Animal African trypanosomiasis (AAT) is one of the biggest constraints to livestock production and a threat to food security in sub-Saharan Africa. In order to optimise the allocation of resources for AAT control, decision makers need to target geographic areas where control programmes are most likely to be successful and sustainable and select control methods that will maximise the benefits obtained from resources invested.The overall approach to classifying cattle-owning communities in terms of AAT vulnerability was based on the selection of key variables collected through field surveys in five sub-Saharan Africa countries followed by a formal Multiple Correspondence Analysis (MCA) to identify factors explaining the variations between areas. To categorise the communities in terms of AAT vulnerability profiles, Hierarchical Cluster Analysis (HCA) was performed.Three clusters of community vulnerability profiles were identified based on farmers’ beliefs with respect to trypanosomiasis control within the five countries studied. Cluster 1 communities, mainly identified in Cameroon, reported constant AAT burden, had large trypanosensitive (average herd size  = 57) communal grazing cattle herds. Livestock  were reportedly the primary source of income in the majority of these cattle-owning households (87.0 %). Cluster 2 communities identified mainly in Burkina Faso and Zambia, with some Ethiopian communities had moderate herd sizes (average = 16) and some trypanotolerant breeds (31.7 %) practicing communal grazing. In these communities there were some concerns regarding the development of trypanocide resistance. Crops were the primary income source while communities in this cluster incurred some financial losses due to diminished draft power. The third cluster contained mainly Ugandan and Ethiopian communities which were mixed farmers with smaller herd sizes (average = 8). The costs spent diagnosing and treating AAT were moderate here.Understanding how cattle-owners are affected by AAT and their efforts to manage the disease is critical to the design of suitable locally-adapted control programmes. It is expected that the results could inform priority setting and the development of tailored recommendations for AAT control strategies. Glossina spp.) and animal African trypanosomiasis (AAT) are an important constraint to livestock production and a threat to food security in sub-Saharan Africa [Tsetse , the use of traps or targets, ground or aerial insecticide spraying, or reducing the risk of exposure through changes in livestock management. The process of privatisation of veterinary services in many sub-Saharan African countries means that farmers and community animal health workers (CAHW) with limited training are often responsible for the treatment of the disease . TraditiIn recognition of the need for co-ordinated actions against AAT, the Pan-African tsetse and trypanosome eradication campaign (PATTEC), funded by the African Development Bank, was established in the year 2000 and has set tsetse elimination as its goal. Although this goal presents a huge challenge that would require extensive resources and there is debate as to whether it is feasible, the last decade has seen renewed interest in the research and development of control options. Governments, charities and philanthropists have made funding available for this purpose, despite this, the reality is that many of the communities afflicted by AAT have insufficient resources available for its control and are not always reached by control programmes. In addition, macro-level decision making may ignore important heterogeneities between communities.In order to optimise the allocation of resources for AAT control, decision makers target geographic areas where control programmes are most likely to be technically, economically, socially and environmentally sustainable and select methods of control that will maximise the benefits obtained from resources invested . To thisA series of interviews were conducted with cattle owners in different agro-ecological zones across five countries in sub-Saharan Africa, namely Burkina Faso, Cameroon, Ethiopia, Uganda and Zambia. Data collected on cattle was mainly on owners’ knowledge and perceptions of AAT. The study sites provided a large variation in environment, AAT eco-epidemiology, cattle management and socio-economic impact of AAT. The overall approach to classifying communities in terms of AAT vulnerability was based on the selection of key variables collected through field surveys followed by a formal Multiple Correspondence Analysis (MCA) to identify factors explaining the variation between areas. Hierarchical Cluster Analysis (HCA) was then performed in order to categorise communities into groups describing their AAT vulnerability profile.A series of surveys were conducted in 17 study areas in five countries in sub-Saharan African during 2013; Burkina Faso, Cameroon, Ethiopia, Uganda and Zambia. A previous review of tsetse density and trypanosome prevalence studies was the basis for the geographic focus of the study, identifying the selected countries as moderate to high risk AAT areas. The countries were also selected to cover a range of eco-regions and AAT epidemiology, in addition the ease of conducting fieldwork in the selected countries was taken into account. Within the countries, study areas were classified in terms of environment, including ecoregion and available information on AAT risk. A brief description of the study areas is given below and in Table T. congolense, and the first reports of trypanocide resistance came from these study areas [The main income in these study areas comes from rain fed agriculture with cattle utilised for draft power. Livestock rearing in extensive systems is also common; however trypanosomiasis is a constraint to livestock production in the area. AAT is endemic, and cattle owners report it as the most important disease in tsetse challenged areas . Resistady areas . The LérThe study was conducted in the Adamawa plateau which is the most important cattle rearing region in Cameroon. Here, white and red Fulani cattle are reared extensively, with a system of communal herding and Gudali (Sahelian Zebu) cattle are also important in the region. There is risk of AAT infection in at least two-thirds of the territory where 90 % of the cattle are found and the disease is one of the biggest limitations to the development of the cattle sector in Cameroon . In 19952 of fertile land in south and southwestern parts of Ethiopia [Tsetse infest around 220,000 kmEthiopia , 17. AATEthiopia . AAT hasEthiopia . The surEthiopia . In thisEthiopia  .T. vivax is the most prevalent species of trypanosome in Ugandan cattle and T. congolense and T. brucei rhodesiense infections also occur [T. b. rhodesiense [T. b. rhodesiense causes human African trypanosomiasis (HAT) or ‘sleeping sickness’ which is fatal if left untreated. The distribution of T. b. rhodesiense in Uganda has increased dramatically in the past 10 years; this is attributed to the restocking of infected cattle into naïve areas following military conflict in the late 1990’s [T. b. rhodesiense cases in the whole of Africa between 2000 and 2009 were from Uganda [In Uganda the ‘tsetse belt’ runs from the highlands in southwestern Uganda across Lake Kyoga to north-eastern Uganda and at least 70 % of the entire country is thought to be infested with tsetse flies . T. vivaso occur , 22. Foldesiense . T. b. rm Uganda . The stu2 whilst Mambwe has a population density of approximately 13.4 people/km2. An increase in pressure on natural resources in the plateau area of the district has led to the relocation and expansion of the human population into the edges of the Luangwa valley expanding the wildlife-livestock interface. HAT cases have also occurred in the valley [The Luangwa valley runs through the Eastern Province of Zambia, with 3.84 million hectares of national park (46.9 %) and 0.41 million ha dedicated game management area the valley is an ecological niche for trypanosomes allowing vector-host interaction due to favourable conditions for tsetse in terms of vegetation, climate and abundance of wildlife hosts . The stue valley .Study areas were selected using random sampling from a sample frame of all communities within selected administrative divisions of each of the study countries. Within 195 communities 1,259 households across the five countries were visited during the course of the study. A household was defined as a group of people who usually reside and eat together. The head of the household was interviewed using a pre-piloted questionnaire which contained general questions about their herd, access to veterinary products and services, and livestock diseases. They were then asked questions specifically about AAT in an attempt to assess the relative importance of the disease to farmers. These questions focused on what is currently known, done and perceived with regards to AAT and its control. The study-unit for this analysis was the community; therefore the results from individual households were aggregated at community level.The vulnerability of a community has been defined as a product of exposure, sensitivity and capacity to adapt when an extreme event takes place . We consThe first step for the MCA was to identify variables likely to be associated with the exposure, sensitivity and adaption to AAT in an area. This was done using existing literature and the available field data. It was based on two principal criteria: firstly, the relevance of variables to the objective of the assessment and secondly the completeness of data collected. The resulting variable selection is detailed in Table MCA is a data reduction technique  which allows complex patterns in a dataset of categorical variables to be identified. Briefly, MCA provides a graphic representation describing the relationships between categories of variables and creates factors which describe the variation in the data. This technique also allows variables exhibiting little variation between the communities to be excluded and those which vary the most between communities to be identified. This technique has previously been used to identify biosecurity profiles of farms , 29.FactoMineR in R v. 3.0.1.MCA was performed on the selected variables at community level using the Indicator method. The coordinates of each community were calculated on three dimensions explaining 47.1 % of the variance and HCA was then performed on the selected dimensions using Ward’s method to aggregate areas into relatively homogeneous subgroups or profiles. These profiles maximise inter-cluster variation and intra-cluster correlation. The analysis was performed using the package Dimensions 1 and 