Scaling graphene down to nanoribbons is a promising route for the implementation of this material into devices. Quantum confinement of charge carriers in such nanostructures, combined with the electric field-induced break of symmetry in AB-stacked bilayer graphene, leads to a band gap wider than that obtained solely by this symmetry breaking. Consequently, the possibility of fabricating AB-stacked bilayer graphene nanoribbons with high precision is very attractive for the purposes of applied and basic science. Here we show a method, which includes a straightforward air annealing, for the preparation of quasi-free-standing AB-bilayer nanoribbons with different widths on SiC(0001). Furthermore, the experiments reveal that the degree of disorder at the edges increases with the width, indicating that the narrower nanoribbons are more ordered in their edge termination. In general, the reported approach is a viable route towards the large-scale fabrication of bilayer graphene nanostructures with tailored dimensions and properties for specific applications. Bilayer graphene nanoribbons are very promising for future nanoelectronics. Here Lopes et al. show a novel approach for the fabrication of quasi-free-standing bilayer graphene nanoribbons on SiC, based on the precise control of the layer-by-layer growth of graphene and a simple annealing step in air. Ion/Ioff ratios compared with monolayer GNR counterparts8et al.The graphene quasi-one-dimensional nanostructure called a graphene nanoribbon (GNR) has attracted much attention owing to its distinct electronic properties, which are strongly dependent on the type of edge  and the width of the ribbon145678Therefore, beyond the conventionally used substrate transfer and patterning of graphene sheets—two technological steps that usually result in structural damage—the direct synthesis of bilayer GNRs on an insulating template, by employing a method that allows exerting control over dimensions and edge structures, would be extremely advantageous for fundamental investigations of the pristine properties of these nanostructures, as well as for applications. In that respect, the epitaxial growth of GNRs on vicinal step facets of SiC(0001) appears as a very promising route1314141517Here we demonstrate a novel approach for the synthesis of isolated bilayer GNRs on SiC(0001) surfaces. It is based on the precise control of the layer-by-layer growth of graphene and a straightforward annealing step in air. With this method it is possible to prepare μm-long bilayer GNRs of different widths (ranging from about 30 to 320 nm) exclusively over step edge regions of a SiC surface. The present results open a new perspective on the tailored fabrication of bilayer graphene nanostructures for different applications in nanoelectronics.R30° periodicity with structure and lattice parameter very similar to those of graphene. It differs from a graphene layer as it is partially bound to the SiC surface through covalent bonding23241718The steps involved in the preparation of bilayer GNRs on SiC(0001) are depicted in 182021After exerting control over the layered graphene growth, the second step that allows the creation of bilayer GNRs consists of a simple annealing in air. We have previously shown that high-quality bilayer graphene can be produced by thermally treating monolayer graphene on SiC(0001) in airn] facet. The discontinuity and even appearance of three layers (at the upper terrace) in the high-resolution GNR image in et al.3027et al.et al.The cross-sectional TEM image in −1—the most prominent features observed in the spectrum of the BL-covered sample)−1. Its low intensity is due to the fact that only a small fraction of the total area illuminated by the laser spot (about 1 μm in diameter) corresponds to monolayer graphene. The remaining area is covered only by the bare BL. For every GNR with width ≥70 nm (width at the upper terrace), synthesized at temperatures ≥1,450 °C for 15 min, the 2D peak can be well fitted by a single Lorentzian, demonstrating that monolayer graphene is formed. For narrower GNRs , a reliable peak fitting could not be done as the signal intensity in those cases was too low. Hence, information about the narrow stripe or isolated patches of few-layer graphene built at the step facet  could not be obtained by this technique. Note that the G peak  cannot be clearly distinguished in the spectra as they lie within the same spectral range as the BL-related signal. The same is valid for the D , which might exist owing to the edges of the monolayer GNRs.Raman spectra taken at the step edge regions of non-annealed GNR samples see  grown fo−1  and the other at ∼1,591 cm−1 , is measured. Such splitting probably occurs due to the existence of different doping levels between the two graphene layers, which leads to the breaking of the inversion-symmetry, resulting in the activation