low-frequency out-of-plane vibrations of h-BN and graphene lattices are significantly modified at heterostructure interfaces. Equipped with an external back gate, we can also detect high-order coupling phenomena between phonons and plasmons, demonstrating that h-BN-based tunneling device is a wonderful playground for investigating electron-phonon couplings in low-dimensional systems.Inelastic electron tunneling spectroscopy is a powerful technique for investigating lattice dynamics of nanoscale systems including graphene and small molecules, but establishing a stable tunnel junction is considered as a major hurdle in expanding the scope of tunneling experiments. Hexagonal boron nitride is a pivotal component in two-dimensional Van der Waals heterostructures as a high-quality insulating material due to its large energy gap and chemical-mechanical stability. Here we present planar graphene/ Especially in low-dimensional systems such as graphene- and carbon nanotube-based devices, electron-phonon scattering has been considered as the major source for limiting conductivity178h-BN) has been acclaimed as a superb gate-insulating material for low-dimensional electronic and optical devices with graphene121617h-BN and its hybrid structures are greatly needed, not only for understanding physical properties of h-BN-based nanoscale devices but also for developing new electronic and optical devices. Although there are several experimental and theoretical reports on h-BN phonons192021222324h-BN-based heterostructures are sparse with a few of experimental reports on Raman-active E2g phononsh-BN relied on optical methods due to a large band gap (≈6 eV) of h-BNHexagonal-boron nitride  finding good agreement with experimental data. Finally, we are able to identify charge-density-dependent excitations with energies higher than 200 meV, suggesting that the overtone excitations can be due to multi-phonon scattering processes combined with charge-density-dependent excitations, plasmonsIn this article, we present electron-tunneling spectroscopy measurements of gated single-layer graphene devices with a thin tructure . In our h-BN-based tunneling devices with both thick h-BN (>20 nm) and SiO2 (300 nm) as gate insulators, and a doped Si substrate as bottom gate. Sample bias (Vb) is applied to the top probe, and tunneling current as well as differential conductance (G = dI/dVb) are monitored through the graphene layer. We use a thick-and-narrow graphite flake as the tunneling probe, which shows superior tunneling characteristics to metal electrodes3334The inset of T = 5.9 K. We apply small AC-excitation voltage  to DC sample bias and measure both I-Vb  of graphene is expected to be at gate voltage Vg = −40 V and the dip at the Fermi level, EF (Vb = 0 mV). Additionally, we can find several kinks in dI/dVb around EF within |Vb| ≤ 200 mV. These features become more prominent in the IETS signal dG/dVb  and Vg = 50 V to 40 V  with a spacing of ΔVg = 2 V. Without any difficulties, we can identify that there exists two types of tunneling features, with and without density dependence. As expected, the feature related to ED changes its position upon varying charge density, as marked with red dotted arrows. However, most spectra features within |Vb| ≤ 200 mV barely change their positions in sample bias with a little variation in intensity even though charged carriers switch polarity from electrons to holes in the full range of gate voltages, −50 V ≤ Vg ≤50 V.To analyze spectrum features in detail, we obtain a series of tunneling spectra while varying the charge density with Vg and Vb reveals a plethora of electronic structures not just limited to EF but also extended to both empty (Vb >0 mV) and filled (Vb <0 mV) states. In the |dG/dVb| gate mapping  state obtained from Vg = 10 V to Vg = 50 V are also plotted  of h-BN, which is Raman-active E2g phonon at the frequency of ≈1365 cm−1 (≈169 meV)19Γ3− (or Γ4+) (ZO) and acoustic Γ4+ (ZA) phonon modes can be assigned to the peaks at P6 (≈103 meV) and P1 (≈17 meV), respectively. We can argue that Γ4+ phonon mode  becomes hardened because 4-layer h-BN is encapsulated with relatively thick top graphite probe and bottom h-BN gate insulators. We can recognize several K-point phonon modes as well: in-plane optical K1,2  and K5 , in-plane acoustic K5  and K1,2 , and out-of-plane optical K6  and acoustic K6  phonon modes. Note that some of phonon modes especially out-of-plane acoustic K6 (ZA) phonons are slightly off from theoretical expectations, which we relate to graphene/h-BN heterostructures. We will discuss how these phonon modes are influenced by nearby layers later. In addition, we tend to assign the peak at ≈186 mV  to the overbending mode of the in-plane optical  phonons of h-BN, as marked with a red star in et al.h-BN crystals at the frequency of 1470 cm−1 (≈182 mV) with resonant Raman measurements, and Kern et al.−1, 185 mV) of LO-phonon branch is formed not at the high symmetry point of Γbut in between Γ and K resulting LO-overbending branch.First, we compare our data with DFPT-calculated phonon dispersions of 4-layer h-BN , which hh-BN . As indiV P10 in  is attriΓ and K: in-plane optical Γ5+  and out-of-plane optical Γ4+  phonon modes, and in-plane optical K1,2  , K5 , and in-plane acoustic K2  phonon modes along with out-of-plane optical/acoustic K6  phonon modes. It is worth mentioning that phonon dispersions for graphite and single-layer graphene are almost identical (Γ6− (or Γ2−)  phonon mode which is solely ascribed to the layered structure of graphite. Note that several excitations could have contributions from both h-BN and graphite/graphene phonons because of the structural similarity of h-BN and graphite/graphene. However, the peak at ≈199 mV (P12) are exclusively from the Γ5+phonon mode of graphite/graphene, which is Raman-active E2g phonon (G peak) at ≈1580 cm−1 (≈196 meV)Γ pointAs mentioned previously, we employed graphite flake as a tunneling probe, therefore we can expect that our data should have signatures of graphite phonons. Indeed, we can identify several graphite phonons at the high symmetry points of dentical . Thus, wΓ4+ ZO, P phonon mM are missing differing from the previous IETS studies with STM tunneling measurements1011M2+ phonon  mode. For example, M3− phonon  mode of graphite/graphene marked with a black star in M3− phonons are observed. Additionally, M3+ phonon  mode, one of the conspicuous phonons with large density-of-states (DOS), is missing in our IETS measurements. M-phonon suppression is observed in h-BN phonons as well. Based on DFPT calculation  and M (M2+) points, which could be linked to the excitation at ≈156 meV (P9). However, the strongest IETS signal in our devices is from the Γ5+ phonon mode at ≈168meV, which has considerably lower phonon DOS compared to other M-point phonons (M2+ (or M3−) phonon (TA) mode at ≈68 meV is also missing in the tunneling spectra given the fact that the excitation at ≈67 meV (P4) is the weakest IETS feature . Even though electrons require momentum change  to interact with phonons at the K point, the electron-phonon scattering rate for the phonons at K point could be higher than the case for M-point phonons, which require multiple electron-phonon scatterings. Note that the excitation at ≈84 meV (P5), which we tentatively assign to the M2+ phonon (ZO) mode, is one of the broadest excitations among twelve peaks with ≈18 mV FWHM. Here, we conclude that the tunneling in our planar h-BN junctions would be less susceptible to momentum-changing scattering events, especially parallel momentums during tunneling process, contrast with STM measurements in which one electrode is an atomically sharp non-planar metal tip, which breaks parallel-momentum conservation71011In both graphene and graphite, all available electronic states of tunneling electrons are located in the vicinity of the h-BN layers form heterostructure, it is expected that the lattice dynamics of graphene and h-BN, especially low-frequency out-of-plane modes, are influenced by the nearby layers. Indeed, we find that graphene/h-BN heterostructure shows phonon dispersions different from those of