2 from the results of the MCA of variables associated with AAT vulnerability are presented in Figs. The rural communities in this study were classified into three profiles or clusters Fig. , which mAll of the communities which kept some trypanotolerant breeds (Boulé) and Métis crossbreeds were found in cluster 2 31.7 %) and these were mainly communities located in Burkina Faso. This cluster is found in the top right quadrant of Figs. 1.7 % andWe then investigated the difference between selected supplementary variables and membership of the different clusters Table . The majWhen asked about diagnosis and treatment, farmers were most likely to be responsible for both in cluster 1 . With between a half and two-thirds of communities reportedly using trained individuals for diagnosis and treatment in clusters 2 and 3. Trypanocidal resistance was reported as a possible reasons for treatment failure in clusters 2 (63.3 %) and 3 (57.5 %). Drug quality was also a concern of farmers in clusters 1 (60.9 %) and 3 (37.9 %). Communities appeared to spend the most diagnosing and curatively treating AAT in cluster 1 with 63.0 % reportedly spending an average of 55 or more US$ in the past two years. Communities in cluster 2 appeared to have incurred the lowest costs with 42.5 % spending less than 15 US$ over the same time period.In terms of herd sizes, cluster 1 communities tended to have larger numbers of cows (82.6 % > 10), castrated males 47.8 % > 2), uncastrated males (71.7 % > 4) and calves (80.4 % > 4). Sheep were kept by around 90 % of these communities but they were less likely to keep goats (43.5 % = zero) and pigs (91.3 % = 0). Cluster 2 was mostly composed of medium sized  herds. Most households in this cluster also kept sheep and goats. Most households in cluster 3 had small herds of up to three adult female cows (57.5 %) with the rest keeping 4 to 10, around half had sheep and were more likely to have goats than households compared to the other two clusters. These communities were the most likely to keep pigs (67.8 %), with 53.3 % of farmers in cluster 2 also keeping pigs. A summary of the results is given in Fig. 7.8 % > 2The results from this study were then compared to previous surveys available in the study areas Table . ClusterHistorically, large-scale efforts to control trypanosomiasis have focussed on elimination of the vector which requires considerable investment and detailed planning. In addition, sustained elimination is only possible when the total tsetse population is addressed, which requires either an isolated population, or the sequential eradication of a full tsetse belt , 33. CooIn areas where sustained suppression is not currently feasible, understanding of how cattle-owners are affected by AAT and their efforts to manage the disease is critical to the design of suitable locally-adapted control programmes . The varFarmers are heavily reliant on chemotherapy for AAT control , 35. TryIn some communities cattle production is unlikely to be sustainable due to the high tsetse-trypanosome burden and lack of herd or community-level control of the disease due to lack of resources or technical capacity . In thesWhere seasonality allows, an alternative management strategy to reduce the risk of AAT is to only graze in tsetse infested areas in certain times of the year . In the Communities in cluster 1 reported no tsetse control, despite the Mission spéciale pour l’éradication des glossines (MSEG) running low level control operations for many years . HoweverIdeally, livestock owners should be encouraged to use trained veterinarians and veterinary assistants to diagnose and treat the disease. However, an estimated 35 million trypanocide doses are administered every year, large numbers of animals can be affected in certain seasons and many communities with the disease are in more remote areas close to national parks or game reserves where access to veterinary services may be reduced . TraininTrypanocide resistance was the main reason attributed to treatment failure in cluster 2, and second most cited reason in clusters 1 and 3. Trypanocide resistance has been reported in most of the regions studied within the five countries , 18, 45.T. b. rhodesiense in newly affected areas of Northern Uganda by block treating >180,000 head of cattle [T. brucei of pigs in the Iganga study area has been reported to be around 8.1 % [T. congolense in the Zambian study area, in addition there are an abundance of wildlife hosts for AAT here [This study only considered AAT and not HAT, in areas where both diseases overlap efforts between animal and public health officers should be coordinated. For example, the Stamp Out Sleeping Sickness (SOS) campaign was a public-private partnership designed to target the cattle reservoir of f cattle . Communind 8.1 % . Pigs weAAT here , 48. HigAAT here .Vulnerability to AAT is dynamic and complex and contains some level of judgement. The current study is solely based on the experience of farmers, who are usually not involved in the design of control programmes but who suffer the impacts of the disease. One identified key failure from reviews of past control programmes was the inability to transfer responsibility for the control of AAT to cattle-owners once the programme has ceased . FurtherThis study identified three clusters of community vulnerability profiles within the five countries studied, based on farmers’ beliefs with respect to trypanosomiasis control. Further work relating this information to entomological, disease prevalence and data from interviews with AAT control managers is needed to triangulate these results. However, these findings already provide an indication of the ways in which community level variables may impact upon the need, suitability and effectiveness of disease control programmes. Detailed descriptive reports at household-level are also being published, which include information on the demand and willingness to pay for novel treatments and diagnostics under development. Results of this work will be used to develop tailored recommendations, to reduce the impact of AAT in these communities. Including the integration of novel treatments and diagnostics with new and existing programmes, should they become available. The results may also be applicable to other communities with similar profiles to those identified here.


The corresponding Grüneisen parameters for the G and 2D peaks were found to be between 1.97 ± 0.15 and 2.86 ± 0.12, respectively. These values agree reasonably well with those obtained from small-strain bubble-type experiments. The results presented are also backed up by classical and ab initio molecular dynamics simulations and excellent agreement of Γ-E2g shifts with strains and the Grüneisen parameter was observed.Thin membranes, such as monolayer graphene of monoatomic thickness, are bound to exhibit lateral buckling under uniaxial tensile loading that impairs its mechanical behaviour. In this work, we have developed an experimental device to subject 2D materials to controlled equibiaxial strain on supported beams that can be flexed up or down to subject the material to either compression or tension, respectively. Using strain gauges in tandem with Raman spectroscopy measurements, we monitor the G and 2D phonon properties of graphene under biaxial strain and thus extract important information about the uptake of stress under these conditions. The experimental shift over strain for the G and 2D Raman peaks were found to be in the range of 62.3 ± 5 cm As is now well established, graphene is the first ever 2-dimensional crystal and is constituted of carbon atoms ordered in a honeycomb hexagonal lattice. The measured or predicted extraordinary properties of graphene, such as, its high carrier mobility, its high thermal conductivity, its high stiffness and strength among other things can or have already found useful applications in the field of electronics and sport1234567810111213et al.–1 (or 130 GPa considering graphene thickness as 0.335 nm). Other true biaxial experiments were attempted by Zabel et al.et al.et al.The effect of uniaxial strain on single layer graphene has been studied experimentally by the imposition of axial loads upon supported graphene flakes on plastic bars which can be flexed up or down to subject graphene to compression and tension, respectively, for strain levels of up to ~1.5%2345et al.As is evident, biaxial deformation (stretching) is particularly relevant for thin films or membranes at all scales. In this context, CVD-grown graphenes that can be produced in macroscopic dimensions are quite important for numerous applications  for which biaxial loading is required. To date the only cited work on CVD-grown graphenes in this area is by Jie 2, etc.) to controllable equibiaxial tensile strain gradients. The principle of this technique is based on the extension along two dimensions of the three-point bending configuration of plastic bars that have been employed for uniaxial loading as mentioned earlier. The added advantage of this approach is the fact that due to the symmetry of the loading procedure the term referring to the shear deformation potential (SDP) becomes zero and therefore the Poisson’s ratio of the underlying substrate has no effect on the measured strain234In this work a new experimental technique has been developed for subjecting 2-dimensional crystals  simulations has been used for the implicit calculation of the Γ-E2g phonon frequency of graphene at finite temperature (and at different strain levels), thus providing a more realistic correspondence to experiments. The numerical results are in good agreement with the experimental measurements.In this work, we have examined several graphene samples within a range of thicknesses from monolayer up to nanographite, and have monitored in detail the response of the Raman phonons. To our knowledge this is the first time that phonon shifts for trilayer graphene are reported under biaxial deformation and this complements the prior work reported under uniaxial strainIn h is the thickness of the plastic bar (substrate–cruciform), δ is the deflection of the bar at its centre, and L is the length between the two opposing pin edges, as shown in where 2g mode the solution of the secular equation under an externally applied strain field yieldsAs argued in earlier publications42g phonon, and G peak of graphene by applying equibiaxial strainεll = εtt) the second term of equation where 2g frequency is related to the applied strain through the corresponding Grüneisen parameter. Furthermore, no splitting of the peak due to lifting of degeneracy is induced and therefore the Poisson’s ratio of the substrate is not required as in the case of uniaxial experiments344Thus for