of the antisymmetric mode Eu  has shown to be given by Imin+(Imax–Imin)cos4(θ), where θ is the angle between the polarization of the linearly polarized photons and the ribbon edge  surfaces. By controlling the layered growth of graphene on SiC(0001) stepped surfaces, monolayer GNRs of different widths (ranging from about 30 to 320 nm) were fabricated on top of the BL by varying the growth conditions. The GNRs are formed exclusively over step edge regions, while the BL is extended over the terraces. Strikingly, by annealing the samples in air, the uncovered BL is selectively etched, while the monolayer GNRs are converted into quasi-free-standing bilayer GNRs. The generalization of this GNR synthesis method to pre-patterned SiC surfaces2) cut from a nominally on-axis 2 inch semi-insulating wafer polished on the (0001) face. The samples were chemically cleaned in n-butyl-acetate, acetone and methanol. The following processing steps, namely the H-etching and GNR growth, were both performed in a furnace equipped with an induction heating system. The H-etching treatment was carried out at 1,550 °C for 15 min in a forming gas atmosphere (95 at.% Ar and 5 at.% H) of 900 mbar and a flow rate of 500 s.c.c.m. The epitaxial monolayer GNRs were prepared at the 1,410–1,500 °C temperature range for 5–15 min in a 900 mbar Ar atmosphere with a flow rate of 500 s.c.c.m.Epitaxial monolayer GNRs were grown on 6H-SiC(0001) substrates (0.5 × 1 cmThe choice for growth duration of 15 min was not arbitrary. The synthesis of a complete monolayer graphene on Si(0001), using our experimental set-up, is achieved after a 15 min long process at a temperature of 1,600 °C19−1.Oxygen intercalation/etching was accomplished by thermally treating the samples for 40 min at 600 °C in air with a preceding heating ramp of 50 °C min+ ion laser for excitation. The spatial resolution (∼1 μm) allowed performing measurements of a single step edge, since the typical terrace width is larger than 1 μm, as determined by AFM. Spectra of all samples were obtained under the same experimental conditions (integration time and incident laser power) to allow a direct comparison of the absolute intensity between them. Furthermore, the Raman signal of a bare SiC substrate was subtracted from all original data. Raman mapping was taken from an 8 × 13 μm2 area around one step edge. Polarized Raman measurements were carried out by coupling a half-wave plate to the microscopy optics, which allows rotating the polarization of the incident and scattered light, but keeping them parallel to each other. An analyser was employed to ensure that the dependence of the spectrometer efficiency on the light polarization does not affect the experiments. Measurements were performed as a function of the angle between the polarization of the linearly polarized laser and the longitudinal direction of the ribbons. In all cases the absolute position of the Raman peaks was corrected by using the emission lines of a Neon lamp.Raman measurements were performed before and after oxygen intercalation using the 482.5 nm-line of a KrAFM measurements were taken using the tapping mode to obtain the height and phase contrast images. The choice for phase contrast is justified as this mode gives information about regions of the SiC surface covered by different numbers of graphene layers.The TEM samples used for the analysis were prepared in cross-sectional geometry using standard procedure of mechanical grinding and dimpling. Afterwards they have been thinned by argon ion beam milling with an energy of 4 keV under an incident angle of 4° using a Gatan precision ion polishing system. The TEM data were collected in a JEM-2100F operated at 80 kV equipped with a field emission gun. Additional TEM images were taken with a JEM-3010 operated at 200 kV.How to cite this article: Oliveira Jr., M.H. et al. Synthesis of quasi-free-standing bilayer graphene nanoribbons on SiC surfaces. Nat. Commun. 6:7632 doi: 10.1038/ncomms8632 (2015).Supplementary Figures 1-3


In this study, we identified bacteria using DMS as a carbon and energy source in terrestrial environments, an agricultural soil and a lake sediment, by DNA stable isotope probing (SIP). Microbial communities involved in DMS degradation were analysed by denaturing gradient gel electrophoresis, high-throughput sequencing of SIP gradient fractions and metagenomic sequencing of phi29-amplified community DNA. Labelling patterns of time course SIP experiments identified members of the Methylophilaceae family, not previously implicated in DMS degradation, as dominant DMS-degrading populations in soil and lake sediment. Thiobacillus spp. were also detected in 13C-DNA from SIP incubations. Metagenomic sequencing also suggested involvement of Methylophilaceae in DMS degradation and further indicated shifts in the functional profile of the DMS-assimilating communities