free-standing graphene and h-BN. h-BN heterostructure. For the sake of simplicity, we consider single-layer graphene is aligned on top of h-BN layer in an AA stacking, with graphene atoms (unit length: 0.245 nm) relaxed to h-BN lattice (unit length: 0.251 nm). To compensate the lattice deformation (strains) due to the structural mismatch, we calculate Grüneisen parameters for free-standing graphene and h-BN, and obtain the phonon modes of the unstrained graphene/h-BN heterostructure (h-BN becomes hardened shifting K6 phonon (ZA) mode to higher frequency (≈46 meV), which can be assigned to the previously-unassigned peak at ≈47 mV (P3). Second, out-of-plane acoustic Γ6−(or Γ2−) phonon (ZA) mode of graphene is blue-shifted to a frequency of ≈36 meV (P2). Third, M2+phonons (ZO) of graphene become hardened, shifting closer to the experimental data at ≈86 meV (P5). Last, graphene LA-phonon branch becomes slightly softened, moving K5 phonons closer to the peak at ≈140 meV (P8).When graphene and tructure   = Ei + α exp^(β Vg), and find E1 ≈ 214 mV (E2* ≈ −201 mV) for the empty (filled) state excitation in a and b, obtaining E2 ≈ 200 mV, E3 ≈ 181 mV (E3* ≈ −183 mV) and E4 ≈ 167 mV (E4* ≈ −170 mV) in positive (negative) Vb. As displayed in E1, E2 and E2* are higher than the highest phonon energy of our systems , suggesting that these overtone modes could be due to multi-phonon scattering events. Interestingly, we are able to find the combinations of two-phonon modes, comparable to each of the baseline excitation: E1 with P3 + P10 (≈215 mV), E2 (E2*) with P3 + P9 (≈204 mV), E3 (E3*) with P3 + P8 (≈183 mV), and E4 (E4*) with P3 + P7 (≈174 mV) sharing P3, out-of-plane acoustic K6 phonons of h-BN as a common excitation.We now discuss charge-density-dependent IETS spectra. As indicated with dotted-yellow boxes in h-BN heterostructures with a thin h-BN layer as a tunneling insulator and observed all the available phonon modes of h-BN at the ΓandK high symmetry points by electrical transport measurements for the first time. We find out out-of-plane motions of graphene and h-BN lattices become hardened when graphene/h-BN heterostructure is formed. Additionally, we observe charge-density-dependent excitations, which could be related to both multi-phonon and plasmon-related scattering processes. With IETS employing h-BN as a tunneling insulator, we have demonstrated that h-BN-based tunneling devices become a powerful experimental playground for exploring not only novel electronic structure2830h-BN hybrid devices as well as other low-dimensional materials such as TMDCs and carbon nanotubes. Moreover, benefited by much-improved-stable tunneling junctions, our tunneling devices make it possible to probe electronic and mechanical properties of nanoscale devices with applications of external variants such as magnetic fields, electro-magnetic waves, temperature, and pressure where previous tunneling experiments have been limited.In summary, we have performed inelastic electron tunneling spectroscopy studies of graphene/h-BN tunneling devices are fabricated using multiple steps of dry-transfer method developed by Dean et al.h-BN (>20 nm) are mechanically exfoliated from high-quality single crystals and transferred on thermally grown 300 nm thick SiO2 on Si. Next, single-layer graphene, thin h-BN (3 or 4 layers) and graphite flakes are sequentially transferred on top of bottom h-BN insulator. For each step, we first prepare Si substrates spin coated with water-soluble Poly-StyrenSulfonic (PSS) acid and PMMA. Desirable flakes mechanically exfoliated on the polymer-stacked Si substrates are examined under an optical microscope and later transferred to pre-defined locations with micro-manipulating transfer stage. Before dissolving PMMA in acetone, we bake out the samples at 200 °C for 2 minutes to promote the adhesion between transferred layers. It is critically important to have clean and flat interfaces for high-quality tunneling devices. To further remove any polymer residues and reduce the number of bubbles formed in 2-D heterostructures, we anneal the samples in the mixture of Ar and H2 (9:1 ratio by flow rate) at the elevated temperature 350 °C for more than 4 hours. In total, we applied dry-transfer method three times and high-temperature annealing four times before loading the samples in the cryostat for measurements. Additional annealing process is performed after fabricating Ti/Au (5 nm/55 nm) electrodes with electron beam lithography.Graphene/h-BN, graphite/graphene and graphene/h-BN heterostructures are calculated using density functional perturbation theory (DFPT)43h-BN are aGR = 0.245 nm and ah-BN = 0.251 nm, respectively, which are close to the experimentally observed values within 1%1h-BN heterostructure, we use the 6 × 6 × 2 supercell, assuming that single-layer graphene is commensurately aligned on top of h-BN layer in an AA-stacking direction. With obtained Grüneisen parameters for free-standing graphene (γGR) and h-BN (γh-BN) layer, we calculate the phonon modes of the unstrained graphene/h-BN heterostructure, as shown in h-BN/graphene heterostructure (aGR = 0.245 nm), the phonon modes of h-BN are increased by <γh-BN > = 1.13% in average compared with the unstrained graphene/h-BN heterostructure. Additionally, for the tensile-strained-graphene/h-BN heterostructure (aBN = 0.251 nm), the phonon modes of graphene is decreased by <γGR> = 1.43% in average, with respect to the unstrained graphene/h-BN heterostructure. Although AB-stacked graphene/h-BN heterostructure is expected to be more stable than its counterpart AA-stacked heterostructureh-BN heterostructures are fabricated using dry-transfer methodh-BN lattices, resulting in the atomic-stacking sequence is arbitrary.Phonon dispersions of How to cite this article: Jung, S. et al. Vibrational Properties of h-BN and h-BN-Graphene Heterostructures Probed by Inelastic Electron Tunneling Spectroscopy. Sci. Rep. 5, 16642; doi: 10.1038/srep16642 (2015).


The first reversible specific capacity of the hollow graphene spheres was as high as 903 mAh g-1 at a current density of 50 mAh g-1. Even at a high current density of 500 mAh g-1, the reversible specific capacity remained at 502 mAh g-1. After 60 cycles, the reversible capacity was still kept at 652 mAh g-1 at the current density of 50 mAh g-1. These results indicate that the prepared hollow graphene spheres possess excellent electrochemical performances for lithium storage. The high rate performance of hollow graphene spheres thanks to the hollow structure, thin and porous shells consisting of graphene sheets.The hollow graphene oxide spheres have been successfully fabricated from graphene oxide nanosheets utilizing a water-in-oil emulsion technique, which were prepared from natural flake graphite by oxidation and ultrasonic treatment. The hollow graphene oxide spheres were reduced to hollow graphene spheres at 500°C for 3 h under an atmosphere of Ar(95%)/H81.05.ue; 61.48.Gh; 72.80.Vp It is regarded as the ‘thinnest material in the universe’ with tremendous application potential. These attractive properties of graphene generate huge interest from different scientific communities in the possible implementation of graphene in different application fields such as biomedicine, reinforced composites, sensors, catalysis, energy conversion and storage device, electronics, and transparent electrodes for displays and solar cells [Since the paper on freestanding graphene was published by Novoselov et al. , the prear cells .4C6 stoichiometry) by density of states calculations [Nowadays, lithium-ion batteries are widely used in various electronic devices, such as notebook computers, cellular phones, camcorders, electric vehicles, and electric tools due to their superior properties such as long cycle life, high energy density, no memory effect, and environmental friendliness. To meet the increasing demand for lithium-ion batteries with high reversible capacity and energy density, much effort has been made to develop new electrode materials or design novel structures of electrode materials -14. Receulations .In this work, the hollow graphene oxide spheres (HGOSs) were fabricated