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).


In addition, the growth temperature was substantially reduced to as low as 100 °C, which is the lowest temperature reported to date for pristine graphene produced by CVD. Our modified CVD method is expected to allow the direct growth of graphene in device manufacturing processes for practical applications while keeping underlying devices intact.There is significant interest in synthesizing large-area graphene films at low temperatures by chemical vapor deposition (CVD) for nanoelectronic and flexible device applications. However, to date, low-temperature CVD methods have suffered from lower surface coverage because micro-sized graphene flakes are produced. Here, we demonstrate a modified CVD technique for the production of large-area, continuous monolayer graphene films from benzene on Cu at 100–300 °C at ambient pressure. In this method, we extended the graphene growth step in the absence of residual oxidizing species by introducing pumping and purging cycles prior to growth. This led to continuous monolayer graphene films with full surface coverage and excellent quality, which were comparable to those achieved with high-temperature CVD; for example, the surface coverage, transmittance, and carrier mobilities of the graphene grown at 300 °C were 100%, 97.6%, and 1,900–2,500 cm It is therefore indispensable to synthesize graphene at reduced growth temperatures, permitting large-area fabrication with relatively low cost. Several groups have demonstrated that a drastic reduction in growth temperature can be achieved by using plasma-enhanced CVD (PECVD), where plasma generates reactive species1719201719Graphene, a two-dimensional monolayer of Additionally, the typical graphene growth routes are mostly performed in low-pressure conditions in order to achieve a high degree of graphene crystallinity. Although the vacuum CVD process has been widely used for graphene growth, atmospheric pressure CVD (APCVD) growth of graphene of appreciable quality is desirable in order to lower the manufacturing cost and increase throughput. Moreover, severe evaporation of metal catalysts in low-pressure conditions, which may lead to deterioration of the graphene quality, can be significantly suppressed by increasing the working pressure. However, oxygen and water molecules in ambient air should be avoided to obtain high-quality graphene. In this study, we developed a modified APCVD route to synthesize graphene on Cu foils, which is called the “oxygen-free APCVD” process. In this method, we extended the graphene growth step in the absence of residual oxidizing species that may influence the formation of amorphous and oxidized carbon layers. Using this method with benzene as a carbon feedstock, large-area, continuous graphene sheets with excellent quality were synthesized on Cu foils at 300 °C. The growth temperature was substantially reduced to as low as 100 °C, which is the lowest temperature reported to date for pristine graphene produced by CVD.2/Si substrate. The OM and SEM images show the same contrast over the entire film area, in contrast to the film grown by APCVD. This reveals the formation of a continuous graphene film. Moreover, the Raman measurement indicates a monolayer graphene with a low defect density , which indicates the formation of a uniform, continuous graphene film with the monolayer coverage of >99% by merging adjacent graphene domains . For the growth time of 30 sec, the defective graphene flakes were synthesized on a Cu foil . As the ecreased , reveali domains . For lon domains .−1  of the device were 1,900 and 2,500 cm2 V−1 s−1, respectively204-derived monolayer graphene at ~1,000 °C by LPCVD1120The quality of our graphene film grown by oxygen-free APCVD was further tested by electrical transport measurements in a field-effect transistor (FET) configuration, which was fabricated on a 300 nm-thick SiOet al. that oxygen influences graphene nucleation and reduces the nucleation densityThe graphene growth from aromatic benzene molecules involves the following steps: (1) benzene molecules are introduced into the furnace by bubbling liquid benzene with argon and hydrogen as the carrier gases at ambient pressure; (2) the carbon sources then undergo adsorption on Cu surfaces and catalytic dehydrogenation at low temperatures to form highly reactive hexahydric ring species, leading to the formation of graphene nuclei; (3) continual growth of graphene takes place when hexahydric ring-containing molecules are incorporated into the edges of graphene nuclei that are chemically active . In prev19212426272 V−1 s−1. Even when the growth temperature was substantially lowered to 100 °C, graphene films were synthesized, but with reduced surface coverage. This demonstration is a significant step toward the direct growth of graphene in device manufacturing processes for practical applications such as CMOS back-end interconnects and flexible devices.We developed a modified CVD route, called the oxygen-free APCVD process, to synthesize large-area, continuous monolayer graphene films from benzene on Cu at as low as 300 °C at ambient pressure. The oxygen-free APCVD process introduces a step consisting of pumping and purging cycles prior to graphene growth, which leads to the absence of oxidizing impurities during growth and thus to the formation of uniform and continuous monolayer graphene films of improved quality. The graphene grown at 300 °C had properties comparable to those of 1,000 °C-grown graphene, with surface coverage of 100%, optical transmittance of 97.6%, and field-effect mobilities of 1,900–2,500 cm−4 Torr and then purged with argon. This process was repeated five times to flush the air contained in the quartz tube . After a mixture of hydrogen and argon  was supplied into the quartz tube, the pump was switched off, and the quartz tube was kept at ambient pressure. To increase the Cu grain size and ensure the removal of native oxide and a smooth Cu surface, the Cu foil was annealed at 1,000 °C for 30 min under a hydrogen and argon atmosphere and was subsequently rapidly cooled down to the desired growth temperature (25–300 °C). Once the desired temperature was reached, the flow rate of hydrogen gas was changed to 20 sccm without flowing argon (purging gas) while the carbon source was introduced by bubbling liquid benzene  at room temperature. The carbon source was supplied to the quartz tube with 5 sccm of argon flow (carrier gas) for 5 min, producing a monolayer graphene. The graphene growth was completed by stopping the supply of benzene, and the furnace was subsequently cooled to room temperature while hydrogen and argon  continued flowing without supplying benzene. For comparison, graphene was synthesized at 300 °C by APCVD  without the introduction of the pumping and purging step. To transfer the graphene onto a heavily p-doped Si substrate with a thermally grown 300 nm-thick SiO2 layer or a glass substrate, poly(methyl methacrylate)  was spin-coated on the graphene/Cu, which was baked at 60 °C for 5 min to remove the solvent from the polymer14Cu foil  was used as a catalytic substrate to grow graphene. The Cu foil was first cleaned with 18% HCl, deionized water, acetone, and isopropyl alcohol and subsequently dried with nitrogen. Then, it was loaded into the hot center of a quartz tube. Prior to graphene growth, the quartz tube was pumped down to ~10p-doped Si substrate with a thermally grown 300 nm-thick SiO2 layer, which were used as the bottom gate and gate dielectric, respectively. After transferring the graphene sheet onto the substrate, source and drain electrodes were deposited using photolithography, followed by electron-beam evaporation of Ti/Au. Then, a second photolithography and an oxygen plasma treatment were performed to pattern the graphene channel. Typical channel width and length were 8 and 40 μm, respectively.Graphene FETs were fabricated using standard photolithography techniques. The substrate was a heavily The surface morphology of graphene was visualized by optical microscopy  and field-emission scanning electron microscopy . Raman spectroscopy  with a laser excitation wavelength of 532 nm was used to characterize the thickness, quality, and uniformity of the graphene films. Optical transmittance was measured using an UV-vis-nIR spectrometer  to examine the thickness and uniformity of the graphene transferred onto a glass substrate. The electrical properties of the graphene were studied in vacuum at room temperature using a semiconductor parameter analyzer  and a four-point probe measurement system .How to cite this article: Jang, J. et al. Low-temperature-grown continuous graphene films from benzene by chemical vapor deposition at ambient pressure. Sci. Rep.5, 17955; doi: 10.1038/srep17955 (2015).