in line with methylotrophy and oxidation of inorganic sulphur compounds. Overall, these data suggest that unlike in the marine environment where gammaproteobacterial populations were identified by SIP as DMS degraders, betaproteobacterial Methylophilaceae may have a key role in DMS cycling in terrestrial environments.Dimethylsulphide (DMS) has an important role in the global sulphur cycle and atmospheric chemistry. Microorganisms using DMS as sole carbon, sulphur or energy source, contribute to the cycling of DMS in a wide variety of ecosystems. The diversity of microbial populations degrading DMS in terrestrial environments is poorly understood. Based on cultivation studies, a wide range of bacteria isolated from terrestrial ecosystems were shown to be able to degrade DMS, yet it remains unknown whether any of these have important roles  Methylated sulphur compounds are significant intermediates in the global biogeochemical sulphur cycle owing to their role in transferring sulphur between the atmosphere, terrestrial and marine ecosystems . Its oxiM, similar to, if not higher than typical DMS concentrations in the upper marine water column  is the major source for DMS , degradaHyphomicrobium and Thiobacillus spp. have been described, but whether these represent the dominant active DMS-degrading populations in terrestrial environments remains unknown  into their DNA  allows relating the function of uncultivated microorganisms to their identities through incorporation of stable isotope labels  using an ethanol-sterilised trowel on 20 January 2010. Hunts Mill is an organic sandy loam soil with 73% sand, 12% silt and 14% clay  fitted with a 30 m × 0.32 mm column (DB-1) and a flame ionisation detector was used to monitor the concentration of DMS in the headspace of the incubation vials. Helium was used as the carrier gas at a temperature of 200 °C. Similarly, an experiment was carried out to assess [12C2]- and [13C2]-DMS uptake by the soil samples. Triplicate microcosms were set up using 1 g of soil sample and amended with [13C2]- or [12C2]-DMS as described above. 13C-labelled DMS was synthesised as described previously  of DMS-degrading bacteria was carried out according to −1. In all, 15–20 μl aliquots of DNA (3 μg of DNA) were subjected to ultracentrifugation and DNA was recovered via gradient fractionation as described previously  according to the manufacturer's instructions. DNA concentrations were estimated using a spectrophotometer  and found to be in a range of 150–200 ng μlPyrosequencing of the hypervariable regions (V1 and V2) of bacterial 16S rRNA genes was performed using a Roche 454 GS Junior  at Micropathology Ltd. . The manufacturer's Universal Tailed Amplicon Sequencing protocol was applied with primers 27 F  and 519 13C-labelled DNA from ‘heavy' and ‘light' fractions were sequenced using the Illumina HiSeq2000 technology  which generated 8–12 Gb of 100 bp paired-end sequence data per sample. Read data have been submitted to the EBI short read archive under study accession PRJEB6310.Multiple displacement amplification was carried out to obtain micrograms of DNA from the ‘heavy' (fraction 7) and ‘light' (fraction 12) DNA of the second time-point SIP incubations. For this purpose, 100 times diluted templates were amplified using the Illustra GenomiPhi V2 DNA amplification kit  according to the manufacturer's instructions. PCR-DGGE analysis of phi29 amplified DNA did not reveal major changes in community fingerprints due to multiple displacement amplification . Metagende novo metagenome assembler . Analysis of variance and least significant difference (LSD) tests were further applied to the key OTUs identified by PCA to find significant differences at the 5% level. Metagenomics profiles obtained by MG-RAST  sediment day−1. Therefore, SIP incubations were set up using the samples from the top layers (0–2.5 cm) of the lake sediment.The top two layers of the lake sediment were found to be the most active in terms of [12C2]- and [13C2]-DMS into soil was also assessed using a total amount of 15 μmoles of DMS amended to the incubations in 20 days. DGGE fingerprinting of the fractions (12C- and 13C- labelled DMS. A distinct pattern was observed in the ‘heavy' part of the [13C2]-DMS fractions (fractions 7 and 8) when compared with the ‘light' part of the same microcosm (fractions 11 and 12) representing the rest of the bacterial community that did not assimilate the [13C2]-DMS. Three dominant bands  of all samples with percentages between 87.5 and 99.5. At the family level, members of the Methylophilaceae were prevalent across both soil and lake sediment 13C-labelled ‘heavy' DNA samples.The ‘heavy' and ‘light' SIP gradient fractions from the three time points were further analysed by pyrosequencing of the 16S rRNA genes. A total of 31 401 quality-filtered, chimera-free sequences were obtained from 39 samples. These