directly from graphene oxide (GO) utilizing a water-in-oil emulsion technique, which were prepared from natural flake graphite by oxidation and ultrasonic treatment. The hollow graphene oxide spheres were reduced to hollow graphene spheres (HGSs) by heat treatment under a hydrogen atmosphere. Compared with the graphene sheets , the pre4 was employed as the oxidant to obtain graphite oxide. Firstly, 1 g of flake natural graphite powder with the mean diameter of 15 μm  was added to 23 mL of cooled (0°C) concentrated H2SO4. Then, 3 g of KMnO4 was added gradually with stirring and cooling, so that the temperature of the mixture was maintained below 10°C. The mixture was then stirred at 35°C for 30 min. After this, 46 mL of distilled water was slowly added to cause an increase in temperature to 98°C, and the mixture was maintained at that temperature for 15 min. The reaction was terminated by adding 140 mL of distilled water followed by 10 ml of 30% H2O2 solution. The suspension was then repeatedly centrifuged and washed twice with 5% HCl solution and then repeatedly with water until sulfate could not be tested with barium chloride. The collected precipitate was dispersed in 450 mL water and sonicated for 2 h. Then, the suspension was separated into the supernatant liquor and a golden colored residue by centrifugation at 5,000 rpm for 10 min. The supernatant was centrifuged again at 15,000 rpm for 5 min to remove the suspended substance. The precipitate was ultrasonicated, collected, and dried in a vacuum oven at 60°C; thus, GO nanosheets were obtained.GO nanosheets were prepared in two steps: the oxidation of flake natural graphite powder via a modified Hummers' method and ultrasonication. KMnO2(5%).GO nanosheets of 0.1 g were dispersed into aqueous ammonia  through agitation and were stirred at 30°C for 1 h to obtain the GO nanosheet suspension. Then, the suspension was slowly poured into hot olive oil  preheated to 90°C and intensely stirred for 30 min at 90°C. Subsequently, with the formation of a water-in-oil emulsion, the viscosity of the emulsion rapidly increased with the appearance of a golden foam. Half an hour later, when the bath temperature was increased to 95°C, the viscosity decreased gradually. With the intensive stirring, water was gradually separated from the oil. In the meantime, emulsion turned clear as olive oil. Finally, the emulsion system was cooled to room temperature. The HGOSs were obtained by centrifugation, washing, and drying. The HGOSs were reduced to HGSs at 500°C for 3 h under an atmosphere of Ar(95%)/Hλ = 1.5406 Å) over the range of 5 to 90° (2θ) at room temperature. The morphologies of the samples were observed by scanning electron microscope . The information of functional groups was measured by Fourier transform infrared spectroscopy instrument .The products were characterized by X-ray diffraction (XRD) on a Rigaku D/max-2500B2+/PCX system  using Cu/K radiation  mixture of ethylene carbonate (EC) and dimethyl carbonate (DMC) from Merck & Co., Inc.  was used as electrolyte. The Celgard 2400 microporous polypropylene film provided by Jimitek Electronic  Co. Ltd was used as separator. The coin-type cells were galvanostatically discharged (Li insertion) and charged (Li extraction) in the voltage range from 0.01 to 3.50 V vs. Li/Li+ at the different current densities. Electrochemical impedance spectroscopy measurements of the electrodes were carried out on an electrochemical workstation  using the frequency response analysis. The impedance spectra were obtained by applying a sine wave with amplitude of 5.0 mV over the frequency range from 100 kHz to 0.01 Hz.The electrochemical performances of the HGSs as anode materials for lithium-ion batteries were measured with the coin-type cells. The lithium sheets were used as both reference and counter electrodes, and composite electrodes comprising active mass , carbonaceous additive , and poly(vinylidene difluoride)  binder were used as working electrodes. The thickness and density of electrode are 50 μm and 1.95 mg cmThe morphology and structure of HGOSs and HGSs were characterized by SEM, and their images are shown in Figure d-spacing of GO increased to 0.765 nm from 0.335 nm of graphite, which is ascribed to the oxide-induced O-containing functional groups and inserted H2O molecules [-1 can be attributed to O-H stretching vibrations of adsorbed water molecules and structural OH groups, and the peak at 1,619 cm-1 can be attributed to O-H bending vibrations. The presence of carboxyl and epoxy functional groups can also be detected at around 1,724 and 1,224 and 1,053 cm-1, respectively [4 in the concentrated sulfuric acid, the original extended conjugated π-orbital system of graphite were destroyed, and oxygen-containing functional groups were inserted into carbon skeleton. Therefore, it is reasonable to believe that GO nanosheets should be regarded as ‘amphiphilic molecules’ and perform a surfactant-like function in a water/oil emulsion system [The structural changes from GO to HGSs were investigated by XRD measurement, and the patterns are shown in Figure olecules ,22 that ectively ,22. Thesn system . Due to 2, these functional groups derived from the intensive oxidation were eliminated, which can be proved by the disappearance of the peaks at 1,724, 1,619, 1,224, and 1,053 cm-1 while an appearance of a new peak at 1,631 cm-1  film formation and the generation of irreversible capacity. From the second cycle, the charge/discharge curve of HGS slope without distinguishable plateaus, which can be attributed to the smaller crystallite structure, high specific surface area [-1, respectively. Obviously, the reversible capacity of HGSs is much higher than that of previously reported graphene nanosheets (672 mA h g-1 at a current density of 0.2 mA cm-2) [-1 in the second cycle, but it was still maintained at 741 mA h g-1 in the fifth cycle. This evidence indicates that the prepared HGSs exhibited stable cyclic performance from the second cycle because of the formed stable SEI film during the first discharge process. The cyclic voltammograms (CV) of the prepared HGSs are shown in Figure Figure ace area , and disace area ,16. For mA cm-2) . The posmA cm-2) . The posmA cm-2) . It is wmA cm-2) , and themA cm-2) ,24-28. TmA cm-2) . The rev-1, 100 mA g-1, 200 m mA g-1, 500 m mA g-1, and 1,000 mA g-1 are shown in Figure -1 for HGSs. These results demonstrated that the prepared HGSs have an intensive potential as a candidate of anode materials with high reversible capacity, good cycle performance, and high rate discharge/charge capability.Cycle performance of HGSs at different current densities of 50 mA gTo investigate the kinetics of electrode process of HGS electrode, its Nyquist complex plane impedance plots are presented in Figure -1 at a current density of 50 mAh g-1. After 60 cycles at different current densities of 50 mA g-1, 100 mA g-1, 200 m mA g-1, 500 m mA g-1, and 1,000 mA g-1, the reversible specific capacity was still maintained at 652 mA g-1 at the current density of 50 mA g-1, which indicated that the prepared HGSs possess a good cycle performance for the lithium storage. The high rate performance of HGSs thanks to the hollow structure, thin and porous shells consisting of graphene sheets.The HGSs have been successfully fabricated from GO nanosheets utilizing a water-in-oil emulsion technique and thermal treatment. The electrochemical performance testing showed that the first reversible specific capacity of the HGSs was as high as high as 903 mAh gHGOSs: hollow graphene oxide spheres; GO: graphene oxide; HGSs: hollow graphene spheres; XRD: X-ray diffraction; SEM: scanning electron microscope; FTIR: Fourier transform infrared; SEI: solid electrolyte interface; CV: cyclic voltammograms.The authors declare that they have no competing interests.RRY designed parts of the experiments and sample preparations and drafted the manuscript. DLZ is the corresponding author and provided a great help for experimental designs. LZB, NNY, and LX took part in sample preparation and characterizations and discussed the results. All authors have read and approved the final manuscript.