The paper presents the results of investigations on resistance structures based on graphite oxide (GRO) and graphene oxide (rGO). The subject matter of the investigations was thaw the sensitivity of the tested structures was affected by hydrogen, nitrogen dioxide and carbon dioxide. The experiments were performed at a temperature range from 30 °C to 150 °C in two carrier gases: nitrogen and synthetic air. The measurements were also aimed at characterization of the graphite oxide and graphene oxide. In our measurements we used (among others) techniques such as: Atomic Force Microscopy (AFM); Scanning Electron Microscopy (SEM); Raman Spectroscopy (RS); Fourier Transform Infrared Spectroscopy (FT-IR) and X-ray Photoelectron Microscopy (XPS). The data resulting from the characterizations of graphite oxide and graphene oxide have made it possible to interpret the obtained results from the point of view of physicochemical changes occurring in these structures. This is2  and WO3 2 [3  can serv2 [3 . It is t2 [3 ,8 or for2 [3 . The app2 [3 ,11.The techniques applied for the preparation of graphite oxide  and its In this paper, the authors present the results of experiments aimed at investigating the responses of resistance structures based on graphite oxide and graphene oxide exposed to different atmospheres. We also present the results of characterization of the layers comprising, among others, an analysis of the composition and topography of the surfaces. The aim of our investigations was to understand the physicochemical phenomena occurring during contact of the sensor structures with the given gaseous atmospheres.x group pollute the natural environment, leading to the formation of smog and affecting the lungs [Our investigations were focused on the detection of three dangerous gases: nitrogen dioxide, hydrogen and carbon dioxide. Gases from the NOhe lungs . Hydrogehe lungs . Carbon he lungs . Both hyhe lungs ,17 and ta-58 nm, Lc-30 nm, d002-0.336 nm and Cdaf-99.5%), supplied by Graphit Kropfmühl AG  was ground and sieved to a particle size <20 µm and oxidized according to the methodology described in Commercial natural graphite powder  was used as an acid. KMnO4 and NaNO3 were slowly added to keep the temperature in the 10–15 °C range. After their addition, the reaction temperature was maintained between 10 and 20 °C. After each reaction, 120 mL of milli-Q water and 50 mL of 3% H2O2 were slowly added. The mixture was stirred for 30 min and then centrifuged, the supernatant being decanted off. The graphite oxide was washed with milli-Q water and centrifuged repeatedly until a neutral pH was achieved. Graphite oxide was finally dried overnight under vacuum at 50 °C and stored in the presence of P2O5 as desiccant. Graphene oxide was obtained from graphite oxide by thermal treatment at 900 °C in a vertical furnace, under a nitrogen flow of 50 mL/min. The residence time at the final temperature was 5 min. Concentrated H2sp bonded carbon network. We used a combination of several measurement methods to reveal the structural evolution from pristine graphite to graphite oxide and next to graphene oxide . The further sections discuss the results obtained using these methods.Both graphite oxide (GRO) and graphene oxide (rGO) possess unique properties that differ from those of pristine graphite because of the structural changes arising due to the introduction of oxygen functionalities into the −5 mbar). The microscope magnification was ×1000 and ×5000. Additionally, the investigations by means of scanning electron microscopy were extended by applying an energy dispersive spectrometer X-ray (EDX) detector. This detector allows for qualitative and quantitative analysis of the sample composition. The measurements were examined by using a Nova NanoSEM 450 microscope  operating under low vacuum (0.3 mbar) at an accelerating voltage of 10–15 kV and using a large-field secondary electron detector.The topography of the surface of graphite oxide and graphene oxide was investigated by means of Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). For measurements using Scanning Electron Microscopy, an INSPECT S50  instrument was used. The measurement parameters were: HV = 5 kV, bias = 0, spot = 3.0 and HV = 2 kV, bias = 1400 V, spot = 3.5 for graphite oxide and graphene oxide, respectively. In order to illustrate both materials, an Everhart-Thornley Detector (ETD) was used. The measurements were made under high vacuum conditions  using the intermittent contact mode. The images were obtained at a resonance frequency equal to 136.281 kHz, both for graphite oxide and graphene oxide. The HA_NC tip was used in those measurements. Moreover, the Raman Spectroscopy (RS), Fourier Transform Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD) and X-ray Photoelectron Microscopy (XPS) were also applied for the characterization of the layers. The Raman spectra were obtained from the N_TEGRA Spectra platform (NT-MDT). In the measurements, the wavelength was equal to 532 nm. The spectra were recorded with a resolution of 2.89 cm−1.FT-IR measurements were made with a Tensor 27 spectrometer . Absorbance spectra of discs with a KBr/sample ratio of 500:1 were collected at a resolution of 4 cmXRD measurements were taken with an X’Pert PRO PW 3040/60 by PANalytical B.V.  diffractometer. Samples of ground graphite, graphite oxide and thermally reduced graphene oxide were deposited onto glass and analyzed by using Co Ka1 radiation with a voltage of 40 kV and a current of 30 mA.3sp hybridization (285.4 eV), hydroxyl and epoxy groups (286.5 eV), carbonyl or quinone groups (287.6 eV), carboxyl groups (288.9 eV) and a satellite peak corresponding to the π–π* transition in the aromatic systems (290.4 eV). The O1s excitation was resolved into two peaks corresponding to C=O groups such as ketone and carbonyl (531.7 eV), hydroxyl and epoxy functionalities (533.4 eV) and a carboxyl group (535.3 eV). Curve fittings were performed using an iterative least squares algorithm (CasaXPS software) with a Gaussian-Lorentzian (70/30) peak shape and Shirley background removal. The resulting spectra represent the binding energies of pyridinic (398.5 ± 0.2 eV), amine (399.4 ± 0.2 eV), hydroxypyridinic (400.5 ± 0.2 eV), quaternary-N (401.2 ± 0.2 eV) and N-oxide (402.9 ± 0.2 eV) groups.The elements present in graphite oxide and graphene oxide as well as their chemical state were identified by XPS analysis. X-ray Photoelectron Microscopy was carried out using a PHI 5000 VersaProbe—Scanning ESCA Microprobe™  and a monochrome Al Kα source (1486.6 eV). The charging of the sample was corrected applying the C1s peak at 284.5 eV as an internal standard. Curve fitting of the spectra was performed using a Gaussian-Lorentzian peak shape after conducting a Shirley background correction. The high-resolution C1s signal was deconvoluted into five individual peaks ascribed to graphitic carbon (284.5 eV), carbon atoms with The elementary composition of graphite oxide and graphene oxide  was determined directly using a Vario Macro Cube automatic elementary analyser .The properties of graphite oxide and graphene oxide as a function of temperature were investigated by the following techniques: Thermogravimetric (TG), Differential Thermogravimetric (DTG) and Differential Scanning Calorimetry (DSC). TG, DTG and DSC curves were obtained using a TG-STA209LUXX unit . The samples were tested in the range from 40 to 1000 °C with a heating rate of 10 K/min and a flow of argon kept at 25 mL/min.The resistance of sensor samples as a function of temperature was tested in a measuring system designed and realized by the staff of the Department of Optoelectronics of the Silesian University of Technology . This system allows presetting and control of the temperature of samples. The measurements were made within the range of temperature from 30 °C to 150 °C. The same system made it possible to measure the resistance at various degrees of temperature in various gaseous atmospheres. It can preset and control the composition of the gaseous atmosphere over the samples (the gas flow: 100 mL/min and 500 mL/min in the undertaken experiments). The measurements were taken in the atmospheres of nitrogen and synthetic air, which were used as carrier gases, as well as in the atmosphere of hydrogen, nitrogen dioxide and carbon dioxide at various concentrations in the carrier gases.The tested sensor structures  differedTo obtain a 1.2% (wt.