biaxial strain the shift of the Raman Eet al.2/Si wafer. The depressions had a square shape with 6 μm sides and 20 nm depth. The reported equi-biaxial strain was 0.066%. The Raman shifts and Grüneisen parameters  for the G and 2D peaks are given in et al.x/Si substrate. Having optically identified spherical graphene bubbles with diameters in the range of 5–10 μm, they reported average biaxial strains of ~1%. Using eqn. 3 the calculated Grüneisen parameters were smaller than those obtained by Metzger et al. but in good agreement with the experimental and theoretical values from uniaxial experiments given by Mohiuddin et alet al.eqn. 3 by measuring the peak shift and then converting to strain using the Grüneisen parameters estimated by Mohiuddin et al. for uniaxial tension . The measurements show that by bending the substrate upwards (at its geometric centre) the strains in the x (εxx) and y (εyy) directions are tensile and equal and this confirms the validity of the design considerations of our apparatus. Certainly, in future experiments the design itself can be easily modified by allowing for opposing pins to be adjustable. This would ultimately allow the application of either equi-biaxial, or non-equal biaxial strain, or even for subjecting the specimen to complex biaxial strain fields (eg. tensile in one direction and compressive in the other). This versatility may also prove very useful for the study in a controllable manner of other 2-dimensional highly anisotropic materials, such as MoS2, black phosphorus, etc.In the experiments presented here, two types of glassy polymers were used as the substrate, PMMA (Poly-methyl methacrylate) and polycarbonate (PC). The PMMA bars can reach a biaxial strain level prior to failure of Several experiments were performed on simply-supported monolayer graphene flakes. In some cases a thin layer of SU-8 was spin coated over the polymer substrate, prior to the deposition of the graphenes, in order to improve the contrast and thus the optical visibility of graphene. The combinations examined were graphene resting on (a) PMMA, (b) PC, (c) PMMA/SU-8, and (d) PC/SU-8. The chosen polymers possess similar mechanical properties and, as the results clearly show, graphene adheres well to all of them. The results for all cases are presented in Prior to the experimental procedure, several Raman measurements were taken at central regions of the graphene flakes. For any given flake the strain distributions were found to be uniform with very small deviations of the Raman peaks. For each case only a negligible or small Raman wavenumber downshift was identified indicating that some of the flakes initially had a small residual tensile strain which apparently only affects the position of the zero strain developed in graphene and not the relationship between Raman frequency and strain in this situation5–1/% and –62.3 ± 5 cm–1/% for the 2D and G peaks respectively. The corresponding Grüneisen parameters were estimated to be 2.86 ± 0.12 and 1.97 ± 0.15 in reasonable agreement with the reported experimental and theoretical values . Despite this increase the lack of any observed splitting reflects the conservation of the E–1/% for the D22, D21, D12, D11 sub-peaks, respectively. The evolution of the 2D Raman peak is also presented for various strain levels. The consistent form of the 2D spectra indicates that the AB stacking is preserved throughout the range of applied strains due to the equi-biaxial strain field. This means that there is no relative slipping between the individual layers that form the bilayer and both are stressed equally as a result of the direct attachment to the polymer. Thus, the strain level achieved here is small for inducing these kind of structural changes as was also pointed out elsewhere–1/% which is similar to values reported for monolayer graphene by us and others15We now turn our attention to bilayer graphene. We note that the only available results for comparison with the literature on shift rates for bilayer graphene are for bilayer graphene bubbles and balloons from ref. Thicker graphene flakes were additionally examined that include trilayer (3LG) graphene, few layer graphene (FLG) and nanographite (NG). To our knowledge, results from biaxially strain-controlled experiments have not been previously reported for graphenes of these thicknesses. The corresponding 2D spectra are presented in For trilayer and few-layer graphene a change in slope is noted at ~0.15%, and for nanographite at ~0.23%. In 2g phonon frequency, we performed classical and ab initio molecular dynamics (MD) simulations on monolayer graphene. From the MD simulations we obtained the frequencies of the E2g phonon by suitable processing of the atomic trajectories and velocities. This approach has the advantages of being applicable at finite temperatures as well as accounting for anharmonic effects. To the best of our knowledge such results have not been reported in the literature. A detailed description of the method is given in ref. In order to gain an insight in the effect of biaxial strain upon the E2g frequencies that correspond to a temperature of T = 300 K for strains up to 2%. All of the potentials produce the expected decreasing linear dependence. We can see that the Tersoff-2010 and LCBOP potentials produce slopes of –60.7 cm–1/% and –59.7 cm–1/%, respectively, which are in very good agreement to the experimental values. The AIREBO potential overestimates the shift rates with a slope value of –78.4 cm–1/%. This performance complements the trends noted in the overall performance of these potentials on describing the dispersion curves of graphene at T = 300 K, as discussed in ref. For the classical MD simulations we employed the AIREBO34ab initio MD simulations were performed with the electronic structure computed using density functional theory (DFT) within the local density approximation (LDA), at a temperature of T = 300 K. Details on the calculations are given in the Methods section. The shift rate of the Γ-E2g mode with strain is computed at –60.5 cm–1/%, and the corresponding Grüneisen parameter is 1.82, which are in the experimentally expected range reported here and by other works  Grüneisen parameters for the G and 2D peaks. Indeed, since the Grüneisen parameter is a universal constant for the phonon characteristics of a perfect 2D crystal (such as graphene) any comparison of the actual values will highlight the problems encountered in this field vis-à-vis the methodology proposed here.Using the method presented above, the shift rates of the G and 2D peaks, as well as the Grüneisen parameters 2D that we present in G, in which case any scatter seems to emanate mainly from experimental error. It seems overall that there is certain discrepancy between γ2D values deduced from uniaxial experiments with supported samples vis-à-vis values obtained from biaxial experiments with suspended samples. This may be another indication of the effect of the underlying substrate on the 2D peak and the Dirac points’ position which has an effect on the derived Grüneisen parameter42D values.In –1/%, respectively. These rates correspond to Grüneisen parameters of –1/%, respectively, whereas for few layer graphenes (from trilayer to nanographites) a reduction of the above rates was observed due possibly to cohesive failure within the flakes. These results are in good agreement with both classical and ab initio theoretical results at finite temperature obtained by a method based on molecular dynamics simulations. The Γ-E2g mode Grüneisen parameter and shift rate from the ab initio calculations are 1.8 and –60.5 cm–1/%, respectively.In the present study a new experimental technique is introduced that employs a device designed for subjecting any 2D crystal to controlled biaxial tensile deformations. The device can be easily handled and possesses all the features of the corresponding uniaxial devices. Graphene flakes of various thicknesses, ranging from monolayer to nanographite, were examined under biaxial strain in tandem with Raman spectroscopy measurements. For the monolayer graphene the 2D and G band shift rates were found to be Cruciform polymer substrates, as schematically shown in λlaser = 785 nm (1.58 eV) excitation was used to obtain the Raman spectra. The Raman measurements were taken at the centre of the flakes to avoid edge effectsThe monolayer flakes were subjected to biaxial strain using the two dimension three-point-bending jig as discussed earlier. The strain was applied incrementally with a step of 0.025% for PC and of 0.028% for PMMA. This small difference is the result of the PMMA polymer that was used being slightly thicker than the PC, and the jig calibration step. At every loading step measurements for the Raman 2D and G peaks were carried out. A laser of T = 300 K and initial equilibration at constant energy  was performed. The corresponding lattice parameter  was computed by performing a subsequent equilibration within the isothermal–isobaric ensemble (NPT) at zero pressure. The lattice constant was taken as the average over these steps. A computational cell was created anew using the corresponding constant lattice for each potential. This cell was used in the NVE simulations from which the trajectories and velocities were acquired for the zero strain level. Transition to the next strain levels was performed by deforming the unit cell with care on uniformity, specifically, the unit cell edges were equally elongated and the angle was maintained at 60° by elongating the tilt of the triclinic cell to half of that of the a1 axis . After each elongation thermal equilibration was performed prior to the main NVE simulations. The time step was 0.05 fs and the NVE simulations were run ~32 ps (for recording the trajectories and velocities used in the follow up kVACS (k-space velocity autocorrelation function) methodThe MD simulations were performed at 300 K using a triclinic computational cell of 20 ×20unit cells  and periodic boundary conditions. The procedure followed for the simulation is as follows. The computational cell was initially relaxed for each potential . Randomized velocities  were attributed to the atoms, within a Gaussian distribution, corresponding to a temperature of ζ valence plus polarization (DZVP) basis set with LDA-optimized Goedecker-Teter-Hutter (GTH) norm-conserving pseudo-potentials42s22p2). A triclinic computational cell of 6 ×5 unit cells  and periodic boundary conditions were used in the x–y plane. A vacuum of 25 Å between periodic images of the graphene is considered in the z-direction. The procedure followed was analogous to that for the classical molecular dynamics simulations. The time step was 0.8 fs and the NVE simulations were run ~20 ps .