sequences were assigned to 9080 distinct OTUs at 97% identity (average 805±327 reads per sample with an average length of 481 bp). Taxonomic analysis revealed distinct community profiles in the ‘heavy' and ‘light' DNA fractions of the soil and lake sediment SIP samples . The cha samples . The fire points . BetaproMethylophilaceae comprised an average of 89.4% (±1.5%) of the sequences from the 13C-labelled ‘heavy' fractions in the first time-point samples , decreasing to 63.2% (±3.7%) and 40.5% (±10.4%) in the second  and third time-point samples , respectively. The mean Methylophilaceae read abundances in the 13C-labelled ‘heavy' fractions were significantly higher than those in the 13C-labelled ‘light' fractions at each time point , the pairwise difference between the Methylophilaceae read abundances was even more pronounced . There was a concomitant increase in the mean read abundance of Hydrogenophilaceae in the 13C-labelled ‘heavy' fractions from 0.2% in the first time point (day 11) to 28.4% (±4.1%) and 52.2% (±12.4%) in the second (day 17) and third time points (day 20), respectively. Similarly, the mean read abundances of Hydrogenophilaceae in the 13C-labelled ‘heavy' fractions from the second and third time points were significantly higher than that of the 13C-labelled ‘light' fractions  to the discrimination of the samples by treatment only . Other Betaproteobacteria also increased in the second and third time point ‘heavy' fractions  compared with the first time point (3.2±0.8%). Likewise, there was a significant pairwise difference between 13C-labelled ‘heavy' and ‘light' fractions  soil samples. Their relative abundances decreased while Thiobacillus (Hydrogenophilaceae) abundance increased in the second and third time-point  samples . Their read abundance increased to an average of 87.6% in the second time-point replicates  and decreased to 80.9% in the third time-point sample . The pairwise differences between the Methylophilaceae read abundances in the 13C-labelled ‘heavy' and ‘light' fractions at each time point were significant . Methylotenera remained the most dominant genus in the lake sediment samples from the three time points .The lake sediment DMS-SIP e points . While T13C2]-DMS soil incubation, respectively  comprised 13% and 80.6% of the soil ‘heavy' and ‘light' fractions, respectively.Average contig lengths were 1279 and 1728 bp for the ‘light' and ‘heavy' fractions of the [ectively . Based oProteobacteria and Bacteroidetes predominated the soil ‘heavy' and ‘light' fractions with 45.8% and 32.8%, respectively. Sphingobacteria dominated the sequences among the Bacteroidetes in the ‘light' fraction (34.7%) while Betaproteobacteria dominated in the ‘heavy' fraction (60.9%). With increasing taxonomic resolution at the family level, unclassified Sphingobacteriales was the most dominant group in the ‘light' metagenome with 5.0% abundance. However, in the ‘heavy' metagenome, Methylophilaceae were the most abundant family (11.1%). According to STAMP comparison, the Methylophilaceae family was also indicated as the most overrepresented family in the ‘heavy' fraction .fraction . STAMP ctagenome . BacilluArchaea and Eukarya comprised 12.8, 2.4 and 2.4% of the ‘heavy' metagenome, and 12.8%, 3.9% and 1.0%, in the ‘light' metagenome, respectively. Kraken analysis showed that of the classified reads in the ‘heavy' fraction of the lake sediment SIP, 71.7% were bacterial, 1.68% were archaeal and 3.3% viral DNA, while in the ‘light' fraction of lake sediment SIP 46.13% were bacterial, 6.6% were archaeal and 4.2% were of viral origin. In all, 23.2% and 43% were Eukaryotic sequences  in the ‘heavy' and ‘light' fractions, respectively.For the lake sediment metagenomes, the average contig lengths were 1230 and 1286 bp for the ‘light' and ‘heavy' fractions, respectively . SimilarBacteroidetes was the most dominant phylum  followed by Proteobacteria which increased from 24.5% in the ‘light' fraction to 38.4% in the ‘heavy' fraction. Within Proteobacteria, the most abundant class in the ‘light' fraction was Gammaproteobacteria (39.8%) whereas Betaproteobacteria was the most abundant in the ‘heavy' fraction with 30.3%.Taxonomic assignments of the lake metagenomic sequences showed differences compared with the soil metagenome. In both fractions, Methylophilaceae, Hyphomicrobiaceae and Bdellovibrionaceae as the most overrepresented taxa  was lower than that derived by amplicon sequencing. Similarly, Kraken analysis estimated the Methylophilaceae fractional abundance to be 45.5% and no marked increase of Hydrogenophilaceae in the lake sediment ‘heavy' fraction . However, the abundance of the sequences representing the ribulose-monophosphate pathway significantly increased from 0.30% to 1.3% in the ‘heavy' fraction . Among the CO2 fixation genes, sequences from the ‘Calvin-Benson cycle' were the most dominant