%) solution the appropriate amounts of graphite oxide and graphene oxide (sensitive layers) were mixed with ethanol  and then transferred to an ultrasonic bath . After 24 h ultrasonication a homogeneous dispersion was obtained, which was deposited on the basic structures. After that the basic structures were placed in an vacuum dryer  and dried at 22 °C under nitrogen atmosphere for 24 h. Next, the dried structures were annealed with temperature ramp 2 °C/min to the final temperature of 100 °C at which were maintained for additional 3 h. Afterwards the temperature was increased up to 150 °C using the same ramping program and kept for 2 h under reduced pressure (20 mbar) to removal of any residual ethanol from the prepared structures. The whole annealing process were conducted in an inert atmosphere of nitrogen to avoid oxidation of the samples.2. The application of the sensitive layer on such large surfaces caused that the obtained results were repetitive for different groups of sensors  of the surface of graphene oxide is presented in The results of tests carried out when applying Atomic Force Microscopy confirm the difference in the development of these structures. The topography  of 0.3363 nm. The intensity of this peak sharply decreases for GRO, and a new 001 peak appears at 2Θ = 13.18°. The d001 interlayer distance calculated for this sample is 0.7795 nm, which is in agreement with its high degree of oxidation [002 amounts to 0.3393 nm.The crystalline structure of synthesized graphite oxide and graphene oxide, as well as that of the parent graphite, were analysed by XRD. As shown in xidation ,24. The 2sp and 3sp hybridization accompanied by some shoulders at higher binding energies due to the presence of oxygen linkages [2sp hybridization. The C1s spectrum consists of a peak at 285.4, 286.2, 287.0, 287.9, 288.9, and 290.4 eV assigned to C–C, C–O, C=O, O–C–O, O=C–OH and π–π* transition in aromatic systems [The oxygen speciation was studied by means of XPS measurements. linkages ,26. The linkages . The speThe O1s core-level spectra for GRO, shown in i.e., the widest) occurs in the range of 3320–3430 cm−1 and is attributed to the C–OH stretching vibrations of a hydroxyl group [−1 and is attributed to the C=C skeletal vibration of non-oxidized graphite [−1 and is attributed to the alkoxy C–O stretching vibration [The FT-IR spectra of the samples of graphite and graphene oxides are presented in yl group ,32,33,34graphite ,33,34. Aibration ,32,34.−1 and is attributed to the C=O stretching vibrations of a carbonyl group (at the edges of planes of the graphite layer) [−1 are characteristic for asymmetric and symmetric stretching vibrations of the C-H bond in CH2 and CH3 groups [i.e., the widest) peak is located at approximately 1220 cm−1 and is attributed to the stretching vibration of the epoxy group C–O–C [−1 and is attributed to the C=C skeletal vibration of the graphene planes [−1, is attributed to C–OH stretching vibrations of a hydroxyl group [−1) is attributed to the stretching vibration of the carbonyl group C=O [The next peak with the lowest intensity is located at approximately 1730 cme layer) ,32,34,353 groups ,34. The up C–O–C ,32,34,36e planes ,33,34,36−1 (D) and 1550–1600 cm−1 (G) [The Raman spectra of graphite oxide and graphene oxide are shown in cm−1 (G) . The ratThe presence of additional elements in the analyzed structures was also confirmed by application of other methods of measurements, for example Scanning Electron Microscopy with the EDX detector. EDX maps are shown in Nevertheless, the analyses are in quite good agreement, but the EDX and EA data confirm the presence of sulphur in the samples. On the basis of the maps, it may be concluded that the samples are very uniform over the entire volume.The properties of graphite oxide and graphene oxide change together with the temperature, affecting directly the properties of sensor structures based on the mentioned materials. Therefore, measurements were accomplished using the TG/DTG, and DSC techniques and also measuring the resistance at various degrees of temperature.Analysing the data from Sensor structures are also characterized by various resistance values. In the course of the oxidation process, the incorporated oxygen overcomes the van der Waals bonds and as a result, the interplanar distances increase. The reduction of graphene oxide eliminates a considerable amount of oxygen and changes the impedance characteristic of the material. A reduction in the amount of groups containing oxygen increases the electronic conduction, resulting in a decrease of resistance.It ought to be mentioned that the resistance of graphite oxide changes in a different way with the rise of temperature than the resistance of graphene oxide. The conductivity of a structure containing graphite oxide increases with the rise of temperature (within the range from 50 °C to 150 °C), both in nitrogen and in synthetic air. The character of these changes is linear. In the case of nitrogen, the rate of changes amounts to 37.7 mΩ/°C, whereas for synthetic air it amounts to 38.6 mΩ/°C. In the structure with graphene oxide, the resistance grows with the increasing temperature. For nitrogen, the rate of changes amounts to 7.8 mΩ/°C while for synthetic air it is equal 8.1 mΩ/°C. The results are presented in The different conductivity characteristics are probably due to the changes occurring in the atomic bonds of materials. The connection of oxygen to graphite (during the formation of graphite oxide) is at the expense of double bonds in the graphite. The effect of this is the degradation of the π set and the change in the mechanism of conductivity of the current in the obtained graphite oxide (from electron to semiconductive). A further reduction results in a partial regeneration of the structure of the π bonds by removing the oxygen from the structure and a return to electron conductivity which is decreasing with an increase in the temperature in the obtained reduced graphene oxide.Subsequent experiments were devoted to investigations concerning the sensor properties of the structures with graphite oxide and graphene oxide in varying gaseous atmospheres. As the first test, the resistance was recorded during an alternate dosing of hydrogen with a concentration of 4% in synthetic air. These investigations were executed at various temperatures within the 30 °C to 150 °C range. The resistance of the graphite oxide structure did not change in spite of changes in the composition of the surrounding atmosphere . Such a character of the changes may be explained by the formation of dangling bonds in the reduction process. Favorable spots for the adsorption of the gases are created in the structure contributing to electric response changes; hydrogen adsorbing onto the structure transfers electrons and as a consequence the resistance of the structure is increasing . This faThe difference between the resistance during the dosing of nitrogen or synthetic air and the resistance during the dosing of hydrogen (4% in a carrier gas) increases . It is cThe sensor structure with graphene oxide was characterized by changes of the resistance during the dosing of nitrogen oxide. In such a case, both during alterdosing nitrogen oxide with various concentrations in the nitrogen, and nitrogen oxide in synthetic air, the resistance of the structure dropped in the