Halococcus salifodinae BIpT DSM 8989T, an extremely halophilic archaeal isolate from an Austrian salt deposit (Bad Ischl), whose origin was dated to the Permian period, was described in 1994. Subsequently, several strains of the species have been isolated, some from similar but geographically separated salt deposits. Hcc. salifodinae may be regarded as one of the most ancient culturable species which existed already about 250 million years ago. Since its habitat probably did not change during this long period, its properties were presumably not subjected to the needs of mutational adaptation. Hcc. salifodinae and other isolates from ancient deposits would be suitable candidates for testing hypotheses on prokaryotic evolution, such as the molecular clock concept, or the net-like history of genome evolution. A comparison of available taxonomic characteristics from strains of Hcc. salifodinae and other Halococcus species, most of them originating from surface waters, is presented. The cell wall polymer of Hcc. salifodinae was examined and found to be a heteropolysaccharide, similar to that of Hcc. morrhuae. Polyhydroxyalkanoate granules were present in Hcc. salifodinae, suggesting a possible lateral gene transfer before Permian times.  Halococcus salifodinae BIpT DSM 8989T was obtained as a viable isolate from Permian rock salt deposits of a mine in Bad Ischl, Austria DNA-DNA h [et al. . These eOther methods: For comparative phylogenetic analyses 16S rRNA gene sequences from validly described Halococcus spp. were obtained from the European Molecular Biology Laboratory (EMBL) web interface and fitted in a subset of aligned archaeal sequences obtained from the Ribosomal Database Project II [http://www.megasoftware.net; [oject II . Phylogeoject II ). The weare.net; ) and Cluare.net;  were useare.net; . Halococcus salifodinae strains can be considered as living fossils and constitute a promising source of novel evolutionary information.From considerations of close genetic relatedness and origin from ancient geological materials it is concluded that


A strong electric field enhancement produced in the 3D framework nanohybrids of graphene nanosheet/Ag-nanoparticles is responsible for this great enhancement of catalytic reaction, due to larger electron collective oscillation in the composite system. Especially the oxygen adsorbed on the graphene and Ag nanoparticles can be excited to triplet excited states, and the electrons on the graphene and the nanoparticles can be effectively transferred to the oxygen, which plays very important role in molecular catalytic reactions. Our results demonstrate the contribution of graphene in plasmon-driven catalytic reactions, revealing a co-driven reaction process.This excellent SERS substrate can be used for future plasmon and graphene co-catalytic surface catalytic reactions, graphene-based surface plasmon sensors and so on.Three dimensional (3D) plasmonic nanostructure is perfect for the surface-enhanced Raman scattering (SERS) and also very suitable for surface catalytic reaction, but how to design and fabricate is still a robust task. Here, we show a 3D plasmonic nanohybrid of vertical graphene-nanosheet sandwiched by Ag-nanoparticles on the silicon nanocone array substrate for enhanced surface catalytic reaction. By SERS detection, we find that this hierarchical nanohybrid structure is highly efficient in the enhancement of catalytic reaction, even at a very low concentration of 10 Noble metallic nanoparticle catalysts and their use in catalytic processes have recently been widely investigated experimentally and theoretically due to their advantages in nanoscience and nanotechnology. Since the first report of plasmon-driven catalytic reaction of p-aminothiophenol (PATP) oxidizing to dimercaptoazobenzene (DMAB) revealed by surface-enhanced Raman scattering (SERS)13456789111213141517181920Graphene, as an atomic-scale honeycomb lattice made of carbon atoms, conducts heat and electricity with great efficiency. Recently, graphene has also been used as the substrate of SERS162324252728Recently, the graphene-nanoparticle hybrids were reported to be able to tune the enhancement of Raman scattering3133−11 M. A mechanism of graphene enhancing plasmon-driven catalytic reaction is proposed and discussed here. This excellent 3D SERS substrate can be used for future graphene and plasmon co-driven surface catalytic reactions, graphene-based surface plasmon sensors and so on.In this work, we fabricated a 3D hierarchical nanostructure of vertical flower-like graphene nanosheets (FGNSs) sandwiched by Ag nanoparticles (Ag-NPs) grown on silicon nanocone arrays substrate, in which silicon nanocone arrays can further optimize the 3D hierarchical hybrid structure and greatly improve its surface-to-volume. Firstly we verified that this hierarchical hybrid structures have a great SERS ability to be a perfect SERS substrate. Further, to demonstrate that this excellent SERS substrate is very suitable for graphene enhancing plasmon-driven catalytic reactions, PATP and 4NBT were separately oxidized and reduced to DMAB. Our results demonstrate that this hierarchical hybrid structure is very efficient in graphene enhancing plasmon-driven catalytic reaction, even at the level of 109/cm2. The FGNSs were uniformly grown on the silicon nanocone array by hot filament chemical vapor deposition using the mixture of H2 and CH4. Furthermore, the structural details of the as-grown graphene nanosheets are acquired from TEM images and EELS, respectively, as shown in The Ag nanoparticles are coated on both sides of graphene nanosheet grown on the Si nanocone arrays, and the distribution of Ag nanoparticles can be tuned by Ag nanofilm with different thicknesses from 2 to 8 nm, in which 6 nm thick Ag nanofilm with 30 nm in average particle diameter is selected as next tested sample because this thickness exhibits the best SERS ability among different Ag thicknesses /I2D(1335) = 1.66, I2D(1432)/I2D(1335) = 2.11, I3D(1396)/I3D(1335) = 3.10, I3D(1432)/I3D(1335) = 4.24. More than twice enhancement in the relative intensity ratio reveals that the 3D structure of graphene/Ag-nanoparticles is much more efficient for enhancing plasmon-driven catalytic reactions than 2D plane structure.In order to further demonstrate the significant effect of graphene nanosheets on this co-driven catalytic reaction, the SERS spectra of three kinds of substrate deposited by Ag nanoparticles using the same Ag film thickness of 6 nm – a pure planar silicon substrate, a CVD single-layer graphene (SLG) coated planar silicon substrate by transferring process and a silicon nanocone array coated by FGNSs – were measured under the same conditions for PATP, as shown in −1 over 1335 cm−1 is gradually decreased. Even at 10−11 M, the Raman profile is at 1432 cm−1 of DMAB can be observed in −11 M. This result is much better than our previous plasmon -driven catalytic reaction at the molecular concentration 10−71−5 M) at 10×10 μm. The ratio of Raman intensities of DMAB at 1432 cm−1 over Raman intensities of graphene at 1335 cm−1 can be seen in 2 at 1361 cm−1 can also be found. By increasing of laser power, Raman peaks of the graphene are significantly enhanced, but the Raman peak of DMAB at 1432 cm−1 only increases a little along with the increase of laser power. By increasing the laser intensity, it seems that the probability of such catalytic reaction is increased slightly. So, it seems that the reduced catalytic reaction is not as good as the oxidized catalytic reaction (from PAPT to DMAB) in this 3D hybrids nanostructure, and the reason for this will be discussed in the next section.It is known that DMAB can also be converted from 4NBT. It is interesting to study the laser power dependent probability and efficiency. The contribution of graphene nanosheet sandwiched by Ag nanoparticles see  to the p2 adsorbs on two Ag atoms of a cluster at the ground state, as shown 2 to the Ag cluster on the triplet electronic transition of O2 adsorbed on the Ag cluster, as displayed in 2, and the electrons on triplet excited O2 transfer to the Ag cluster. This is a very good process for the plasmon-driven catalytic reaction. The lowest energy of transition (S1) from singlet oxygen at ground state to the triplet excited oxygen is 1.7726 eV. The S2 transition (2.2234 eV) can also result in the triplet excited state for oxygen, since the related energy is also lower than the laser frequency (532 nm) that we used. So, it is occurred that plasmon and graphene co-catalytic surface catalytic reactions in our experiments, in which graphene play a key role