in both fractions (36.8% and 43.9%). However, the number of genes for ‘CO2 uptake' and ‘carboxysome' increased significantly in the ‘heavy' fraction . The abundance of genes related to sulphur metabolism was also investigated .Analysis of the MG-RAST subsystems category ‘carbohydrate metabolism' in soil fractions revealed the over-representation of ‘central carbohydrate metabolism', ‘COtagenome . Within stigated . In comp2 fixation' were significantly more abundant in the ‘heavy' metagenome  was not found in metagenomes. Similarly, the relative abundance per recA copy of several genes encoding enzymes involved in inorganic sulphur compound oxidation/assimilation was higher in the ‘heavy' fraction, including flavocytochrome c sulphide dehydrogenase, dissimilatory sulphite reductase, adenylylsulphate reductase and sulphate adenylyltransferase. Similar changes in gene frequency were detected in the metagenome of the ‘heavy' fraction of the lake sediment microcosm  in the ‘heavy' soil metagenome were exclusively affiliated with Methylophilaceae and Thiobacillus, but originated from a wide diversity of bacteria in the ‘light' soil metagenome. In the lake sediment ‘heavy' metagenome, recA sequences were mainly originating from Methylotenera, Bdellovibrio and other bacteria while the diversity of recA in the ‘light' fraction reflected a more diverse community  released by Thiobacillus spp. High abundance of Methylophilaceae representatives might be due to the ability of some strains to use formaldehyde as a carbon source  was substantial compared with that of the cooperating Methylophilaceae populations  suggested that these methylotrophic Betaproteobacteria are metabolically versatile  but also implicated a group of methylotrophs not previously realised as DMS degraders, uncultured Methylophilaceae. These findings reemphasise the significance of members of the Methylophilaceae as widely distributed and versatile methylotrophs (The combination of SIP, 454 sequencing of gradient fractions and metagenomic sequencing proved useful for identifying microbial populations actively involved in DMS cycling in two terrestrial environments. It detected known DMS degraders (lotrophs , which h


Impaired regulatory T cell (Treg) function is thought to contribute to ongoing inflammatory responses in sarcoidosis, but underlying mechanisms remain unclear. Moreover, it is not known if increased apoptotic susceptibility of Tregs may contribute to an impaired immunosuppressive function in sarcoidosis. Therefore, the aim of this study is to analyze proportions, phenotype, survival, and apoptotic susceptibility of Tregs in sarcoidosis.n = 58) were included at time of diagnosis. Tregs were analyzed in broncho-alveolar lavage fluid and peripheral blood of patients and healthy controls (HC).Patients with pulmonary sarcoidosis  contains supplementary material, which is available to authorized users. Sarcoidosis is a multisystem granulomatous disorder of unknown cause, often affecting the lungs . The disFailure of immune regulatory mechanisms to limit duration of inflammation has been suggested to contribute to persisting granulomatous responses in sarcoidosis , and mayTregs are an indispensable subset of T cells with strong immunosuppressive capacities on a wide range of immune cells, including Th cells, B cells, and antigen presenting cells . They haSee for review [The role of Tregs in the pathogenesis of sarcoidosis remains controversial. An impaired immunosuppressive function of sarcoidosis peripheral blood (PB) Tregs has been reported consistently , 16–18. r review :), it rer review .+FoxP3high) proliferating Tregs are found [+FoxP3high Tregs are described to be both more suppressive and more sensitive towards apoptosis in healthy individuals [Pro- and anti-apoptotic pathways play an important role in Treg homeostasis . Intrigure found . The sigividuals . Althougividuals , it remaTherefore, the aim of this study is to analyze proportions, phenotype, survival, and apoptotic susceptibility of sarcoidosis Tregs. Our results demonstrate that in patients with active pulmonary sarcoidosis, Tregs show impaired survival and enhanced apoptotic susceptibility towards CD95L.Patients with pulmonary sarcoidosis were included at time of diagnosis. The diagnosis of sarcoidosis was made conform the guidelines of the ATS/ERS/WASOG .Exclusion criteria were use of immunomodulatory medication 3 months prior to study inclusion; respiratory tract infection 4 weeks prior to study inclusion; concomitant pulmonary disease (including chronic obstructive pulmonary disorder and asthma), autoimmune diseases, malignancies, human immunodeficiency virus seropositivity, pregnancy, and allergies.For this study, in total 58 newly diagnosed sarcoidosis patients donated PB and/or BALF. Due to limitations in the number of cells isolated per patient, we were not able to perform