Hypoxia effects on pulmonary artery structure and function are key to diseases such as pulmonary hypertension. Recent studies suggest that growth factors called neurotrophins, particularly brain-derived neurotrophic factor (BDNF), can influence lung structure and function, and their role in the pulmonary artery warrants further investigation. In this study, we examined the effect of hypoxia on BDNF in humans, and the influence of hypoxia-enhanced BDNF expression and signaling in human pulmonary artery smooth muscle cells (PASMCs).2+ responses to serotonin: an effect altered by HIF1α inhibition or by neutralization of extracellular BDNF via chimeric TrkB-Fc. Enhanced BDNF/TrkB signaling increased PASMC survival and proliferation, and decreased apoptosis following hypoxia.48h of 1% hypoxia enhanced BDNF and TrkB expression, as well as release of BDNF. In arteries of patients with pulmonary hypertension, BDNF expression and release was higher at baseline. In isolated PASMCs, hypoxia-induced BDNF increased intracellular CaEnhanced expression and signaling of the BDNF-TrkB system in PASMCs is a potential mechanism by which hypoxia can promote changes in pulmonary artery structure and function. Accordingly, the BDNF-TrkB system could be a key player in the pathogenesis of hypoxia-induced pulmonary vascular diseases, and thus a potential target for therapy. Samples were from lobectomies or pneumonectomies for focal non-infectious indications such as localized tumors, and were classified as being non-PH or PH based on clinical data. The limited PH data set was entirely mild-moderate, where mean PAP clinically estimated by transthoracic or transesophageal echocardiography ranged from 25 mmHg (mild) to 48 mmHg (moderate), given high anesthesia risk for non-transplant surgeries in severe PH. Samples were limited to areas distant from the focal pathologies as identified by the pathologist and verified under gross microscopy. Since the samples and clinical data were de-identified, the protocol was considered minimal risk Human Subjects Research.2 after which the source tissue was removed and cells plated in 60mm dishes , 8 well Labtek culture chambers or 96 well plates (imaging and proliferation assays). Prior to experimentation, cells were serum-deprived for 24h, and all cells were used between passages 1 and 4 to ensure maintenance of PASMC phenotype, verified by markers such as smooth muscle actin and myosin, and transgelin (SM22), but absence of fibroblast specific protein.Samples were transported rapidly in ice-cold Hank’s Balanced Salt Solution (HBSS). The PA segment was partitioned for testing of endothelium-intact PA, and for analysis of endothelium- and adventitia-denuded PA smooth muscle. The latter was used as tissue or for isolation of PASMCs, which involved mincing and growth of explants in DMEM with 10% FBS/1% ABAM. Tissue explants were maintained for 5–7 days at 37°C in 95% air/5% CO2 for 48h in a hypoxia chamber (Biospherix C-chamber). The chamber was flushed 3 times for 10 min each with high flow 1% O2 prior to use. For BDNF exposure, intact PA or isolated PASMCs were incubated for 24 or 48 h (see protocols) at 37°C in regular growth media (vehicle control) or 100 pM recombinant human BDNF . This concentration was based on previous reports of circulating serum BDNF levels of ~20 ng/ml which translates to <1 nM. ; i.[in situ by exposing PASMCs to known Ca2+ levels with the ionophore A-23187.We have previously published techniques for imaging of cellular [Ca2+]i. Briefly,Proliferation of PASMCs was assayed at 48h (with or without preceding interventions) using the fluorescent CyQuant NF kit (Invitrogen) as previously described. Cells wepost hoc analysis using SigmaPlot , and statistical significance was established at P < 0.05. Values are means ± SE.PA tissue or PASMC experiments were performed in samples from 3–7 patients (no pooling), with 3 repeats/sample. Data were analyzed and results were compared using one way ANOVA with Student-Newman-Keuls or Dunnet’s 2 which is known to induce HIF1α; see below)i responses to 1 μM serotonin  but not eliminated. Furthermore, [Ca2+]i responses to 5HT were diminished in the presence of the HIF1α inhibitor  enhanced the amplitude of i in PASMCs in the presence of hypoxia would suggest an opposing, vasoconstrictive effect that may offset any beneficial effects of BDNF on endothelium. Furthermore, the enhancing effect of BDNF on smooth muscle proliferation demonstrated in these data and reported by others [2+]i regulation, BDNF can activate mechanisms such as IP3 and influx pathways such as TRPCs, both of which are well-known to be important in PASMCs  hig hig22] hy others  would onn PASMCs ,57,58,592+]i responses to serotonin, including enhancement of Ca2+ influx as well as enhanced SR Ca2+ release. Furthermore, the enhancing effect of BDNF on agonist responses is probably not specific only to serotonin. For example, we have previously demonstrated in human airway smooth cells that BDNF can increase baseline [Ca2+]i and responses to agonists such as Ach. [2+]i regulatory proteins such as CD38, Orai1, IP3 and RyR receptors. i by altering expression of regulatory proteins: a topic yet to be examined.There are likely a number of mechanisms to explain how BDNF increases i and proliferation that can potentially contribute to hypoxia effects on arterial contractility and remodeling. While locally produced BDNF may be particularly important, the potential exists for circulating BDNF to further influence PA. Future studies should examine the mechanisms by which hypoxia can modulate endothelial vs. PASMC BDNF/TrkB signaling and the overall, in vivo effect on PA contractility and remodeling, especially in the context of diseases such as PH.In conclusion, our study demonstrates enhancement of BDNF and TrkB expression and signaling in PASMCs following hypoxia, resulting in increased PASMC [Ca


Maize streak Reunion virus (MSRV) is a member of the Mastrevirus genus in the family Geminiviridae. Of the diverse and increasing number of mastrevirus species found so far, only Wheat dwarf virus and Sweetpotato symptomless virus 1 have been discovered in China. Recently, a novel, unbiased approach based on deep sequencing of small interfering RNAs followed by de novo assembly of siRNA, has greatly offered opportunities for plant virus identification.Samples collected from maize leaves was deep sequencing for virus identification. Subsequently, the assay of PCR, rolling circle amplification and Southern blot were used to confirm the presence of a mastrevirus.Maize streak Reunion virus Yunnan isolate  was identified from maize collected from Yunnan Province, China, by small RNA deep sequencing. The complete genome of this virus was ascertained as 2,880 nucleotides long by conventional sequencing. A phylogenetic analysis showed it shared 96.3 % nucleotide sequence identity with the isolate of Maize streak Reunion virus from La Reunion Island. To our knowledge, this is the first identification of MSRV in China. Analyses of the viral derived small interfering RNAs (vsiRNAs) profile showed that the most abundant MSRV-YN vsiRNAs were 21, 22 and 24 nt long and biased for A and G at their 5’ terminal residue. There was a slightly higher representation of MSRV-YN siRNAs derived from the virion-sense strand genome than the complementary-sense strand genome. Moreover, MSRV-YN vsiRNAs were not uniformly distributed along the genome, and hotspots were detected in the movement protein and coat protein-coding region.A mastrevirus MSRV-YN collected in Yunnan Province, China, was identified by small RNA deep sequencing. This vsiRNAs profile derived from MSRV-YN was characterized, which might contribute to get an insight into the host RNA silencing defense induced by MSRV-YN, and provide guidelines on designing antiviral strategies using RNAi against MSRV-YN.The online version of this article (doi:10.1186/s12985-015-0384-3) contains supplementary material, which is available to authorized users. Geminiviridae are taxonomically classified into seven genera  based on insect vector, host range and genome organization , which was abbreviated to MSRV-YN.In a PCR using primer pair F/R1 to investigate the incidence of MSRV-YN among the 22 maize samples collected from Yuanmou county, Yunnan province, amplicons of the expected size, about 700 bp, were generated in ten of the 22 tested samples, revealing an infection rate for isolate MSRV-YN of 45.5 % (data not shown). To our knowledge, this is the first report of a isolate of MSRV in China.Back-to-back primers F/R designed based on the assembled contigs were used to amplify the full-length genomic sequences of MSRV-YN from a crude extract of total DNA from the maize sample, yielding a fragment of about 3.0 kb. This fragment was then cloned and sequenced by conventional Sanger sequencing and assembled by SeqMan , generating a complete sequence 2880 bp long (GenBank: KT717933). A BLASTn search using the completed genome of MSRV-YN in NCBI revealed significant similarity (98 %) to the isolate of MSRV from La Reunion Island (GenBank: JQ624880).To further prove the presence of MSRV-YN, the aforementioned DNA was used as template for RCA amplification followed by restriction fragment length polymorphism (RFLP) analyses, which is a nonsequence-specific method that potentially allows the identification of all circular DNA viruses. Three different RCA-amplified products from randomly selected MSRV-YN positive samples were digested with the BamHI restriction