in enhancing plasmon-driven catalytic reactions.It has also been shown that surface plasmon resonance can activate the oxygen to the triplet excited state, which plays very important role in plasmon-driven catalytic reactionsIt has been found that the reduced catalytic reaction (from 4NBT to DMAB) is not as good as the oxidized catalytic reaction (from PAPT to DMAB). The reason is that in the oxidized reaction, the hot electrons generated from plasmon decay play an important role only in decreasing the reaction barrier, where the hole electrons temperately attach to the molecules, and then the neutral potential energy surface (PES) being ion PES12345678911121314151112131415In summary, we designed and fabricated a 3D plasmonic hybrid nanostructure of flower-like graphene nanosheets sandwiched by Ag nanoparticles on silicon nanocone arrays, which has been applied for graphene enhancing plasmon-driven catalytic reactions and showed the more superiority than 2D graphene-nanoparticles hybrids. Our experimental results reveal that the sensitivity of the co-driven catalytic reaction in this 3D plasmonic hybrid nanostructure can be improved by fourfold over that of the plasmon-driven catalytic reaction. The mechanism of plasmon and graphene co-driven catalytic reaction has been theoretically revealed for the first time. Our results are very valuable for designing and fabricating excellent SERS substrates based on graphene and plasmon co-driven surface catalytic reactions, graphene-based surface plasmon sensors, and so on.2 and SF6 are used as the etching gas to fabricate the Si nanocones. Geometry of Si nanocones, for example, the height, and density as well as the aspect ratio, were modulated through changing etching parameters. Samples of the Si nanocone array were fabricated at the conditions: SF6/O2 = 22/7, pressure of 6 mTorr, ICP power of 800 W, and the etching time of 7 minutes. An as-formed silicon nanocone array served as the substrate to grow the few-layer graphene nanosheets by hot filament chemical vapor deposition (HFCVD). The Ar, CH4 and H2 were fed into the HFCVD system with the ratio of 40:10:1 for the growth of the petaloid graphene nanosheets (PGNSs). And, the growth processes were carried out at the temperatures of 1000 °C, where the pressure was maintained at 2 kPa. The positive bias voltage was added on the filament during growth. The bias current was measured with the ammeter. The structure, morphology and the chemical bond-state of the obtained samples were characterized by the scanning electron microscopy (SEM), and transmission electron microscopy (TEM), and high resolution transmission electron microscopy (HRTEM) (at 200 kV), the electron energy loss spectroscopy (EELS), and the Raman spectra, respectively. And then, the Ag nanospheres were prepared by the 6 nm thick Ag nanofilm deposited on the PGNSs using the E-beam evaporation.The silicon nanocone arrays are fabricated by the maskless etching in the inductively coupled plasma (ICP) system, where O−5 M PATP or 4NBT ethanol solution for 4 hours, and then which was rinsed with the ethanol to get rid of the non-adsorbed molecules. The 10−5 M solution was gradually diluted to 10−6 M and up to 10−11 M by Multiple dilutions.SERS measurements were performed with 532 nm laser with the different powers from 0.1 mW to 1 mW. The objective lens is used to focus the excitation laser on the samples, and to collect Raman spectra with the 1μm laser spot. The as-fabricated graphene nanosheets/Ag-nanoparticles hybrids structure substrates are immersed in 102/(V*s) and 1×106 m/s, respectively. The ground state geometry of graphene-O2 system was optimized with density functional theory (DFT)FDTD was used to demonstrate the plasmon intensity and field distribution in our substrate, which revealed the contribution of graphene to electronic field enhancement. The diameter of Ag nanoparticles is set at 30 nm and the graphene sheet is set at 1 nm (3 layers). Fermi energy, mobility and Fermi velocity of graphene in the calculation are 0.4 eV, 1500 cmHow to cite this article: Zhao, J. et al. Three Dimensional Hybrids of Vertical Graphene-nanosheet Sandwiched by Ag-nanoparticles for Enhanced Surface Selectively Catalytic Reactions. Sci. Rep.5, 16019; doi: 10.1038/srep16019 (2015).


Nanofluids with enhanced thermal properties are candidates for thermal management in automotive systems, with scope for improving energy efficiency. In particular, many studies have reported on dispersions of nanoparticles with long-term stability in the base fluid, with qualitative evaluations of the dispersion stability via either the naked eye or optical instruments. Additives such as surfactants can be used to enhance the dispersion of nanoparticles; however, this may diminish their intrinsic thermal properties. Here, we describe molecular dynamics simulations of nanofluids containing graphene sheets dispersed in ethylene glycol and water. We go on to suggest a quantitative evaluation method for the degree of dispersion, based on the ratio of the total number of nanoparticles to the number of clustered nanoparticles. Moreover, we investigate the effects of functional groups on the surface of graphene, which are expected to improve the dispersion without requiring additives such as surfactants due to steric hindrance and chemical affinity for the surrounding fluid. We find that, for pure graphene, the degree of dispersion decreased as the quantity of graphene sheets increased, which is attributed to an increased probability of aggregation at higher loadings; however, the presence of functional groups inhibited the graphene sheets from forming aggregates. Thermal management is a crucial factor in heat transfer equipment, and determines the energy efficiency. In automotive systems, working fluids such as water, ethylene glycol (EG), propylene glycol, engine oil, mineral oil, kerosene oil, and silicon oil are indispensable for effective thermal management; however, enhancements in the thermal conductivity of these fluids are desirable for improved energy efficiency . DispersThe concept of a “nanofluid” was first reported by Choi  at Argon2 nanotubes =90. Therefore, the degree of dispersion would be Gact/Ntotal=90/100=0.9.where Figure Chemical exfoliation is a promising method to form graphene sheets from graphite. During this process, various types of oxide functional groups are formed on the surface of graphene sheets, which can influence the dispersion stability in a base fluid. These functional groups can induce hydrogen bonding, stabilizing the dispersion, and providing active sites that enable the formation of heterogeneous structural composites. We investigated three functional groups at the surface of graphene sheets: alcohol and amide (amine) groups, as shown in Fig. Figure On the other hand, we compared a value of the mean square displacement (MSD), or mean square fluctuation between graphene sheets with and without functional groups. In statistical mechanics, it demonstrates the measure of the spatial extent of random motion measuring the portion of the system “explored” by the random walker, and is defined asT is the time for average, and x0 is the reference position of the particle. Typically, this reference position will be the time-averaged position of the same particle. Figure where We investigated the degree of dispersion of nanofluid formed of graphene sheets with and without functional groups at graphene loadings in the range 1–25 wt.%. Figure The degree of dispersion of the functionalized graphene sheets exhibited a much weaker dependence on the graphene loading (see the red triangles in Fig. The concept of a nanofluid was first suggested in 1995, and nanofluids are promising candidates for thermal management in automotive systems. We carried out MD simulations of nanofluids formed of graphene sheets dispersed in a 1:1 mixture of EG and water. In this study, we have suggested an evaluation method to quantify the degree of dispersion of nanoparticles in a base fluid based on the ratio of the total number of nanoparticles to the number of nanoparticles that exist in clusters.Furthermore, we clearly quantify that the functional groups at the surface of graphene sheets improved the degree of dispersion due to steric hindrance and chemical affinity for the surrounding fluid, without requiring additives. With non-functionalized graphene sheets, the degree of dispersion decreased as the graphene loading increased because of an increased probability of aggregation, whereas the functionalized graphene sheets did not tend to aggregate, even at high loadings.