case of dosing nitrogen dioxide. The structure with graphite oxide did not change its resistance in spite of changes of dosing gases. Exemplary characteristics are presented in In contact with the structure, nitrogen dioxide behaves like a donor of electrons, reducing the resistance of the structure ,21,41. T2 and the carrier gas: nitrogen and synthetic air.During the experiments, neither the structure with graphite oxide nor the structure with graphene oxide changed their resistance due to their contact with carbon dioxide in a distinct way. Insignificant changes of resistance were recorded , but mosThe paper deals with a technique for obtaining graphite oxide by applying Hummer’s method and the technique of getting (on this graphite oxide) reduced graphene oxide. The paper presents the results of investigations concerning the chemical and physical properties of the obtained materials.viz. XPS, EDX and AE methods, which confirmed a considerable reduction in the amount of oxygen in the graphene oxide. Changes caused by the reduction were also confirmed by Raman’s Spectrometry and the methods TG/DTG as well as DSC.The investigations performed by means of the SEM and AFM techniques have proved that the surfaces of graphite oxide were evidently more developed than the surfaces of reduced graphene oxide. The thermal reduction involved a separation of numerous functional groups from graphite oxide, resulting in a change of the chemical composition of the obtained material. These changes were confirmed by three methods of measurements, independent of each other, 2 in atmospheres of synthetic air . The investigations have proved that a resistance structure with a layer of reduced graphene oxide can react to NO2 already at a concentration of ppb in an atmosphere of carrier gas. This provides options for its practical use in sensing of nitrogen dioxide obtained by means of the method suggested in this paper.Investigations on the sensitivity of the electric properties of the resistance structures based on graphite oxide and reduced graphene oxide to chosen gases were also performed. Generally, it may be assumed that the possibility of using graphite oxide for gas sensors is rather limited. It seems that there are considerable perspectives for applying reduced graphene oxide for detecting NO


PD-L1 expression was significantly associated with poorer five-year-EFS . Further studies in osteosarcoma are needed to determine if targeting the PD-L1:PD-1 axis improves survival.Osteosarcoma patient survival has remained stagnant for 30 years. Novel therapeutic approaches are needed to improve outcomes. We examined the expression of Programmed Death Ligand 1 (PD-L1) and defined the tumor immune microenvironment to assess the prognostic utility in osteosarcoma. PD-L1 expression in osteosarcoma was examined in two patient cohorts using immunohistochemistry (IHC)  and expression was validated using quantitative real time PCR (n = 21) and western blotting (n = 9). IHC was used to determine the presence of tumor infiltrating lymphocytes and antigen-presenting cells (APCs) in the tumor. Expression of PD-L1 was correlated with immune cell infiltration and event-free-survival (EFS). The 25% of primary osteosarcoma tumors that express PD-L1 were more likely to contain cells that express PD-1 than PD-L1 negative tumors . Expression of PD-L1 was significantly associated with the presence of T cells, dendritic cells, and natural killer cells. Although all immune cell types examined were present in osteosarcoma samples, only infiltration by dendritic cells (28.3%  Osteosarcoma is the most common primary malignant bone tumor in children and young adults1246789In recent years, numerous studies have sought to define the interplay between the immune system and tumor, with the potential to target this interaction for therapeutic intervention. In many malignancies, both innate and adaptive immune cells play a role in the tumor microenvironment, with communication between natural killer (NK) cells, antigen presenting cells (APCs) such as macrophages and dendritic cells (DC), and lymphocytes allowing for effective tumor control1213Immune cells are initially attracted to tumor cells by the presence of tumor specific antigens. Cancers with higher mutational loads present greater numbers of tumor specific neoantigens and are frequently associated with robust immune infiltration111617191822Tumors escape immunosurveillance by expressing immune checkpoints, such as programmed death ligand-1 (PD-L1)24272930323334Previous studies have begun to examine the role of PD-L1 in osteosarcoma. Utilizing quantitative real-time PCR (qRT-PCR), PD-L1 mRNA expression was associated with lymphocyte infiltration22PD-L1 expression was quantified in osteosarcoma patient and standard cell lines by qRT-PCR. Three quarters of patient derived cell lines, all the osteosarcoma standard cell lines , and two thirds of tumor specimens demonstrated expression of PD-L1 mRNA greater than the negative MCF7 control cell line; however, only a small subset expressed PD-L1 at the same level as the positive control, breast cancer cell line MDA-MB-231. Specifically, three of the sixteen cell lines , 19% , and fouPD-L1 protein expression was validated with western blotting: 3 of 10 patient samples (30.0%) and 4 of 10 osteosarcoma patient derived cell lines (40%) demonstrated detectable levels of PD-L1 protein . mRNA anPD-L1 protein expression was subsequently examined using IHC  on an osThe presence of six groups of tumor infiltrating immune cells was examined by IHC in whole slides . PresencPD-L1 positive tumors were associated with infiltration by multiple immune cells types as compared to PD-L1 negative tumors, which demonstrated infiltration by fewer immune cells types. Tumors expressing PD-L1 were significantly more likely to demonstrate infiltration by all immune cell types compared with PD-L1 negative tumors . Tumors expressing PD-L1 compared to those that were PD-L1 negative were more likely to demonstrate infiltration by CD3+ T-cells , CD56+ NK cells , CD68+ macrophages , and CD1a+ DCs . In addition, immune cells were significantly more likely to express PD-1 in PD-L1 positive tumors compared with PD-L1 negative tumors  .IHC indicated that PD-L1 expression and immune cell infiltration is heterogeneous within the whole slide samples examined. Two osteosarcoma tumor maps, containing slides from multiple areas of tumor, were examined in order to assess the consistency of PD-L1 expression and tumor infiltrating immune cells across whole tumors. Four slides were selected from the map for patient A, containing tissue from four locations across the tumor mass . Six sliPatient A’s tumor was PD-L1 negative across all sections examined  and pativs. 69.4%, p = 0.01). In contrast, although the TMA cohort showed a trend for poorer survival with PD-L1 expression , this result did not achieve statistical significance  and CD68+ macrophages   . Infiltr= 0.032) .In this study we demonstrated that PD-L1 was expressed in up to 25% of osteosarcoma samples, as determined by IHC using whole slide tumor sections. It is noteworthy that only 6.8% of osteosarcoma tumors, examined using a TMA containing 1 mm cores, were PD-L1 positive. It seems likely that this discrepancy between whole slides and the TMA is a result of the heterogeneity of PD-L1 expression in osteosarcoma—although PD-L1 positive cells were robustly stained, they made up a small fraction of cells examined on whole slide tumor sections. It is also possible that creation of the TMA, which is created to maximize areas of viable tumors, may bias against areas that are PD-L1 positive. These results suggest that IHC using a 1mm TMA may underestimate the number of PD-L1 positive osteosarcoma tumors.