endonuclease, which proved to have a single restriction site based on the information of the obtained complete genome sequence of MSRV-YN. As expected, a single DNA fragment of about 3.0 kb was obtained, the size expected for a mastrevirus. Meanwhile, no band was ever found in the negative control from healthy maize leaves Fig. . Next, SMastrevirus, revealed that MSRV-YN shares <63 % genome-wide pairwise identity with known Mastrevirus species, except that for a close identity with MSRV (GenBank: KJ437669); complete genome identity and amino acid identities for MP, CP, Rep, RepA are 96.3, 99.0, 98.0, 95.1 and 93.3 %, respectively  in Nicotiana benthamiana [Cabbage leaf curl virus (CabLCV) in Arabidopsis thaliana [The identification of viral small interfering RNAs (vsiRNAs) mapping to specific viral genome provides strong evidence for presence of the virus. We characterized the profile of vsiRNAs derived from MSRV-YN Fig. . A totalthamiana  and Cabbthaliana .Fig. 4ChA. thaliana, AGO2 and AGO4 predominantly favored small RNAs starting with the 5’ -terminal A, AGO1 preferentially recruited sRNAs initiating with a 5’ -terminal U, while AGO5 predominantly bound small RNAs that with a 5’ -terminal C [Previous studies have indicated that the recruiting of sRNAs into specific AGO complexes mainly depends on the identity of the first 5’ -nucleotide of sRNAs , 17. To rminal C , 17. It rminal C , 15, 18.rminal C , 18, 19.When the vsiRNAs strand polarity was evaluated using the 20–25 nt vsiRNAs, a slightly higher representation of MSRV-YN siRNAs derived from the virion-sense strand (positive vsiRNAs) was unexpectedly observed, accounting for about 63.46 % of the total vsiRNAs, irrespective of length Fig. , suggestCucumber mosaic virus satellite RNA could be targeted more efficiently by DCL4 [A genome-wide view of vsiRNAs revealed that the vsiRNAs of MSRV-YN were not uniformly distributed along each genome segment, with the most abundant siRNAs matched both positive and negative strands of the MP and CP-coding region Fig. , indicat by DCL4 . Since M by DCL4 . A greatSetaria barbata and Rottboellia sp. in Nigeria, suggesting expanded host and geographical ranges for this virus [Currently, mastreviruses are receiving more and more attention since they have been associated with many serious crop diseases. Of the 29 currently recognized mastrevirus species, 23 include viruses that can infect monocotyledonous hosts and 6 include viruses that infect dicotyledonous hosts . To dateis virus . This fiRecent advances in deep sequencing have greatly accelerated the accuracy and rate of virus discovery. Our study, the first to use deep sequencing to characterize siRNAs derived from MSRV-YN, should provide guidelines on designing antiviral strategies using RNAi against MSRV-YN.Zea mays) leaf samples were collected from five maize fields in Yuanmou County, Yunnan Province, China, the distance between which ranged from 100 m to 2000 m more than. These samples displaying a range of disease symptoms such as yellowing and chlorotic streaks along leaf veins (data not shown) were stored at −80 °C. Total RNA was extracted from 0.1 g of symptomatic maize leaf according to the manufacturer’s instructions . The purified RNA preparations were evaluated using a spectrophotometer  and agarose gel electrophoresis. The better quality RNA described as YM was shipped with dry ice to the Beijing Genomics Institute (BGI) for small RNA library construction and sequencing.In May 2014, 22 maize (k-mer) required for joining two sRNAs into a contig [Sequencing was performed according to the manufacturers’ protocol by using the high-throughput Illumina HiSeq-2000 sequencing technology. Data processing was conducted using a custom bioinformatics pipeline. Raw data were analyzed using an in-house Perl script, and 18–28 nt reads consisting of trimmed sRNA sequences were collected for subsequent analysis. The Velvet program  was useda contig . The assa contig .A CTAB-based extraction method  was usedThe rolling circle amplification of circular DNA was conducted using TempliPh 500 Amplification Kit  according to the manufacturer’s protocol. From 10 to 100 ng of total deoxyribonucleic acids was dissolved in 5 μl sample buffer, denatured at 95 °C for 3 min and then immediately cooled down on ice for 10 min. Subsequently, 5 μl reaction buffer and 0.1 μl enzyme mix were added, and the reaction was run at 30 °C for 18 h. The reaction was then stopped at 65 °C for 5 min. The RCA products were digested with the single restriction enzyme BamHI, and fragments were then separated on 1 % agarose gels, excised and recovered using a Gel Extraction Kit (Axygen Scientific Inc). For Southern blot analysis, 20 μg of total DNA preparations and 20 ng RCA recovered products were used and hybridized with a virus-specific probe according to standard Southern blot protocols. The probe was synthesized using the primer pair F1/R1  were predicted using the ORF finder function of the Snap Gene software . NucleotSmall RNA mapping was conducted by Bowtie 1.0 with zero mismatch, and results were exported to Microsoft Excel for further analysis. The program MISIS  was used


Transmission electron microscopy (TEM) photograph and X-ray photoelectron spectroscopy (XPS) analysis of nanoporous graphene nanosheets indicated that the structure of nano-pore was irregular and the oxygen-containing groups in the surface were limited. More importantly, the as-prepared MMMs presented better separation performance than that of pristine EC membrane due to simultaneous enhancement of C3H6 permeability and ideal selectivity. The ideal selectivity of the MMMs with 1.125 wt‰ nanoporous graphene content for C3H6/C3H8 increased from 3.45 to 10.42 and the permeability of C3H6 increased from 57.9 Barrer to 89.95 Barrer as compared with the pristine membrane. The presumed facilitated mechanism was that the high specific surface area of nanoporous graphene in polymer matrix increased the length of the tortuous pathway formed by nanopores for the gas diffusion as compared with the pristine graphene nanosheets, and generated a rigidified interface between the EC chains and fillers, thus enhanced the diffusivity selectivity. Therefore, it is expected that nanoporous graphene would be effective material for the C3H6/C3H8 separation.Nanopore containing graphene nanosheets were synthesized by graphene oxide and a reducing agent using a facile hydrothermal treatment in sodium hydroxide media. The as-prepared nanoporous graphene was incorporated into ethyl cellulose (EC) to prepare the mixed matrix membranes (MMMs) for C Currently, the separation is mainly achieved by fractional distillation at cryogenic temperature343H6/C3H8 system using nanofillers, and most nanofillers are spherical shape like silica materials and C60, or square zeolitic like imidazolate framework ZIF-8 13143H6/C3H8 was 6.12 and the permeability of C3H6 was 0.098 Barrer with a nanocomposite membrane that contains 30 wt% silica particles under 2 bar feed absolute pressure and 35 °C temperature. Koros et al.3H6/C3H8, and a permeability of 56.2 Barrer for C3H6 with 48.0 wt% ZIF-8 loading, which were 150% and 258% higher than the pure 6FDA-DAM membrane respectively for selectivity and permeability.In recent years, a few studies have been reported about employing mixed matrix platform to the Cet al.26As mentioned above, most available literatures focused on the spherical and square shape nanofillers to enhance the gas separation performance. It is inevitable to generate the ‘sieve-in-a-cage morphology’ between these nanofiller materials and polymer interface due to the low aspect ratio of the spherical and square shape nanofillers and the weak interfacial adhesion. Such voids decrease the selectivity of the MMMs, even the permeability is increased71719202122233H6 diffusion coefficient. Furthermore, massive oxygen functional groups on the surface of graphene nanosheets generate better interfacial bonding strength with EC polymer28293H6/C3H8 separation performance, light reduction graphene oxide (L-rGO) containing oxygen-containing groups such as carboxyl, hydroxyl group and reduction graphene oxide (rGO) were synthesized and incorporated into EC polymer to prepare MMMs for C3H6/C3H8 permeation. The morphology and microstructure of the as-prepared graphene material were confirmed, and the physical properties of MMMs in terms of microstructure, crystallization, tensile property and thermal stability  is selected in this study as the polymer matrix based on its comparatively large free volume and relatively low glass transition temperatures, which might provide high gas permeability and a superb Clity see  were invated see .4 is inadequate for the chemical reduction of the vast oxygen groups on the GO nanosheets. However, intensive deoxygenation processes can be completed with abundant NaBH4 reduction and NaOH hydrothermal treatment, as shown in Different methods of treatment on GO nanosheets may result various morphologies, and these changes can be directly observed through TEM characterization. 