In principle, accurate guideline recommendations should lead to optimal management based on a secure diagnosis. However, current IPF diagnostic guidelines do not meet the needs of a major sub-group (possibly the majority) of patients with idiopathic pulmonary fibrosis (IPF). A great many IPF patients have HRCT appearances of “possible UIP”. A surgical biopsy is very often impracticable due to age, disease severity, co-morbidities or patient refusal. A guideline-based diagnosis cannot be made in these patients, although the diagnosis is often obvious. Inflexible diagnostic criteria, although essential for treatment trials, must necessarily be structured around an inflexible diagnostic algorithm. With this approach, non-standardised information  must be omitted, including observed disease behaviour prior to and on treatment, findings on bronchoalveolar lavage, likelihoods in relation to age and a wealth of ancillary clinical information. However, when a diagnosis cannot be made using guideline criteria, a probable or highly probable “working diagnosis” of IPF can and should be made in most IPF patients by means of clinical reasoning, integrating all available non-standardised information. Idiopathic pulmonary fibrosis (IPF), the most progressive and most prevalent of the idiopathic interstitial pneumonias (IIPs), requires a radically different management from the other IIPs due to its prognostic implications and the advent of IPF-specific therapies. With regards to their management, the remaining IIPs can largely be conceptualised as forms of immune dysregulation; however, this model has failed in IPF .It might be supposed that the accurate diagnosis of IPF should depend upon the application of evidence-based diagnostic guidelines. Yet, in at least half of IPF patients, the diagnosis cannot be made using current American Thoracic Society/European Respiratory Society/Japanese Respiratory Society/Latin America Thoracic Association (ATS/ERS/JRS/ALAT) guideline criteria . The ‘faCurrently, in roughly half of IPF patients, ATS/ERS/JRS/ALAT 2011 diagnostic criteria for IPF are not satisfied and the disease remains unclassifiable. In this setting, clinicians must speculate on the best course of action – whether to manage the disease according to a diagnosis of alternative disorders or to apply IPF-validated anti-fibrotic therapies. In a nutshell, the ATS/ERS/JRS/ALAT 2011 IPF diagnostic guidelines are ‘broken’.How has it come to this? The pace of recent events is partly to blame. In 2009/2010, when the ATS/ERS/JRS/ALAT guidelines were formulated, there was no universally accepted therapy known to delay disease progression and it was generally agreed that best management consisted of participation in a novel treatment trial , was avaNevertheless, recent events are only partly to blame. There is a second issue related to the decline and fall of ‘eminence-based medicine’. Fortunately, the age of the opinion hegemony – a handful of experts driving best medical practice based on their anecdotal views – is over. However, we have fallen into a new and equally damaging trap – the belief that logic and common sense must be discarded and only data formalised within an ‘evidence base’ can be taken into account. It may seem surprising that the Evidence-based Renaissance Group recently published a perspective entitled, Evidence-based Medicine, a Movement in Crisis . HoweverWhatever else is said about the ATS/ERS/JRS/ALAT 2011 IPF guidelines, it cannot be doubted that, for the first time, an evidence-based ethos was energetically applied to the diagnosis and management of IPF. However, it was telling that nothing useful was said about that great desideratum, multidisciplinary diagnosis. Certainly, the phrase ‘multidisciplinary diagnosis’ makes cameo appearances from time to time, but what is described in the document as multidisciplinary diagnosis is unrecognisable to most practising clinicians, radiologists and histopathologists. The casual reader, focusing on tables and key conclusions, might be excused for imagining that the process of multidisciplinary diagnosis consists of amalgamating a histological grade and a high-resolution computer tomography (HRCT) category, each reached in isolation, providing a definition of possible, probable or definite IPF. In this model, the clinician has no particular role, apart from the exclusion of a primary cause of pulmonary fibrosis. The problem, here, is that diagnosis for trial purposes, enshrined in the 2011 guideline, differs radically from enlightened multidisciplinary diagnosis.In treatment trials, it is essential that diagnosis is rigorous and standardised in order to minimise diagnostic variation between treatment arms. In essence, this means the use of simplistic clinical exclusion criteria and HRCT data, and a requirement for a diagnostic surgical biopsy when HRCT appearances are inconclusive. Rigid diagnostic criteria require the exclusion of all information not available in all patients. Observed disease behaviour, on and off therapy, does not feature as a possible influence on the likelihood of IPF in the ATS/ERS/JRS/ALAT 2011 guidelines because, at enrolment into a trial protocol, observations of disease behaviour are not always available. Bronchoalveolar lavage is not a part of the diagnostic algorithm in all countries and therefore it was relegated to an undefined ‘weak negative’ role in the ATS/ERS/JRS/ALAT 2011 guidelines. This is relevant because, in clinical practice, hypersensitivity pneumonitis exposures are not, in reality, a simple ‘present/absent’ dichotomy. Often, there is real difficulty in assigning significance to exposure. The whole question of what constitutes occult connective tissue disease is too complex to summarise concisely, but frequently contributes to the warp and weft of multidisciplinary diagnosis.For trial purposes, diagnosis must necessarily be ‘dumbed down’. Multidisciplinary diagnosis is a different matter altogether. Trial diagnostic criteria require the consideration only of data standardisable in all patients. By contrast, multidisciplinary diagnosis requires the consideration of all potentially relevant information in every individual patient. Trial diagnostic criteria confine expert judgement to the isolated views of the radiologist and histopathologist, with subtle clinical interpretation disallowed. Multidisciplinary diagnosis consists of debate and the integration and reconciliation of a huge amount of ancillary clinical information. It is sometimes supposed that the major value of multidisciplinary diagnosis consists simply of broadening expertise. However, it can also be argued that the true value of a multidisciplinary team lies in bringing together trained minds accustomed to civilised disagreement and the scrutiny of logic and common sense. Above all, the use of logic, common sense and review of all data (which differs widely in its completeness in every patient) cannot be validated using an evidence-based approach in which inflexible diagnostic criteria are required.any fool can make a rule and any fool will mind it”.With the use of a multidisciplinary approach, a diagnosis of IPF can often be made, based on very high probability, in patients who lie outside guideline criteria. Anti-fibrotic therapy in this large patient subgroup can be instituted with the same confidence as in ‘definite IPF’, as defined in the 2011 guidelines. In other cases, in which IPF is merely the most likely of several possible diagnoses, an eventual working diagnosis of IPF and the introduction of anti-fibrotic therapy can be justified by subsequent disease progression despite immunomodulatory therapy. In this area, medicine is as much an art as a science. The evidence-base apparatchik need not despair – the time will come for the multidisciplinary approach in IPF to be subjected to evidence-based evaluation. The accuracy of a flexible approach to diagnosis, based on the integration of clinical reasoning and morphologic (CT and pathology) pattern recognition, can be examined against subsequent natural history and treatment course, given that the value of making a diagnosis is in the provision of this information. However, the answer does not lie in recreating new rigid diagnostic criteria for IPF to the detriment of clinical reasoning. As Thoreau, father of the pragmatic philosophy movement once said, “


Sarcoidosis is a multisystem immuno-inflammatory disorder of unknown etiology that most commonly involves the lungs. We hypothesized that an unbiased approach to identify pathways activated in bronchoalveolar lavage (BAL) cells can shed light on the pathogenesis of this complex disease.We recruited 15 patients with various stages of sarcoidosis and 12 healthy controls. All subjects underwent bronchoscopy with lavage. For each subject, total RNA was extracted from BAL cells and hybridized to an Affymetrix U133A microarray. Rigorous statistical methods were applied to identify differential gene expression between subjects with sarcoidosis vs. controls. To better elucidate pathways differentially activated between these groups, we integrated network and gene set enrichment analyses of BAL cell transcriptional profiles.Sarcoidosis patients were either non-smokers or former smokers, all had lung involvement and only two were on systemic prednisone. Healthy controls were all non-smokers. Comparison of BAL cell gene expression between sarcoidosis and healthy subjects revealed over 1500 differentially expressed genes. Several previously described immune mediators, such as interferon gamma, were upregulated in the sarcoidosis subjects. Using an integrative computational approach we constructed a modular network of over 80 gene sets that were highly enriched in patients with sarcoidosis. Many of these pathways mapped to inflammatory and immune-related processes including adaptive immunity, T-cell signaling, graft vs. host disease, interleukin 12, 23 and 17 signaling. Additionally, we uncovered a close association between the proteasome machinery and adaptive immunity, highlighting a potentially important and targetable relationship in the pathobiology of sarcoidosis.BAL cells in sarcoidosis are characterized by enrichment of distinct transcriptional programs involved in immunity and proteasomal processes. Our findings add to the growing evidence implicating alveolar resident immune effector cells in the pathogenesis of sarcoidosis and identify specific pathways whose activation may modulate disease progression.The online version of this article (doi:10.1186/s12931-016-0411-y) contains supplementary material, which is available to authorized users. Sarcoidosis is a heterogeneous disease characterized by noncaseating granulomatous inflammation affecting multiple organs. The lung is the most common site involved in this systemic disease