ΔCT method, found that the 84.2% of primary osteosarcoma tumors express detectable PD-L1 mRNAA previous study, using a 2Our results validated pervious PD-L1 mRNA expression reported in osteosarcoma323334In this study, we have shown that PD-L1 expression in primary tumors may be a prognostic marker for poorer survival. In addition to identifying PD-L1 as a potential prognostic marker that may be present in up to 25% of osteosarcoma cases, we demonstrated that PD-L1 positive osteosarcoma tumors have higher numbers of TILs and APCs than their PD-L1 negative counterparts. Although infiltration by TILs is significantly higher in PD-L1 positive tumors and their presence trends with worsened survival, there is no significant association between TILs and patient survival. On the other hand, presence of either of the APCs examined, was significantly associated with worsened survival.in vitroTumor associated macrophages (TAMs) are present in multiple solid tumors, including breast, gastric, and ovarian cancers394041Although previous studies may suggest that the opposite may be the case, we found that the presence of CD68+ TAMs in primary tissue may be significantly associated with worse survival in osteosarcoma. In previous studies, by capturing both monocytes and mature (M1 and M2) macrophages with CD14 staining, the role of mature macrophages may have been confounded. In our current study CD68 staining captures both pro-inflammatory M1 and pro-tumor M2 macrophages. In order to clarify the role of macrophages in osteosarcoma, future studies should correlate the presence of CD68+/CD80+ M1 and CD68+/CD163+ M2 macrophages to osteosarcoma survival.It is also possible that osteoclasts, a subset of CD68+ bone specific macrophages, may impact osteosarcoma patient survival. Presence of osteoclasts in osteosarcomas have been linked to conflicting outcomes in past studiesPresence of a second APC, CD1a+ DCs, is also associated with poor survival in osteosarcoma. With the recent advent of DC vaccines as a cancer treatment, multiple groups have shown that DC vaccines have varying levels of effectiveness in animal models of osteosarcoma51525357A major limitation of this study is the small number of samples available in this single institution analysis of the tumor immune microenvironment in osteosarcoma. Our sample size was further limited by the lack of associated clinical data for a subset of tumors. Although the non-significant correlative outcome results in the TMA may be explained by the limited sample assessed in each core, and the limited sample number, these represent two different patient cohorts and we cannot rule out the possibility that the prognostic correlation is purely a random occurrence in a small patient population. Further studies are necessary to validate that these results can be recapitulated in a larger multi-institutional cohort.In conclusion, we have shown that tumor infiltration by either CD68+ TAMs or CD1a+ DCs may be associated with poorer survival in osteosarcoma. Furthermore, PD-L1 is expressed in a subset of primary osteosarcoma samples at the time of biopsy, and it may be prognostic for increased mortality. PD-L1 expression is associated with TILs and APCs, including PD-1 positive immune cells. Due to its expression in a significant number of osteosarcoma tumors and an abundant presence of immune cells, the PD-L1:PD-1 axis may be a promising target for immunotherapy in osteosarcoma, particularly given osteosarcoma known genetic complexity. Further evaluation of PD-L1 protein expression is warranted in a larger cohort of patients with osteosarcoma. Currently a phase II study of pembrolizumab (anti-PD-1 antibody) in patients with advanced sarcomas, including osteosarcomas, is ongoing, the results of which may be informative.2 humidified atmosphere at 37 °C.Both osteosarcoma standard cells lines  and isolated cells were cultured in Eagle minimum essential medium supplemented with 20% fetal bovine serum (FBS) and antibiotics. PD-L1 negative MCF7 breast cancer cells, PD-L1 positive MDA-MB-231breast cancer cells, and PD-L1 negative 3T3 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. Primary osteosarcoma cultures were generated using standard collagenase disaggregation of surgical specimens from seven osteosarcoma patients, as has been described previously2 were constructed using tumors collected from a second cohort of patients . The tumors were collected in accordance with an IRB approved research protocol with patient/guardian informed consent as described previously−ΔΔCT method. MCF7 and MDA-MB-231 cell lines were used as a negative and positive control for PD-L1 mRNA expression, respectively. Due to lack of detectable amplification of PD-L1 in the negative control, the CT value for MCF7 was set to 40 for calculations.RNA was extracted from flash frozen osteosarcoma tumor samples from 15 patients using Trizol reagent (Invitrogen) and converted to cDNA using Superscript II transcriptase (Life Technologies). qPCR was performed using commercially available TaqMan primers for PD-L1 . Relative gene expression was normalized to an internal control, GAPDH, and calculated using the 2Tumors, collected from osteosarcoma patients were used to construct a TMA, as has been described previously60Multiple slides were selected from tumor maps that were previously generated for clinical purposes using osteosarcoma specimens obtained with parental consent from two patients enrolled in an IRB-approved bio-banking protocol at Montefiore Medical Center. Samples were analyzed from multiple locations within the tumor mass and surrounding tissue at the time of definitive surgery following neoadjuvant chemotherapy. A set of slides was generated for each patient and this was related to the specimen map, which provided each slide’s location within the tumor.Immune staining was optimized for proper antigen retrieval and antibody concentrations using immune tissue for positive controls and brain tissue for negative controls. Osteosarcoma tumor slides were baked at 60 °C for one hour and antigen retrieval was performed using either tri-citrate buffer, pH 6.0 or tris-EDTA buffer pH 8.0 at 100 °C for 20 minutes. Slides were de-paraffinized using xylene, rehydrated along an ethanol gradient, and blocked using dual endogenous enzyme-blocking reagent (Dako) and a subsequent normal goat serum block (Santa Cruz). Slides were incubated overnight at 4 °C in primary antibody followed by a one hour room-temperature incubation with secondary antibody. Antibody concentrations and information can be found in Slides were scanned and graded by three independent reviewers without previous knowledge of patient outcomes or demographics. Slides were graded with a quartile score based on the percentage of positive staining cells present in the core/slide . If scores between the three graders were discordant the average, rounded to the nearest whole integer, was used. A tumor with a score of 1 or higher, representing ≥1% staining, was considered positive for PD-L1 expression or immune cell infiltration. Images of IHC staining were obtained at 20× magnification using the Pannoramic Scanner and captured using Pannoramic Viewer (3DHISTECH Ltd).Patient demographic data from each cohort was reported as frequencies for categorical variables and means with standard deviations for continuous variables. The association between the expression of PD-L1 and the presence of immune cells was assessed using Fisher’s exact test or chi square analysis. Kaplan Meier curves were generated assessing the association between EFS and PD-L1 expression or immune cell infiltration, and the Log-rank (Mantel-Cox) test was utilized (GraphPad Prism software). Survival curves were generated using expression thresholds of negative, defined as no staining (grade 0) and positive defined as at least 1% staining .How to cite this article: Koirala, P. et al. Immune infiltration and PD-L1 expression in the tumor microenvironment are prognostic in osteosarcoma. Sci. Rep.6, 30093; doi: 10.1038/srep30093 (2016).