2 regions in the graphene were gradually weakened and resulted in a defected surface. After that, these defected sites increasingly expanded and finally generated nanopores in the intensive hydrothermal treatment process. Therefore, an increased ID/IG intensity ratio gradually emerges from light chemical reduction (L-rGO) to chemical reduction (rGO), and then hydrothermal treatment (nanoporous rGO). Combined with the results of TEM images, it can be concluded that nanoporous rGO nanosheets would contain large amount of nanopores as compared with L-rGO and rGO nanosheets.Variation on the morphologies of graphene nanosheets can also be indirectly measured by the Raman spectra. As observed in 32−1 wavenumber are ascribed to the C–O, C=C and O–H groups in the L-rGO with a relative higher intensity in comparison with the rGO and nanoporous rGO nanosheets34−1 in rGO and nanoporous rGO results from the greatly decrease of the oxygen functional groups, indicating the completion of chemical reduction process.The micro-structure of graphene nanosheets were also characterized by FTIR spectroscopy. As shown in In order to further confirm the difference of micro-structure of L-rGO, rGO and nanoporous rGO nanosheets, XPS measurement was conducted . As showXRD is performed to investigate the effects of graphene nanosheets on the EC polymer chains. As shown in 3840The cross-section morphologies of three MMMs are presented in 424445The TEM image of nanoporous rGO MMMs solution further verifies the nanoscale morphology of nanoporous rGO nanosheets. As observed in 3H6 permeability, C3H6/C3H8 ideal selectivity gradually increase with the increase of graphene nanosheets content from 0 to 1.125 wt‰, while C3H8 permeability decreases with the content of graphene nanosheets increase from 0 to 0.375 wt‰ and then remain unchanged after 0.0375 wt‰ of graphene nanosheets loading. However, when the loading of the graphene nanosheets in the EC polymer matrix increases to 1.5 wt‰, both C3H6 permeability and C3H6/C3H8 ideal selectivity decrease, indicating that the graphene nanosheets are theoretically impermeable to all atoms and molecules, and greater loading in the MMMs could cause the agglomeration of the graphene nanosheets, resulting in the decrease of the effective surface area4748493H6 permeability of 66.05, 76.34 and 89.95 Barrer, respectively. Meanwhile, it is clearly showed that the ideal selectivity of pure EC polymer membrane is 3.45 and the permeability of C3H6 and C3H8 is 57.9 and 16.78 Barrer, which is consistence with the related literature3H6 permeability of 1.55-fold and C3H6/C3H8 ideal selectivity of 3.02-fold confirm the fact that the nanopores blended into the MMMs enhance the C3H6 permeability as compared with the pure EC membrane gas permeation performance. However, the blending of graphene nanosheets into the EC polymer matrix hinders the C3H8 permeability. Among three kinds of MMMs, nanoporous graphene MMMs have C3H6/C3H8 ideal selectivity and C3H6 permeability advantages over that of L-rGO and rGO MMMs, which indicates that oxygen-containing functional groups decrease the C3H6 permeability. A reasonable explanation of the gas separation mechanism is proposed as shown in In order to investigate the structure of graphene nanosheets and their loading content on the membrane permeation performances, three kinds of MMMs are fabricated with different nanofiller loading. 3H6/C3H8 diffusivity selectivity of L-rGO MMMs, rGO MMMs and nanoporous rGO MMMs are higher than that of pure EC membrane. It is notable that the nanoporous rGO MMMs has the highest C3H6/C3H8 diffusivity selectivity among L-rGO MMMs and rGO MMMs. The lack of nanopores in the nanosheets hinders the further enhancement of C3H6 diffusivity coefficient, even L-rGO nanosheets and rGO nanosheets generate a rigid interface with EC polymer chains and increase the tortuous pathway of the gas diffusion to enhance the diffusivity selectivity. We can conclude that the nanopores in the nanosheets further increase the tortuous pathway of the C3H6 diffusion with less resistance but restrain the diffusion of C3H8 molecules.To further explore the effect of nanopore in the graphene nanosheets on the gas permeation performance, the diffusivity coefficient (D) and solubility coefficient (S) of the membranes with the optimal 1.125 wt‰ of nanofiller loading are compared in 3H6/C3H8 solubility selectivity of L-rGO MMMs, rGO MMMs and nanoporous rGO MMMs with the optimal 1.125 wt‰ of nanofiller loading. The solubility of C3H6 and C3H8 in the polymer membranes depends on their relative condensability, characterized by critical temperature, the interaction between polymer and gases, the fraction and amount of free volumes in glassy polymers. The critical temperatures of C3H6 and C3H8 are in the following order: C3H6(364.76 K) < C3H8(369.8 K). It is believed that higher condensability has the higher solubility of gas in the polymer matrix5253543H6 exhibits greater solubility than C3H8. This is thanks to the high electron cloud on the double bond with high polarity and flat structure, resulting in more affinity of C3H6 to generate interaction with polymer, and thus enhancing the C3H6 solubility. Moreover, the incorporation of graphene nanosheets increases the density of polar groups such as -COOH and -OH in the MMMs. As a result, it creates polar spaces between the interface of nanofillers and polymer. This formation of polar spaces would result in the enhancement in the solubility coefficient of condensable gases3H6/C3H8 molecule to enhance the ideal selectivity of MMMs, due to the existence of nanopores and the formation of rigid interface.The diffusivity selectivity increases by 5.24-fold in comparison with 2.66-fold for L-rGO MMMs and 2.93-fold for rGO MMMs, although the loading of nanoporous graphene nanosheets in the polymer matrix cannot efficiently increase the solubility selectivity of the gases due to the decrease of the free volume of the glassy polymer. The incorporation of nanoporous graphene nanosheets into the EC polymer matrix mainly affects the diffusivity selectivity of C3H6/C3H8 permeation. The permselectivity of nanoporous rGO MMMs exhibits a significant increase with the simultaneous enhancement of diffusivity selectivity and solubility selectivity, especially the 5.24-fold increase of the diffusivity selectivity. This result indicates that nanoporous rGO nanosheets are superior in terms of enhancing the diffusivity selectivity of C3H6 gas molecule. The ideal selectivity for C3H6/C3H8 and the permeability coefficient for C3H6 exhibit a significant increase of 3.02-fold and 1.55-fold in comparison with the pristine EC membrane, respectively. This study shows that nanoporous rGO nanosheets can be effective nanofiller in the polymer matrix to enhance the C3H6/C3H8 permeability.In summary, nanoporous rGO nanosheets were successfully fabricated by sodium hydroxide using a facile hydrothermal treatment. The morphology and micro-structure of the nanoporous rGO material were confirmed by TEM and XPS measurement. Nanoporous rGO nanosheets were incorporated into the EC polymer matrix to prepare the MMMs for the C4), sodium hydroxide (NaOH), sodium dodecylbenzene sulfonates (SDBS), hydrochloric acid (HCl 36~38%), acetone and all other types of reactants were bought from Shanghai Chemical Co., Ltd. (China). The propylene (purity > 99.5%) and propane (purity > 99.9%) were purchased from KODI Gas Chemical Industry Co. Ltd. .EC  was purchased from Shanghai Reagent Corporation (China). Graphite oxide was obtained from Nanjing XFNANO Materials Tech Co. Ltd. (China). Sodium borohydride .In this case, GO colloidal solution (0.1 g in 150 mL deionized water) was obtained using a mild ultrasonic exfoliation method for 2 h. The above colloidal solution was centrifuged to remove the impurities under 8000 r min4 mass ratio of 1:2 at 80 °C for 0.5 h, and then the above L-rGO was collected by filtration. Subsequently, 10 mL SDBS (0.086 mol L−1) and 20 mL NaOH (12.5 mol L−1) were successively added dropwise to form a 60 mL solution. After stirred for 0.5 h, the reaction solution was decanted into a 100 mL Teflon-lined stainless steel autoclave and conducted the hydrothermal treatment at 180 °C for 3 h. Eventually, the reaction solution was cooled to ambient temperature, and the resulting solution was consecutively filtered and washed with sufficient deionized water (five times) and ethanol, and the nanoporous rGO nanosheets were placed in ethanol at a given concentration (0.012 g in 15 mL ethanol).The nanoporous rGO nanosheets were synthesized with the treatment of sodium hydroxide under hydrothermal condition. Firstly, GO colloidal solution (0.1 g in 150 mL deionized water) obtained from an mild ultrasonic exfoliation method for 2 h was chemically reduced to L-rGO using the GO to NaBHA series of different concentration of L-rGO, rGO and nanoporous rGO ethanol solutions were prepared via sonication for 1 h. Dried EC (in a vacuum situation at 40 °C for 24 h) was dissolved in methylbenzene to form a solution with the concentration of 19.7 wt%. Under a nitrogen atmosphere and at ambient temperature, the above dispensed graphene ethanol solutions were then added dropwise to the EC solutions with stirring, The resulting casting solution had a mass ratio of graphene to EC of 0.375, 0.75, 1.125 and 1.5 wt‰ respectively. After 24 h dissolution, the casting solutions were filtered with stainless steel filter, and still degassing for 24 h. The above casting solution was then cast onto the polyimide (PI) ultrafiltration supported membrane using a micrometer film applicator under RH 40% at 298 K. The final membranes were subsequently preserved for gas separation tests. All membrane thicknesses were approximately 15–20 μm.How to cite this article: Yuan, B. et al. Propylene/propane permeation properties of ethyl cellulose (EC) mixed matrix membranes fabricated by incorporation of nanoporous graphene nanosheets. Sci. Rep.6, 28509; doi: 10.1038/srep28509 (2016).


Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.2 The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.5 Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.1 TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,7 Alzheimer's disease (AD)9 and Parkinson's disease (PD).11 TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.14Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.16 Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,22 indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.29 The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.32 One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.37 The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,40 and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.46 Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.28Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.51 Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,51 indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.The goal of this study is to better understand the role of TDP-43 in neuroinflammation, a key feature of ALS. To gain insight into the roles of microglia and astrocytes in TDP-43-mediated neurotoxicity, we knocked down TDP-43 in primary cultured microglia and astrocytes using siRNA against TDP-43, and we then used biochemical approaches to detect the expression levels of COX-2 and iNOS, two markers of neuroinflammation. RNAi-mediated depletion of TDP-43 in microglia resulted in a greatly increased protein level of COX-2 with or without LPS treatment, whereas iNOS protein level was unchanged . In contTo characterize the molecular mechanism underlying the upregulation of COX-2 in TDP-43-deficient microglia, we examined the changes in cell signaling pathways accompanied by upregulation of COX-2 expression in TDP-43-deficient microglia. Given that the classical MAPK signaling pathway could regulate COX-2 expression and is significantly associated with various neurodegenerative diseases, we hypothesized that TDP-43 might be involved in the regulation of MAPKs. To test this possibility, we detected the total and phosphorylated levels of MEK, ERK, JNK and p38 in TDP-43-deficient microglia. As shown in β inhibitor) and SB203580 (p38 inhibitor), had no effect on TDP-43-mediated COX-2 expression  , and theWe next investigated whether the COX-2 inhibitor celecoxib could alleviate neuronal death driven by TDP-43-depleted microglia. We performed the conditioned medium assay as described in Although initially, most studies on ALS focused on the selective loss of motor neurons themselves, increasing efforts to understand the role of glial cells in disease pathogenesis have come to the forefront of the field. The present study reveals microglia-mediated COX-2-PGE2 production as the molecular determinant of TDP-43-associated neurotoxicity, emphasizing the important contribution of microglia to non-cell-autonomous motor neuron degeneration in TDP-43-linked ALS .51 On the basis of this finding, it is possible that other types of cells surrounding motor neurons are involved in the initial toxic effect of motor neurons. Consistent with this notion, microglia have recently been suggested to have a role in ALS initiation,52 but the contribution of microglia to TDP-43-linked ALS is still not fully understood. In this study, we show that loss of TDP-43 in microglia strikingly triggers the increase of COX-2 , indicating that signaling molecules other than NF-κB are required for this regulation. Relatedly, our results show that knockdown of TDP-43 in microglia resulted in the activation of AP-1 , anti-COX-2 , anti-GAPDH , anti-phospho-MEK (S217/221) , anti-MEK , anti-phospho-ERK (Santa Cruz Biotechnology), anti-ERK (Santa Cruz Biotechnology), anti-phospho-JNK (Epitomics), anti-JNK (Santa Cruz Biotechnology), anti-phospho-p38 (Epitomics), anti-p38 (Santa Cruz Biotechnology), anti-phospho-c-Raf (S338) , anti-c-JUN , anti-phospho-Glycogen Synthase (S641) (p-GS) (Epitomics) and anti-MAP2 (Santa Cruz Biotechnology). The secondary antibodies used included horseradish peroxidase-conjugated sheep anti-mouse and anti-rabbit antibodies . The antibody-bound proteins were visualized using an ECL detection kit  or Alexa Fluor 488 (green) donkey anti-mouse IgG .The anti-TDP-43 antibody was described previously,μM), BAY (Beyotime) (25 μM), SP600125 (10 μM), SB216763  (10 μM), SB203580 (10 μM), celecoxib (Sigma) (50 μM), or PBS (Gibco) or LPS (Beyotime) (0.1 μg/ml). The following sequences were used for siRNA targeting mouse TDP-43 (si-TDP-43): 5′-GGATCTGAAAGACTATTTC-3′ siRNA targeting rat TDP-43 (si-TDP-43): 5′-CCAATGCTGAACCTAAGCA-3′. For cell viability analysis, the cells were stained with PI and visualized using fluorescence microscopy. The dead cells with PI-positive staining were counted and the percentage of viability of cells was quantified.Mouse motor neuron cell line (NSC-34 cells) were cultured in Dulbecco's modified Eagle's medium (DMEM)  containing 10% fetal bovine serum (FBS) (Gibco) with penicillin (100 mg/ml) and streptomycin (100 mg/ml). Mouse microglial cell line (BV2 cells) were cultured in similar culture medium, but using 10% heat-inactivated FBS instead. For RNAi experiments, cells were transfected with siRNA against TDP-43 using the RNAiMAX (Invitrogen) transfection reagent according to the manufacturer's instructions. Subsequently, cells were treated for 24 h with DMSO  or U0126   for 15 min at 37 °C, and then the cells were dissociated with a plastic pipette. Subsequently, cells isolated from cortex tissue were plated on poly-D-lysine-coated 24-well plates  and cultured at 37 °C, 5% CO2. Mixed glial cells were cultured in DMEM/F12 with 10% heat-inactivated FBS and penicillin (100 mg/ml) and streptomycin (100 mg/ml), and the culture medium were replaced every 3 days. After 14 days, microglia were separated from mixed glial cells by shaking at 150 rpm for 2 h at 37 °C. Primary cultured astrocytes were similarly dissociated from the cortex of new born ICR mice and cultured in DMEM with 10% FBS and penicillin (100 mg/ml) and streptomycin (100 mg/ml). Cortical neurons were similarly dissociated from the cortex of ICR mouse embryos at embryonic day 17 (E17), and motor neurons were similarly dissociated from the spinal cord of ICR mouse embryos at E17 as previously described.68 In brief, the dissociated neurons were cultured in neurobasal medium (Gibco) with 10% FBS, 1 × B27 (Gibco) and glutamine  for 12 h. Subsequently, culture medium were change to neurobasal medium with 1 × B27 and glutamine (0.05 mg/ml). After 5 days, the neurons were subjected to experiments. The purity of motor neurons were shown in Primary cultured microglia were prepared as described elsewhere.Cells were harvested and lysed in cell lysis buffer (50 mM Tris-HCl (pH 7.6) with protease inhibitor cocktail , 150 mM NaCl, 0.5% sodium deoxycholate, and 1% Nonidet P-40). Then, the proteins were separated by 10% or 12% SDS-PAGE (polyacrylamide gel electrophoresis) and transferred onto polyvinylidene difluoride membranes .