all experiments on all patients. Furthermore, 47 healthy controls donated PB and 5 healthy controls underwent bronchoscopy with BAL for this study.The Medical Ethical Committee of the Erasmus MC Rotterdam approved this study. Written informed consent was obtained from every participant before study inclusion. Further subject characteristics are shown in Additional file Bronchoscopy with BAL and mucosal biopsy was performed as previously described . BALF ceBALF cells and PBMCs were stained for extra- and intracellular markers using the following antibodies: CD3-APC-eFluor780(SK7), CD4-AF700(OKT4), CD45RO-FITC(UCHl-1), CD95-APC(DX2), FoxP3-APC(PCH101) (eBiosciences) and CD25-PE(M-A251), CD25-PE-Cy7(M-A251), CD127-V450(hIL7R-M21) anti-CTLA-4-BV421(BNI3) (BD biosciences) and CD45RA-PE-Texas Red(MEM-56) (Invitrogen). Fixable Aqua Dead Cell Stain kit for 405 nm  was used as live-dead marker. Cells were measured on a Flow cytometer LSRII (BD Biosciences).+CD25+Regulatory T Cell Isolation Kit . Th cells were labeled with carboxyfluorescein succinimidyl ester  according to manufacturer’s protocol. CFSE-labeled Th cells were stimulated with Anti-Biotin MACSiBead™ Particles  and co-cultured with unlabeled Tregs. Th cell division was quantified based on serial halving of CFSE intensity, using algorithms provided by FlowJo software (Treestar).Th cells and Tregs were sorted from PBMCs using a human CD4Cytokine measurements in supernatants of co-cultures were performed using the Bio-Plex Pro Human Cytokine 17-plex Panel (Biorad), run on a Luminex 100 System (Luminex Corporation), according to manufacturer’s protocol.low-intCD127highTh cells and CD25int-highCD127lowTregs were isolated from PBMCs using the BD FACSAria Cell Sorter (BD Biosciences) and cultured with 20 ng/ml recombinant human IL-2 . Survival was determined with DAPI Nucleic Acid Stain (Life Technologies)-negative cells. To measure Treg survival and apoptosis in co-culture with Th cells, CD4+ T cells were isolated from PBMCs using the human CD4+ T cell isolation kit , cultured with 20 ng/ml hIL-2 and examined using the FITC Annexin V Apoptosis Detection Kit I (BD biosciences). To assess Treg apoptotic susceptibility, CD4+ T cells were cultured either with 20 ng/ml hIL-2 or with hIL-2 and 500 ng/ml soluble CD95L .CD25p < 0.05 was considered statistically significant.Comparisons were performed using a Mann–Whitney U test or Wilcoxon matched pairs test. Correlations were analyzed using Spearman’s rank-order correlation test. p-values were two sided, and analyses were performed using IBM SPSS Statistics 21. int-highFoxP3high Tregs in BALF and PB of active sarcoidosis patients and healthy controls by flow cytometry. No evidence was found for a relative deficit of Tregs in BALF of untreated patients with active sarcoidosis compared with healthy control BALF , CD25 and cytotoxic T lymphocyte antigen 4 (CTLA4)  by flow+ Treg suppressive capacity on autologous Th proliferation and cytokine production was significantly less in sarcoidosis compared with healthy controls   Fig. . Importants Fig. .+ Tregs were detected in sarcoidosis. Importantly, these activated CD45RO+ Tregs highly express CD95 in patients compared with controls, suggesting altered apoptotic susceptibility.Altogether, increased proportions of circulating, activated CD45ROlow-intCD127high Th cells and CD25int-highCD127low Tregs from PB of patients and healthy controls. Proportions of CD25int-highFoxP3high Tregs, as measured in the PB of our study subjects . These data underlined previously published data that CD4+CD25int-highCD127low (sortable) can be used as surrogate marker for CD4+CD25int-highFoxP3high Treg isolation and functional studies [We questioned whether sarcoidosis-derived circulating Tregs would display altered survival. To investigate survival of sarcoidosis Th cells and Tregs, we isolated CD25 studies .upper plot)). Moreover, increased proportions of apoptotic Tregs  were observed in sarcoidosis ).Isolated cells were cultured with IL-2 and spontaneous survival was measured at 72 hours. Both healthy- and sarcoidosis-derived Tregs showed decreased survival compared with isolated Th cells , indicating that CTLA4 expression levels are differentially regulated between compartments. Thus, despite adequate Treg proportions and expression of FoxP3-downstream effector molecules, sarcoidosis-derived PB Tregs fail to suppress autologous Th cell responses.To the best of our knowledge, it thus far remained unclear what mechanism(s) underlies Treg dysfunction in PB of active sarcoidosis patients. We did not find differences in FoxP3 expression or diminished expression of its downstream molecules CD25 or CTLA4 in circulating Tregs of sarcoidosis patients. Interestingly, we found that Tregs in lymph nodes from sarcoidosis patients have reduced CTLA4 expression compared with controls  sarcoidosis patients.