with over 90 % of patients developing pulmonary sarcoidosis . AlthougTo our knowledge, the transcriptional state of immune effecter cells residing within lung airspaces has not been systematically surveyed in sarcoidosis. We hypothesized that an unbiased approach to identify pathways differentially activated in bronchoalveolar lavage (BAL) cells of sarcoidosis patients could shed light on the pathogenesis of this complex disease and reveal previously unrecognized targets for future therapeutic intervention.N = 25) were recruited from patients undergoing routine clinical evaluation for diagnosis of sarcoidosis without acute pulmonary symptoms (Table N = 12) composed of individuals with no history of lung disease and no medication usage at time of bronchoscopy was recruited as controls. These healthy individuals volunteered to undergo bronchoscopy as part of an Institutional Review Board-approved research program. The entire protocol was approved by the East Carolina University & Medical Center Institutional Review Board (#05-0581) and written informed consent was obtained from all participants.Sarcoidosis subjects .Transcriptional profiling was performed on a subset of the study cohort: 15 subjects with sarcoidosis and the 12 controls. For each subject, total RNA from BAL cells was extracted using the RNeasy mini kit  and its integrity verified by the Agilent 2100 Bioanalyzer . Amplified biotin-labeled cRNA was generated from 2 mg of total RNA and hybridized to Affymetrix Human GeneChip U133A 2.0 microarrays  and processed according to the manufacturer’s instructions. Background adjustment and quantile normalization of log-transformed intensities was performed using RMA software [t-test [Initially, we used a form of multidimensional scaling known as correspondence analysis  to detert-test  coupled t-test . An FDR t-test .P-values from this analysis were corrected for multiple testing using an FDR threshold < 1 %. We then undertook a more unbiased and comprehensive approach by applying Gene Set Enrichment Analysis (GSEA) to the entire microarray dataset using over 1300 canonical pathways derived from multiple resources including KEGG, Reactome, Pathway Interaction Database, and Biocarta among others [We applied two levels of functional enrichment analysis to the expression dataset. First we focused on the subset of differentially expressed genes FDR < 1 %) and used Webgestalt software  to identg others . These pg others . To dispg others , 16. A s % and usT) values for genes of interest were normalized to a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as previously described [P-values were calculated using two-tailed Student’s t-test of log2-transformed data .We confirmed differential expression of selected genes by qPCR using the ABI Prism 7300 Detection System . RNA from BAL cell samples were analyzed in duplicate using primer sets for proteasome subunit beta type 8 (PSMB8) , proteasome subunit beta type 9 (PSMB9)  and interferon gamma (IFNG) . Threshold cycle (Cescribed  and relaescribed . Data arN = 15); 2) a validation group of patients that were not part of the original microarray study . P-values were calculated using the nonparametric, two-tailed Mann–Whitney test because the cytokine data were not normally distributed based on Shapiro-Wilk’s normality test .We evaluated the protein expression of two candidate genes identified from the microarray experiments, IL6 and CCL5 (RANTES), in BAL fluid of sarcoidosis and control subjects using ultrasensitive kits  as per manufacturer’s instructions. The plates were read using the MSD Discovery workbench analyzer and software package. Two groups of sarcoidosis patients were studied: 1) subjects who also underwent microarray analysis of their BAL cells , but found no significant enrichment in the control group : 1) lung biopsy tissue from patients with sarcoidosis (N = 6) and control subjects (N = 6) as reported by Crouser et al. [N = 38) and healthy subjects (N = 20) from Koth et al. [To extend and compare our findings to previous important work in this field, we obtained publicly available microarray data from two published studies  periN = 86 gene sets) compared to lung tissue (N = 32) or PBLs (N = 47). This finding suggests that airspace immune effector cells may provide a more robust and consistent signal compared to the more heterogeneous cell types obtained from lung tissue biopsies or the lack of proximal association with involved end organ as is the case with circulating leukocytes. Secondly, we found that in both BAL cells and lung tissue biopsies of sarcoidosis patients, T cell-associated gene sets were significantly upregulated but in contrast, these pathways were downregulated in the PBL dataset. While the implications of this observation are not clear, the known critical role played by of activation of Th1 and T cell receptor signaling in the pathobiology of sarcoidosis highlights the importance of sampling cells from affected target organs to understand mechanisms of disease pathogenesis. Finally, the enrichment of the proteasome pathway in sarcoidosis was found only in our BAL cell analysis. Given the importance of this molecular machinery in modulating adaptive immunity and thereby having a potentially novel role in sarcoidosis, our approach of interrogating immune effector cells in the airspaces shows promise for helping unravel previously unrecognized processes in this complex disease.Using an FDR < 1 % cutoff to designate significant enrichment, we identified 32 gene sets upregulated in lung tissue and 47 gene sets upregulated in PBLs of patients with sarcoidosis  as well as an independent group of subjects with sarcoidosis . Subject characteristics of the independent sarcoidosis group are available at Additional file We assessed whether the immuno-inflammatory signal observed in BAL cells of patients with sarcoidosis was also present in their BAL fluid by measuring protein levels of two differentially upregulated genes: IL6 and CCL5. As shown in Fig. The etiology of sarcoidosis continues to be debated, with much investigation into potential instigators of the observed exaggerated and aberrant immune response. Since the lung is the most common site of organ involvement, we hypothesized that investigating the airspace immune cell transcriptome would provide insight into disease pathogenesis and reveal putative novel therapeutic targets.We compared the transcriptional profiles of lung BAL cells in sarcoidosis patients versus control subjects and found significant differences between the two groups, most prominently the selective enrichment of distinct immune-related pathways. As confirmation of our unbiased approach, we identified a number of immune modulators previously implicated in sarcoidosis, including IFNG , IL12 2, and IL1Our data are consistent with studies of whole blood and lung tissue transcriptome which demonstrated upregulation of interferon signaling and activation of immuno-inflammatory pathways –32, 35. By integrating pathway and network analysis of BAL cell expression profiles, we constructed a modular overview of differentially activated processes in sarcoidosis that linked key putative drivers of disease. In particular, our analysis highlighted a potentially important connection between adaptive immunity and the proteasomal apparatus. The proteasome is part of the ubiquitin-proteasome system, which controls intracellular protein degradation and turnover. Activation of proteasome complex components suggests that sarcoidosis induces a proteolytic environment for substrate degradation. We found upregulation of constitutive proteasome catalytic subunits and the inducible proteasome subunits, PSMB8, PSMB9 and PSMB10. The inducible subunits form the immunoproteasome, which is upregulated by oxidative stress and proinflammatory cytokines, particularly IFNG . Key funOur findings are consistent with a recent report by Sixt et al. , who fouOur study has a number of limitations. Since the number of subjects was relatively small, our analyses should be considered exploratory and require confirmation in larger cohorts. However, our sample size was similar to previous reports leveraging tissue gene expression profiling to elucidate disease mechanisms in sarcoidosis , 36. SinWe have outlined a systematic framework to investigate the role of airspace immune cells in the pathogenesis of sarcoidosis. We found that this complex disease is associated with the activation of diverse yet distinct pathways and identified key members of the proteasome apparatus as potential modulators of host immune response. Future work is required to further delineate these relationships and determine their influence in disease progression.


This unique feature stems from the fact that the permittivity of doped graphene can be close to zero at a certain photon energy. The electrical controllability of evanescent coupling strength can enable novel design of optical devices. As a proof-of-concept, we designed a waveguide-type optical modulator of a novel operation principle: transmission modulation depends on the electrically controlled existence of a guided-mode of the waveguide, not the variation of the ohmic loss of graphene, resulting in a low insertion loss and a small device footprint.Electrically tunable permittivity of graphene provides an excellent tool in photonic device design. Many previous works on graphene-based photonic devices relied on variable absorption in graphene, which is naturally small in the optical region, and resonant structures to enhance it. Here we proposed a novel scheme to control evanescent coupling strength by inserting two graphene layers to a frustrated total internal reflection (FTIR) configuration. The resulting structure behaves in a drastically different way from the original FTIR: optical transmission though the structure can be electrically controlled from ~10 Recently, graphene29136Among the many graphene-based optical devices, optical modulators have attracted much interest because of their broad optical bandwidth and Si waveguide compatibility5614In this work, we propose a novel scheme to modulate optical transmission by combining the tunable permittivity of graphene, which can be very close to zero, and the frustrated total internal reflection (FTIR)EF) of graphene is properly controlled to make its permittivity negativeEF , yielding a reflection coefficient of approximately one, thus, greatly reducing the transmission. Because the EF of graphene can be controlled by applying a gate voltageWhen light from a high-index medium is refracted on the surface to a lower-index medium, total internal reflection will occur if the angle of incidence exceeds a certain limit known as the critical angle. If the low-index medium is very thin and another high-index medium is added to the end of