β-actin: 5′-GACCTGACTGACTACCTC-3′ and 5′-GACAGCGAGGCCAGGATG-3′, mouse iNOS: 5′-TCCCAGCCTGCCCCTTCAAT-3′ and 5′-CGGATCTCTCTCCTCCTGGG-3′, and mouse COX-2: 5′-CAGGCTGAACTTCGAAAC A-3′ and 5′-GCTCACGAGGCCACTGATACCTA-3′.Total RNA from BV2 cells was extracted with TRIzol Reagent (Invitrogen); subsequently, the RNA was reverse-transcribed into cDNA using PrimeScript RT Master Mix . Real-time PCR analysis was performed using SYBR Green Real-Time PCR Master Mix (Takara) using a CFX96 Real-Time System  and the following primers: mouse μl of 400 μl medium were measured with PGE2 ELISA kits  according to the manufacturer's instructions.Primary cultured microglia and BV2 cells were transfected with siRNA against TDP-43. After transfection, the cells were treated with U0126 or celecoxib for 24 h, and then the levels of PGE2 in 50 μg/ml of puromycin. After 5 days, survived BV2 cells were selected and the BV2 stable cell line expressing AP-1 reporter construct was generated. Subsequently, the BV2 cells stably expressing AP-1 constructs were transfected with siRNA against TDP-43. After 48 h, the cells were treated with U0126 for 24 h, and then the cells were harvested. Finally, the AP-1 promoter activity was measured using the luciferase assay kit  according to the manufacturer's instructions.BV2 cells were plated on 24-well plate and incubated for 16 h at 37 °C. Then the cells were transfected with Cignal lentiviral AP-1 Reporter (luc)  according to the manufacturer's instructions for 24 h. Subsequently, the culture medium were replaced with fresh medium. After another 24 h, the cells were cultured in culture medium supplement with 2.5


The films are grown under industrially safe conditions, i.e., the flammable gases (H2 and CH4) are diluted to well below their lower explosive limit. The high quality, spatial uniformity, and low density of domain boundaries are demonstrated by charge carrier mobility measurements, scanning electron microscope, electron diffraction study, and Raman mapping. The hole mobility reaches as high as ~5,700 cm2 V−1 s−1 in ambient conditions. The growth process of such high-quality graphene with a low H2 concentration and short growth times widens the possibility of industrial mass production.We demonstrate the growth of continuous monolayer graphene films with millimeter-sized domains on Cu foils under intrinsically safe, atmospheric pressure growth conditions, suitable for application in roll-to-roll reactors. Previous attempts to grow large domains in graphene have been limited to isolated graphene single crystals rather than as part of an industrially useable continuous film. With both appropriate pre-treatment of the Cu and optimization of the CH Due to its outstanding properties676913141517181920152223262728292 at typical growth conditions is a particular problem (the explosive limit of H2 is 4–75% (volume concentration) at room temperature in air). Although there are several reports on graphene growth using no H213322222934353637382V−1s−1, which is not qualified for “electronic-grade” applications2-free growth removes a useful second parameter for control over graphene quality, coverage, and domain size. Thus, the growth of graphene with a high charge carrier mobility is still a challenge under industrially safe CVD conditions.In addition, the safety issue of the CVD process is usually ignored, despite its importance in industrial production. Further economic development would desire the growth at large scale by roll-to-roll reactors, preferentially under intrinsically safe and atmospheric pressure conditions. The flammability of H2 (2%) in 80 min. The average domain size reaches ~1 mm, which is the largest achieved to date for continuous graphene. The hole mobility reaches as high as 5,700 cm2V−1s−1, the highest value among the reported mobilities using similarly low concentrations of H2. We achieve this by combining electropolishing and non-reductive annealing of the Cu foils with optimizing the CH4 concentration. This work brings the current growth method of graphene a large step towards scalable and safe production.In this paper, we demonstrate the growth of continuous monolayer graphene with millimeter-sized domains using diluted H4 and H2 that are diluted to well below their low explosive limit (LEL). 4, respectively. The as-grown samples are baked in air (at 200 °C for 1 min) to visualize the graphene-covered regions. In 60 min, the graphene partly covers the Cu substrate with ~70% coverage. Some isolated domains reach as large as ~1.5 mm , transmission electron microscope (TEM), OM, and Raman spectroscopy.2V−1s−1 (mean ± standard deviation) based on the Drude model12 cm−2. As a reference, we also measure a graphene sample grown by the typical low pressure CVD using H2 annealing as well as a commercial graphene sample. The mobility and carrier concentration of the reference samples are  and  respectively. Compared to the reference samples and the reported values for continuous monolayer graphene, the mobility of our graphene is notably higher than all other results using similarly low concentrations of H2 . The intensity ratio of the 2D peak to the G peak (I2D/IG) is ~2. The 2D peak is of Lorentzian shape with one single component and the full width at half maximum (FWHM) is ~26 cm−1. These are the fingerprints of high quality monolayer grapheneThe SEM image in 2 area. The I2D/IG, ID/IG, and FWHM of the 2D peak reaches 2.3  ±  0.2  drops from 320–100 nm over a 50 μm range as shown in We now discuss the essential factors for synthesizing the continuous graphene films while still achieving millimeter-sized domains under 2% HThe effect of electropolishing on graphene nucleation is shown in 2 diluted in Ar), as can be seen in 2  which is found to drastically reduce the nucleation density, by around 30-fold, compared with annealing in reductive gas  are electropolished in a home-built cell. Two pieces of 10  ×  10 cm2 (2%), and 0.20–0.80 sccm CH4 (50–200 ppm) for 10–80 min, followed by cooling down to room temperature under the same Ar and H2 flow (without CH4). The gas pressure is maintained at 1 atm.The polished Cu foil is loaded into a 2-inch quartz tube furnace, which is then heated up to 1030 °C at ~20 °C/min under 1 atm with 400 sccm (standard cubic centimeter per minute) Ar. The Ar flow rate is then increased to 4,000 sccm to anneal the Cu for 5 min. Graphene growth is carried out by flowing 3,920 sccm Ar, 80 sccm HThe post-growth graphene on Cu foils are baked at 200 °C for 1 min in air on a hot plate to oxidize the Cu areas that are not covered by graphene. The colour contrast between the graphene-covered area and exposed area can visualize individual domains2(300 nm)/Si wafer using a poly(methyl methacrylate) support layer and 0.05 M aqueous solution of (NH4)2S2O8 as Cu etchant2 (300 nm) /Si wafers with 50 × 50 μmFor charge carrier mobility measurement, the graphene film is transferred onto a SiO2(300 nm)/Si wafer. For electron diffraction study, the graphene film is transferred onto a 3 mm-diameter holey carbon copper grid (Agar Scientific No. AGS147).The samples are characterized by optical microscope (Nikon ECLIPSE LV150N), scanning electron microscope , transmission electron microscope , Raman spectroscopy , and optical profilometer (Wyko NT1100). For Raman characterization, the graphene film is transferred onto a SiOHow to cite this article: Wu, X. et al. Growth of Continuous Monolayer Graphene with Millimeter-sized Domains Using Industrially Safe Conditions. Sci. Rep.6, 21152; doi: 10.1038/srep21152 (2016).