Changes in relative humidity, along with other meteorological factors, accompany ongoing climate change and play a significant role in weather-related health outcomes, particularly among children. The purpose of this review is to improve our understanding of the relationship between ambient humidity and child health, and to propose directions for future research.A comprehensive search of electronic databases  and review of reference lists, to supplement relevant studies, were conducted in March 2013. All identified records were selected based on explicit inclusion criteria. We extracted data from the included studies using a pre-designed data extraction form, and then performed a quality assessment. Various heterogeneities precluded a formal quantitative meta-analysis, therefore, evidence was compiled using descriptive summaries.Out of a total of 3797 identified records, 37 papers were selected for inclusion in this review. Among the 37 studies, 35% were focused on allergic diseases and 32% on respiratory system diseases. Quality assessment revealed 78% of the studies had reporting quality scores above 70%, and all findings demonstrated that ambient humidity generally plays an important role in the incidence and prevalence of climate-sensitive diseases among children.With climate change, there is a significant impact of ambient humidity on child health, especially for climate-sensitive infectious diseases, diarrhoeal diseases, respiratory system diseases, and pediatric allergic diseases. However, some inconsistencies in the direction and magnitude of the effects are observed. Their relatively immature immune systems increase the risk of serious consequences from a variety of infectious diseases However, although pediatric health care professionals have written about the effects of climate change on child health, relatively limited research has been conducted specifically on the impact of ambient humidity on child health. To our knowledge, almost all of the studies investigating the association between climate change and child health mainly focus on temperature or rainfall, while ambient humidity is usually considered as a dispensable and alternative variable.Additionally, to date, no systematic review has specifically focused on the relationship between ambient humidity and child health. In order to fill this knowledge gap, the primary objective of this review is to improve our understanding of how humidity, both directly and indirectly, can influence child health and to promote additional research in this field by presenting and summarizing the findings of all relevant studies related to the impact of ambient humidity on child health.Relevant peer-reviewed journal databases , OvidSP and EBSCO host) were searched using the following MeSH  terms and keywords: “climate change”, “humidity”, “wet”, “dew point”, “child”, “infant”, “adolescent” and “pediatric”. The search terms and keywords were adapted to suit the requirements of each database to facilitate a comprehensive search. After selection of the full text, the references of the identified articles were reviewed and the necessary hand search was performed to recover potentially relevant studies not included in the major databases.the Convention on the Rights of the Child proclaimed by the United Nations, we defined children as the group of people 0–18 years of age The search sought to identify journal articles published in English up to March 2013. In accordance with The following eligibility criteria were used in this study:Types of studies: the present review only included peer-reviewed original articles. Thus, reviews, books, meta-analyses and conference abstracts were excluded.Target population: only the studies involving children between 0–18 years of age were considered.Research factors: only studies in which humidity was one of the exposure indicators of interest were considered.P-values or 95% confidence intervals (CIs) to have been reported.Outcome measures: the reported results of the included studies should have included at least one quantitative effect estimate associated with humidity. An outcome measure was reported as either: percent change, regression coefficient, correlation coefficient, relative risk or odds ratio. We also required Three of the authors  independently assessed the potential relevance of all publications, identified during the database search, based on the information provided in the titles and abstracts. When reviewers disagreed, a discussion was held to obtain consensus. After screening by titles and abstracts, the full texts of the remaining articles were then obtained and critically reviewed by the first author.We extracted data, from the final selected references, using a pre-designed data extraction form. The quantitative estimates of each study that met the eligibility criteria were carefully retrieved and the following key characteristics were captured: study region, target population, study design, humidity exposure variable, health outcomes and adjustment of confounders. All data extraction was performed by the first author (Gao) and cross-checked by another researcher (Sun). Any errors identified were corrected through discussion among all authors.To date, the use of quality assessment tools to appraise observational studies included in systematic reviews is less well established than in reviews of randomized controlled trials, and there is no existing generally accepted quality assessment tool for observational and cross-sectional studies P-value was given. As no accepted gold standard exists for judging the quality of observational studies, in the present review we did not exclude any identified studies based on the quality assessment, which used an adapted version of the STROBE checklist.As in a previous systematic review, an adapted version of the STROBE checklist Table 1 Due to heterogeneity in the target populations, outcome measures and design of studies, we did not conduct an overall summary estimate . Instead, we summarized the evidence in a descriptive synthesis.A total of 3787 records were identified from the electronic database searches, and 10 additional studies were retrieved by reviewing references. After screening