Electrostatic interaction between the most electronegative element fluorine (F) and hydrogen (H) is one of the strong interactions in hydrogen bonding, and here we report the selective binding of ammonia/ammonium with F in fluorographene (FG) resulting to a change in the impedance of the system. Very low limit of detection value of ~0.44 pM with linearity over wide range of concentrations (1 pM–0.1 μM) is achieved using the FG based impedance sensor, andthisscreen printed FG sensor works in both ionized (ammonium) and un-ionized ammonia sensing platforms. The interaction energies of FG and NH3/NH4+ are evaluated using density functional theory calculations and the interactions are mapped. Here FGs with two different amounts of fluorinecontents −~5 atomic% (C39H16F2) and ~24 atomic% (C39H16F12) - are theoretically and experimentally studied for selective, high sensitive and ultra-low level detection of ammonia. Fast responding, high sensitive, large area patternable FG based sensor platform demonstrated here can open new avenues for the development of point-of-care devices and clinical sensors.Single molecule detection using graphene can be brought by tuning the interactions  Helicobacter pyloribacterial stomach infection. The amount of exhaled air available will be minimal and hence high sensitive ammonia breathe sensors are highly demanding, but lacking at present. Apart from ammonia gas sensors, analysis of ammonia (ionized/un-ionized) level in blood is also of interest in medicine. Blood ammonia monitoring has of interests in sports medicines (0.1 to 10 ppm) and pediatrics (>100 μmol/L) too34Several articles have been reported in the past discussing the development of various gas sensors, and ammonia is one of the gases taken seriously due to its natural and industrial origin1234Presently various sensing platforms are existing for ammonia: metal oxide based sensors6791114via doping and defects. Further, robustness and availabilities of different detections mechanisms of the graphene based devices (electronic transducers (field effect transistor), electrochemical  sensors etc.) attract the development of graphene based sensors1617Graphene based biosensors are receiving tremendous scientific attention due to the engineering possibilities of graphene by bringing specificity and sensitivity in the device et al. have reported Density Functional Theory (DFT) calculations based graphene oxide (GO) – ammonia interactionset al. reported a reduced GO (RGO) based conductometric ammonia sensor with the LOD of 200ppm and also mentioned its some extend of selectivity towards ammoniaTang 0.25)x)252627Relatively high thermal stability of fluorographene (300–400 °C)via residual groups. FG based screen printed electrode is developed and its efficacy in ammonia sensing using impedance spectroscopy is demonstrated. Large area patternability of chemically derived FG is shown using a soft-lithography process, indicating the possibilities of FG based different sensor geometries.In the present study, we report the interactions of FG and ammonia (unionized and ionized (ammonium)) using DFT calculations. The interactions between graphene and ammonia are also explored to compare the strength of FG-ammonia interactions. The FGs in the present study are derived from fluorinated graphene oxide  and the role of residual functional groups in the interactions is not studied. Later, using Fourier Transform-Infrared (FT-IR) studies, we found that the interaction between fluorine and ammonia is prominent than other hydrogen bonding interactions via sp3 hybridization. FG is an atomically layered material as it is evident from the electron microscope images given in the supporting information, 4+). The DFT studies reveal that FG is having strong interactions with ammonia compared to graphene  due to the strong hydrogen bonding between C-F in FG and N-H in ammonia. The optimized structures of complexes are shown in 4+  is higher than the G-NH4+, the difference is less than that in the case of ammonia. This might be due to the enhanced electrostatic interaction between positively charged of ammonium ion and graphene (π electrons).FG has a carbon honeycomb lattice like in graphene with covalent fluorine (F) attached to carbon 4+) and graphene/FG were calculated from HOMO and LUMO energies of the corresponding complexes. The calculated energy gaps are shown in 3/NH4+ and FG is less than that of NH3/NH4+ and graphene. This can be due to the strong interactions of NH3 or NH4+ with FG, making the energy differences between HOMO and LUMO smaller. Further, the electrostatic potential maps of the interactions are shown in 3/NH4+ has strong electrostatic interaction with negatively charged 2p orbital of fluorine on FG, leading to a hydrogen bonding (electrostatic) interaction. The interaction maps show considerable orbital overlapping of NH3 and NH4+ with FG, while little interaction between NH3 and graphene is observed as it is reported by other researchers4+ shows a better interaction with graphene due to its positive charge.The optimized structures and electrostatic interactions between ammonia/ionized ammonia (NH19 Nuclear Magnetic Resonance (NMR) spectrum, as shown in the supporting information, −1 in theoretical FG is due to the =CH stretching of the end terminated hydrogen while in the case of FG experimental spectrum it is due to the –OH stretching from residual functional groups. In −1is clear in both theoretical and experimental spectra. The FG has been exposed to ammonia gas (pressure ~1 psi) for different exposure times, and the corresponding FT-IR spectra are shown in The theoretical prediction of interaction between FG and ammonia is experimentally validated using FT-IR and micro Raman spectroscopy. Chemically derived (explained in the method section) FG powder (low resolution transmission electron microscope (TEM) image and high angle annular dark field TEM images are shown in 25−1, respectively. The G band of graphene originates from the vibration of in-plane sp2 carbon. The D band is attributed to the defects in graphene. There are no considerable changes in the positions and intensity ratios of D and G bands before and after the exposure of ammonia indicating that the graphene structure is not disturbed after the ammonia exposure The micro-Raman spectra of graphene and FG before and after exposing to ammonia are shown in via the electrostatic hydrogen bonding is utilized for the development of an ammonia sensor, as explained in the following section. Electrochemical impedance spectroscopy (EIS) was used to study the binding of ammonia on FG modified screen printed electrodes  increases with increase in ammonium ion concentration. The increase in Rct is attributed to the binding of ammonium to the FG via hydrogen bonding. In order to confirm that the observed change in impedance is due to surface modification of SPE and not due to superimposed effects, ratio of charge transfer resistance for the desired concentration (Rct(Ci)) and charge transfer resistance of the blank FG electrode (Rct(Co)) is plotted against the logarithm of ammonia concentration (ct(Ci)/Rct(Co) = 1.723 + 0.055 log CNH3. This shows that the sensor works in a linear concentration of ammonia in the range of 1 pM to 0.1 μM with a correlation coefficient of 0.998. Sensitivity of the sensor can be deduced from slope of this curve, resulting to a value of ~0.055 M−1. The limit of detection (LOD) of the sensor has been calculated according to Long and Wineforder methodIt is evident from the ntration . Figure where S.D. is the standard deviation of the blank, a is the linear coefficient, and b is the angular coefficient (sensitivity). The calculated LOD is found to be ~0.44pM.ct (ΔRct) values and the logarithm of ammonia concentrationsreveals a linear detection range for ammonia concentrations in the range of 1 pM to 0.1 μM with 4.4 × 10−13M (0.44 pM) limit of detection. The linear relationship could be characterized using the linear equation: ΔRct (kΩ) = 3.832 + 0.293 log CNH3 (kΩ). This FG modified SPE exhibits a sensitivity of 0.293 kΩ M−1 with a correlation coefficient of 0.997 and standard deviation of 0.018 kΩ  and 5 mM[Fe(CN)6]3− (ferricyanide). The experimental set up is shown in supporting information Further, EIS measurements are also conducted by direct purging of ammonia in to the phosphate buffer solution (PBS)  containing the mixture of 5 mM4− and 5 mM[Fe(CN)6]3 over the frequency range 105–0.01 Hz with 5 mV as the alternating current amplitude. Using the redox probe (5 mM [Fe(CN)6]3−/4−), change in charge transfer resistance (Rct) at electrode/electrolyte interface has been investigated in electrochemicalimpedance.Electrochemical impedance studies (EIS) were conducted using a Biologic potentiostat, model SP-300. Low-cost, screen-printed electrodes  spectrometer. The micro-Raman spectra of GO and FGO samples were studied using LabRamXploRA Raman spectroscope (excitement wavelength 632 nm).http://pubs.acs.org.TEM images of FG, XPS and NMR data of FG, schematics of screen printed electrode and ammonia sensing set up are provided in the supporting file. Theoretically evaluated bond lengths observed in free graphene and FG is also provided in the supporting file. SEM image and Raman mapping of large area printed FG microelectrodes is provided. Direct ammonia sensing data is also provided. Ammonia sensing results  with high fluorine containing graphene is also provided. This material is available free of charge via the Internet at How to cite this article: Tadi, K. K. et al. Fluorographene based Ultrasensitive Ammonia Sensor. Sci. Rep.6, 25221; doi: 10.1038/srep25221 (2016).