the structure, part of the incident light can tunnel through the low-index medium and transmit to the other end; this is known as FTIREF = 0.9 eV and the thickness of the SiO2 layer tgap, was 10 nm. The spectra were calculated using the rigorous coupled-wave analysis (RCWA) method. As expected, the presence of graphene drastically modified the behavior of the FTIR. At the photon energy Eph of 1.304 eV (λ = 951 nm), reflection is approximately 100% over a wide incident angle range. This is attributed to the near-zero permittivity of graphene at that photon energy when EF = 0.9 eV, which is the result of the light incident at an oblique angle on the epsilon-near-zero (ENZ) material, as previously reportedEph ~ 1.215 eV (corresponding to λ = 1021 nm), transmission is high with little dependence on angle over a wide incident angle range. Eph > 1.304 eV, the real part of the permittivity of graphene is positive and approaches zero at Eph = 1.304 eV. This region is known as the locus of Brewster angle in the  planeEph < 1.215 eV, there is the leaky plasmonic mode due to the two graphene layers. These leaky modes can be excited by the plane wave incident from the high-index medium (Si) because their dispersion curves lie above the light line (black solid line). Using the dispersion curves of the leaky modes, we calculated the Eph that satisfies the phase-matching condition as a function of the angle of incidence. The plots are shown in Eph < 1.215 eV) is very flat near the light line due to the thinness of the graphene layerEph ~ 1.215 eV has little dependence on the angle of incidence over a wide angle range. k for the two leaky modes is near zero, which explains why the optical loss of the GA-FTIR structure is so small, as seen in To understand the behavior of the GA-FTIR, we calculated the dispersion curves of leaky modes in the GA-FTIR structure using the method previously described by J. Hu and C. R. MenyukEph = 1.304 and 1.215 eV are presented in Eph = 1.304 eV, transmission of <1% was obtained for angle of incidence θ > 15°, as demonstrated in Eph = 1.215 eV, transmission >98.5% was observed over an angle range of 0° < θ < 75°, where reflection and absorption were both <1% as shown in To illustrate the unique performance of the GA-FTIR structure for a wide range of angle of incidence, the reflection and transmission spectra for tgap = 40 nm but the same chemical potential used in the previous calculations (EF = 0.9 eV). The behavior of the structure is similar to that with tgap = 10 nm. High reflection occurred at the same photon energy, Eph = 1.304 eV, as that for the structure with tgap = 10 nm because EF was the same. However, the photon energy for high-transmission shifted closer to that of the ENZ point (Eph = 1.283 eV) because of the corresponding shift of the second leaky mode in the dispersion curve, as seen in tgap = 10 and 40 nm and different chemical potentials of graphene but with a fixed angle of incidence of θ = 60°. The choice of the angle of incidence was arbitrary. Changing EF shifts the wavelength  at which the minimum (near-zero) permittivity of graphene occurs and thus, the wavelength of the maximum reflectance (Rmax). Similarly, the wavelength of the edge of the second leaky mode, where the minimum reflectance (Rmin) occurs, also shifts with EF. Therefore, the reflectance of the GA-FTIR at a certain wavelength can be modulated by electrically controlling EF . Rmax tends to increase with EF because the minimum permittivity of graphene tends to zero as EF increases, according to the Kubo formulaRmin tends to decrease as EF increases. Consequently, the modulation depth, defined as Rmax/Rmin, increases as EF increases. For the structure where tgap = 10 nm, shown in Rmax ~ 98.9% and Rmin ≤ 10−5 are achieved at EF = 0.9 eV. In this case, the ratio of the modulation depth to the insertion loss, which is frequently used as a figure of merit (FOM) for modulators, is ~1040. To the best of our knowledge of graphene-based modulators, this FOM is higher than the best value reported in the literature28We investigated the tunability of the spectral behavior of the GA-FTIR. tgap = 40 nm, shown in tgap = 10 nm because of the wider gap. On the other hand, the change in chemical potential (ΔEF) that is required for full modulation of reflectance is smaller because the difference in photon energy between the ENZ point and the second leaky mode edge is smaller, as mentioned above. For example, if the operating wavelength is λ = 951 nm, then ΔEF = 0.07 eV (from 0.9 to 0.97 eV) is needed for the full modulation of reflectance when tgap = 10 nm. Whereas, a smaller ΔEF , e.g., ΔEF = 0.02 eV (from 0.9 to 0.92 eV), is required for the full modulation of reflectance when tgap = 40 nm.Changing the gap size does not fundamentally alter the behavior of the structure. For the structure where 2 layer, and a semi-infinite Si medium on the bottom. EF = 0.9 eV and an operating wavelength of 951 nm. The operating wavelength and EF were chosen to obtain wide-angle high reflectance from the GA-FTIR scheme. As a result, the top slab supports the guided mode, and thus a launched TM wave propagates along the top slab waveguide, resulting in a high-transmission state (on-state). EF = 0.97 eV at the same operating wavelength. In this case, the GA-FTIR scheme offers very low reflectance over a wide range of angle of incidences and, thus, the top slab no longer supports the guided mode, so a launched wave leaks into the bottom Si medium, resulting in a low-transmission state (off-state). For comparison, TM wave propagation in a structure in which the two graphene layers and the SiO2 gap are replaced by a Si medium, as shown in the inset of In this work, we propose a waveguide-type optical modulator based on the GA-FTIR scheme. The wide-angle reflectance modulation properties of the GA-FTIR structure can be utilized to control the existence of a guided mode if one of the two semi-infinite high-index (Si) media is replaced with a slab waveguide, as depicted in 2 layer was 10 nm thick, the Si optical waveguide was 400 nm high and 500 nm wide, and the device was 4 μm long. EF = 0.9 and 0.97 eV, and Building on the results of the 2D optical modulator calculations, we investigated a more practical device structure, shown in EF = 0.524 and 0.54 eV), which are appropriate for an operating wavelength of 1550 nm. The modulation performance of the device depicted in In this work, we proposed a novel scheme to control optical tunneling in a FTIR configuration by introducing two graphene layers doped to have the ENZ effect. In addition to the incident angle independent high reflection due to the ENZ graphene layer, very flat dispersion characteristics of the leaky mode supported by two plasmonic coupled graphene layers provides strong optical tunneling modulation over a wide incident angle range, without requiring any complex resonant structures. The electrical controllability of evanescent coupling strength provided by the proposed scheme, which is unique to this scheme to the best of our knowledge, can enable novel design of optical devices. As a proof-of-concept, we designed a waveguide-type optical modulator of a novel operation principle: transmission modulation depends on the electrically controlled existence of a guided-mode of the waveguide.F . In practice, the breakdown of a gap material, SiO2 in the gate structure limits the maximum achievable value of EF: the carrier density in graphene is related to the chemical potential EF as NG = EF2/(πħ2VF2), where ħ is the reduced Plank constant and VF is the Fermi velocity, and from the simple capacitive model, the electric field in the gap layer is given by qNG/εoεSiO2 , which should be smaller than a certain value to avoid the breakdown of the gap material. This limitation can be overcome with combining chemical doping and electrostatic doping induced by a gate voltage. Chemical doping of up to EF = 2.0 eV have been successfully demonstratedF = 0.9 to 0.97 eV, we can dope graphene chemically up to EF = 0.9 eV and apply varying gate electric field from 0 to ~8.3 MV/cm (assuming VF = 106 m·s−1), which lies within SiO2 breakdown electric field of ~13 MV/cmThe operating wavelength of the proposed scheme can be varied over a wide range by changing the chemical potential of graphene, E2O3 could increase the maximum achievable chemical potential. The relative permittivity of Al2O3 is 1.752 and the breakdown electric field of high quality Al2O3 film formed by the atomic layer deposition can be as high as 30 MV/cm2O3 adopted for the gap material, EF up to ~0.83 eV can be achieved by electrostatic doping alone, induced by a gate voltage, which is much higher than the required value of EF for device operation at λ = 1550 nm and will allow the operating wavelength as short as ~1182 nm.The use of a better insulator with higher permittivity and breakdown electric field such as AlVF =106 m·s−1 used for the calculation in this work is a conservative choice and likely represents the lower limit, corresponding to the case where electron - electron interactions are weakVF ~ 2.49 × 106 m·s−1 and ~1.49 × 106 m·s−1 were achieved with quartz and BN substrates, respectivelyVF ~ 1.19 × 106 m·s−1, electrostatic doping alone can achieve EF ~ 0.54 eV and enable device operation at λ =  1550 nm, avoiding breakdown of SiO2.Another way to achieve higher chemical potential for a given gate voltage is to increase the Fermi velocity: a two fold increase in Fermi velocity would reduce the required gate voltage by a factor of four to achieve the same chemical potential. The value of 2 gap material, the maximum achievable chemical potential by the gate voltage is ~0.45 eV even for the conservative value of Fermi velocity of VF = 106 m·s−1, which is large enough to allow device operation at λ = ~2 μm.As mentioned early, the operating wavelength of the proposed scheme can be varied over a wide range and the allowed shortest operating wavelength is limited only by the maximum achievable chemical potential of graphene. For the GA-FTIR scheme with SiO2 were assumed to be 3.42 and 1.452, respectively, and the permittivity of graphene was calculated using the Kubo formulaVF was 106 m·s−1; and the mobility μ was 1 m2·V−1·s−1.In our numerical analysis, the permittivity of Si and SiOThe spectral response of the scheme and its angle dependency were calculated by using the commercial R-Soft DFMOD RCWA software. The finite element method commercial software COMSOL was used to calculate the frequency domain field distribution of the 2D waveguide type structure. For 3D structure, the FDTD commercial package from Lumerical was used.How to cite this article: Tran, T. Q. et al. Tunable Wide-Angle Tunneling in Graphene-Assisted Frustrated Total Internal Reflection. Sci. Rep.6, 19975; doi: 10.1038/srep19975 (2016).