Go = 2e2/h (=12.91 kΩ−1), and in some devices partially quantized including a series of with (n/7) × Go, steps are observed. Such an organic memory device exhibits reliable memory operation with an on/off ratio of more than 10. We attribute the quantized conductance to the existence of a 1-D electron waveguide along the conductive path. The partial quantized conductance results likely from imperfect transmission coefficient due to impedance mismatch of the first waveguide modes.Quantized conductance is observed at zero magnetic field and room temperature in metal-insulator-metal structures with graphene submicron-sized nanoplatelets embedded in a 3-hexylthiophene (P3HT) polymer layer. In devices with medium concentration of graphene platelets, integer multiples of  Go = 2e2/h  in a suspended graphene nanoconstriction at cryogenic temperature of 4.2 K. The observed quantized conductance was attributed to the graphene zigzag or armchair edges that provide effective boundary conditions for the quantum mechanical wave functions. In 1998, Frank et al. [Go conductance per nanotube. Calculations by Sanvito et al. [Graphene  has attrk et al. , 11 obseo et al. , 14 are o et al. –17 in tao et al.  showing Here, we present experimental observation of integer and partially quantized conductance at room temperature in metal-insulator-metal (MIM) structures consisting of graphene nanoplatelet (GNP) ribbons suspended in a 3-hexylthiophene polymer (P3HT) layer interposed between metal copper and gold electrodes.2-bonded carbon atoms densely packed in a honeycomb crystal lattice. The number of stacked layers is typically between 5 and 30. The lateral dimension of the flakes ranges between 100 nm and a few micrometers. The graphene platelet ribbons are substantially flat, but when made thin by exfoliation, they can be curved, curled, or buckled indicating a single flake or a stack of a few carbon sheets [Our graphene-based MIM devices have been fabricated on a thermally oxidized silicon wafer using standard semiconductor processes. The thermally oxidized silicon wafer was first cleaned using acetone and subsequently rinsed in isopropanol and deionized water. The wafer was then patterned photolithographically and deposited with a bottom gold (Au) electrode using electron-beam physical vapor deposition (EBPVD). GNP powder provided by PPG Industries  was firsn sheets , 22. Ultn sheets –22. The n sheets –26. We nFour types of samples have been manufactured with regard to GNP content in the polymer: (i) no GNP, (ii) 0.05 mg/ml, (iii) 0.1 mg/ml, and (iv) 0.2 mg/ml of GNP concentration.Go or repeatable fractions of Go, respectively, as function of applied voltage. Figure υ = 0.225 V/s (V(t) = υ × t) along the positive x-axis from 0 to 2 V. The current is observed to increase gradually and ohmically in the low-voltage region, and then abruptly increase at three voltages, specifically at 0.75, 1.2, and 1.87 V to 10, 162, and 409 μA, respectively. The initially high-resistance state (HRS) of about Roff = 100 kΩ is reduced during the voltage sweep to a final low-resistance state (LRS) of Ron = 5 kΩ. Figure Go. Conductance increases with voltage discretely, and instead of displaying flat plateaus as in quantum Hall effects, it displays sloped rises whose slopes decrease with the subsequent two steps. To characterize this behavior in terms of energy consumed, we have calculated the energy consumed between individual discrete jumps. For the first slope we obtain, assuming a linear voltage ramp rate υ:From these four types of devices, only devices with 0.1 mg/ml show integer and partial quantized conductance as shown in Figs. V1 = V(t1) = 0.72 V, V2 = V(t2) = 1.22 V, and υ = 0.225 V/s) we obtain Estep(1) = 1.03 × 1015 eV. As this number is many orders of magnitude higher than kT, thermal effects can be ruled out for being responsible for the sloped conductance. Assuming that somewhere in the device a one-dimensional filament is being formed, one could attribute the slope to progressive thickening of the conductive filament constituting a waveguide. Another possible explanation is that the transmission coefficient of the electron waveguide is a function of the applied voltage. When the transmission coefficient is close to unity, it causes an integer quantized conductance jump. In Fig. Go instead of nGo as in Fig. V = 1.3 V to V = 1.45 V, it can be seen that the conductance changes abruptly from 1Go to 2Go and above V = 1.5 V rises from 2Go to 3Go. In addition, from V = 0.0 V to V = 1.3 V, a sequence of smaller conductance steps within the 0Go to 1Go interval is also observed, indicating that the partial quantized conductance coexists with integer quantized conductance. The best fit to the partial quantized conductance within the 0Go to 1Go interval can be obtained by assuming a fraction vGo with v = 1/7, 2/7, 3/7, 4/7, 5/7, and 6/7, although 3/7 is slightly off the experimental value. The resolution of the partial conductance steps between 1Go and 2Go is always much less pronounced and appears to follow the same multiples of 1/7. Steps with 8/7, 9/7, 11/7, and 13/7 can be clearly identified. In the third conductance step, from 2Go to 3Go, only a rudimentary partial quantized conductance can be discerned. The behavior shown in Fig. Icc is imposed during the set operation. Such a case is shown in Fig. Icc = 0.1 mA. It can be seen that the hysteresis is now pronounced and readily detectable as a difference in logic state “0” for off-state and “1” for on-state. The conductance observed during the set transition of 1.4 V has been extracted and is plotted in Fig. Using the data from Fig. Icc), which is commonly used in resistive random access memory devices less the device be damaged. Icc is the maximum current that is allowed to flow through the device. In resistive random access memory, devices under the Icc limitation quantized conductance is also observed when a fresh device is subjected to negative bias. In Fig. Vreset = −0.7 V can be observed.As we consider Au/P3HT/Cu cells for potential memory applications, we have performed the set operation under imposition of the so-called compliance current  than the ionization energy for Au (9.23 eV).” As shown in Fig. + ions migrate across the polymer and reach a GNP platelet where they are reduced to Cu atoms. Under a positive bias applied to the Cu electrode, Cu+ ions may be injected into the polymer and form a conductive filament between the Cu electrode and graphene platelets whose other end makes a contact with the Au electrode. As a result of this, the Cu+ ions could aggregate more densely in the polymer than along the graphene surfaces and form tiny nanofilaments connecting the graphene layers to each other or to the electrodes. One observes that when a negative bias is applied to the Cu electrode, a strong enough field is able to ionize the neutralized Cu atoms of the nanofilaments and rupture them, disconnecting them from the graphene nanoplatelets.Following the explanation adopted for Cu and Au nanofilaments and quantum point contacts in mesoscopic physics , 31, we  devices , 33. TheGo), for wider constrictions, second and higher modes are transmitted. Each mode contributes a unit Go to the conductance if the transmission coefficient of the mode through the constriction is unity (perfect transmission without backscattering) [2). At a GNP concentration of 0.1 mg/ml, we find a 50 % chance that one graphene nanoribbon will span the entire dielectric distance. At a GNP concentration of 0.05 mg/ml, not a single case of bridging has been observed. On the other hand, at concentration of 0.2 mg/ml, the very high measured conductance value points to multiple graphene nanoribbons in parallel bridging the two metal electrodes. While the highly conductive devices with GNP concentration of 0.2 mg/ml do not display quantized conductance effects, they are a good material candidate for stretchable transparent electrodes [The 1-D conducting bridge forms a constriction of width of the order of the Fermi wavelength of the electron. For a width approximately half of the Fermi wavelength, one transverse mode will contribute to conductance (1ttering) , 31. Yetectrodes . On the The non-unity transmission coefficients are caused by the sensitivity to the electrostatic environment. Likewise, in memristive devices, in contrast to conventional resistors and capacitors, the resistor and capacitor  are voltIn summary, we have observed integer and partial quantized conductance occurring in graphene nanoplatelets doped organic polymer films at room temperature. The quantized conductance is likely a result of the formation of a 1-D waveguide with a voltage-tunable transmission coefficient. The device shows a distinct I-V hysteresis and lends itself for memory applications


Halomonas spp. CAM2 by introducing single layer graphene coatings. The use of this nanomaterial has been poorly explored for antifouling application.Biofouling, the colonization of artificial and natural surfaces by unwanted microorganisms, has an important economic impact on a wide range of industries. Low cost antifouling strategies are typically based on biocides which exhibit a negative environmental impact, affecting surrounding organisms related and not related to biofouling. Considering that the critical processes resulting in biofouling occur in the nanoscale/microscale dimensions, in this work we present a bionanotechnological approach to reduce adhesion of biofilm-producing bacteria Our study revealed that graphene coatings modify material surface energy and electrostatic interaction between material and bacteria. Such nanoscale surface modification determine an important reduction over resulting bacterial adhesion and reduces the expression levels of genes related to adhesion when bacteria are in contact with graphene-coated material.Halomonas spp. CAM2. Hydrophobic-hydrophilic interaction and repulsive electrostatic force dominate the interactions between Halomonas spp. CAM2 and material surface in saline media, impacting the final adhesion process. In addition no bactericide effect of graphene coatings was observed. The effect over biofilm formation is localized right at coated surface, in contrast to other antifouling techniques currently used, such as biocides.Our results demonstrate that graphene coatings reduce considerably adhesion and expression levels of adhesion genes of biofilm-producing bacteria  Marine fouling is the accumulation of micro and macroorganisms on underwater surfaces, which provide a favorable mechanism to survive in the environment. The economic impact of fouling on shipping vessels, oceanographic sensors, power plants and aquaculture systems, among others, has been estimated to be in the range of 50 billion euros per year –3. In paCurrent techniques to prevent (antifouling) and fight fouling (fouling-release) include physical  and chemical methods . Although the choice of the right strategy will depend on the cost and application possibilities, antifouling coatings are probably the most cost-effective method for boats and other surfaces. They are typically based on the controlled release of organic solvents into the environment to kill the colonizing microorganism. However this approach offers a non-localized solution, affecting surrounding organisms related and not related to fouling. As a consequence, the use of certain biocides has been restricted by some countries and in Europe a large amount of data has been gathered as part of the Biocidal Products Directive  .The critical processes at the biointerface resulting in biofouling occur in the nanoscale/microscale dimensions: it follows therefore that surface properties which could control biofouling need to be on the same length scales . Search Graphene is a one-atom thick carbon sheet that has emerged as a new carbon compound with multiple applications in a wide range of industrial processes and products. While graphene is a promising candidate in electronic applications, its use for biological applications, such as antifouling, has been poorly explored. There are many reports of bacterial interaction with graphene oxide (GO), formed by micro- or nano-sized flakes of functionalized graphene in powder, solution or coating –18, whicHalomonas spp. CAM2, which is used as a model marine bacterium.In this report we present a physico-chemical and biological approach to reduce fouling formation in its initial growth stage as biofilm, by introducing graphene coatings that reduce bacterial adhesion to coated surfaces. Nanoscale behavior is discussed in the particular case of biofilm-producing 2 samples was carried out to evaluate their composition, microstructure, topography with atomic resolution, graphitic quality and contamination.Micro and nanoscale characterization of as-grown graphene on Cu and graphene transferred onto SiO2 process is described in materials and methods section.Scanning electron micrographs of single layer graphene (SLG) grown on Cu showed some contrast at micrometer scale that could be identified as graphene domains Fig. b. Atomic2 samples prior to bacteria contact was screened by SEM, optical microscopy and STM. Wrinkles in CVD graphene grown on Cu are formed by differential thermal expansion, as the metal contracts more than the graphene during post-growth cooling  = 2−ΔΔCt.Relative quantification of each target gene  encoding for adhesin in each experimental sample, and control versus reference gene rDNA16S) was performed in according with Livak Eq. 6S was pe


TrkB is a high affinity receptor for the brain derived neurotrophic factor (BDNF) and its phosphorylation stimulates activation of several intracellular signalling pathways linked to cellular growth, differentiation and maintenance. Identification of various activators and inhibitors of the TrkB receptor and greater understanding their binding mechanisms is critical to elucidate the biochemical and pharmacological pathways and analyse various protein crystallization studies. The data presented here is related to the research article entitled “Brain Derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling”  Specifications TableValue of the data•Three dimensional molecular model of the cyclic peptide cyclotraxin B was generated for the first time to determine its interactions with the TrkB receptor.•Future studies to determine pharmacological, biochemical or interaction studies of TrkB receptor or related molecules will be facilitated by the interaction and binding parameters reported here.•Binding interactions of CTXB with TrkB will determine its potential usage in pharmacological studies and development of new derivatives and ligands.1The data shown here elucidates molecular modeling of cyclic peptide cyclotraxin B which is a TrkB inhibitor. Active site of the extracellular D5 domain of the TrkB receptor which is primarily involved in this interaction was also modelled. Finally the cyclic peptide inhibitor was also identified and docking carried out with the extracellular D5 domain of TrkB. The interacting amino acid residues that are involved in docking are identified and various interaction parameters provided in detail using a combination of molecular modeling and molecular docking computational tools.22.1via MOPAC  method Cyclotraxin B (CTXB) is a cyclic peptide chain of 10 amino acid linked by a disulphide bridge 2.2The primary structure of TrkB receptor and its various domains were examined 2.3in silico assessment of the potential binding of selected CTXB residues to different regions on the TrkB D5 surfaces. This binding interactions were assessed in terms of probability of CTXB participating residues binding to the TrkB D5 receptor surface and theoretical scores determined using the PepSite2 server The protein motif was subjected to PepSite2 is a computational tool that scans the surface of a given protein for patches or grooves that are likely to influence binding of individual amino acid residues or peptides up to ten amino acids and provides a score that reflects the propensity of the peptide to bind to that region. The PepSite score is expressed in relative units and the higher scores reflects superior binding. We applied PepSite in a sliding window of 10 residues to assess the binding of the TrkB-D5 domain and CTXB peptide sequence (CNPMGYTKEG). The scores of the CTXB binding to different regions of TrkB D5 domain are presented  [1].2.4The protein-peptide docking was performed with the PatchDock software available in the public domain