by titles and abstracts, 76 articles were selected for examination of the full text. Finally, 37 papers remained for inclusion in this systematic review Fig. 1. Studies found that ambient humidity plays an important role in the occurrence and prevalence of pediatric infectious diseases. A Brazilian study found that among the children hospitalized with community-acquired pneumonia, overall viral and Chlamydia trachomatis infections correlated with monthly mean relative humidity In Japan, one study indicated that for each 1% increase in the weekly mean relative humidity, the number of mumps cases increased by 1.4% (95% CI: 0.5–2.4%) among children P = 0.004) Existing studies suggest that ambient humidity has a significant impact on malaria risk and diarrhoeal diseases among children. A prior study found that the clinical malaria risk increased non-linearly with the increase in mean relative humidity of the previous month Many pediatric respiratory system diseases are sensitive to relative humidity. An Argentinean study reported that respiratory syncytial virus (RSV) and influenza virus A had a positive correlation with mean monthly relative humidity among children hospitalized due to acute lower respiratory viral infections In Germany, a study found that human rhinovirus positively correlated with relative humidity among children who were hospitalized with acute respiratory tract infections (ARIs) Childhood asthma and other pediatric allergic diseases are important adverse consequences of humidity. In Greece, the results of a study showed that relative humidity was an implicated meteorological variable for younger asthmatic children In one Turkish study of 21 bronchial asthma children, results indicated that increasing relative humidity leads to an increase in total daily complaints of asthma patients On the contrary, a study in Taiwan indicated that relative humidity was negatively correlated with asthma admission rates of pre-school and school-age children Dew point, another measure of ambient humidity, is the temperature at which water vapor in the air becomes saturated and water droplets begin to form. To date, relative humidity and dew point are two widely used indicators for the amount of moisture in air. Accordingly, some other studies also investigated the association between ambient humidity and child health by choosing dew point as the indicator. A negative association between the peak week of child-related influenza visits and dew point was reported by Charland and colleagues, although the relationship was not significant In addition, ambient humidity may also play a role in other diseases among children. For instance, the results of an American study showed a positive association between relative humidity and the likelihood of headache among children The purpose of the present systematic review was to summarize key findings related to the effects of ambient humidity on child health, and identify the relationship between humidity exposure and common pediatric diseases. The systematic review of the 37 included publications performed herein demonstrates that ambient humidity generally plays a significant role in the incidence and prevalence of childhood climate-sensitive diseases, especially for gastrointestinal, respiratory system, and allergic diseases. Considering the expected continuation of climate change, the adverse influence of ambient humidity on children may become increasingly serious. However, some inconsistencies or even contradicting directions and magnitudes of the effects were also observed.Almost 73% of the final included studies were conducted in high-income countries or regions, indicating that more attention should be paid to the developing countries of Asia and Africa. Because of low-income, a large population and poor infrastructure, people in these countries are usually more vulnerable to climate-sensitive diseases. The results of the quality assessment indicate that the combined application of advanced statistical methods, such as time-series analysis combined with Poisson regression, usually tends to provide more reliable results , since adjustments could be made for potential confounders, including long-term trends, seasonality and air pollution.Because of their physiologic, metabolic and cognitive immaturity, and greater sensitivity to certain exposures, children are often the most vulnerable to adverse health effects from environmental hazards Findings from the present review suggest that the occurrence and prevalence of childhood climate-sensitive infectious diseases are significantly associated with ambient humidity. It is predicted that climate change will lead to more frequent and abundant rainfall, increasing temperature and higher concentrations of water vapor, which will cause favorable conditions of ambient humidity and concomitantly favor increases in the occurrence and development of infectious diseases I. ricinus depends on the relative humidity, and below a relative humidity threshold of 86%, the tick dehydrates and cannot continuously seek a host Ambient humidity can affect tick abundance and seasonal tick activity. It has been reported that the host seeking activity of For gastrointestinal infectious agents such as rotaviruses and EV71, changes in ambient humidity have been reported to facilitate viral resistance, thereby enhancing the virulence and increasing the efficiency of transmission through contaminated air, fomites and environmental surfaces Anopheles vector abundance and the extrinsic cycles that the parasites perform inside mosquitoes. Thus, meteorological factors may affect malaria incidence and constitute driving forces for malaria epidemics Malaria is a climate-sensitive vector-borne illness. Meteorological factors  have considerable impact on Many studies demonstrate the effects of relative humidity on pediatric respiratory tract infections. In tropical and subtropical areas , it has been suggested that high humidity and temperature prolong the activity and stability of microorganisms in large-particle aerosols, and permit year-round transmission of the microorganisms The possible effects of ambient humidity on childhood asthma and other allergic diseases are of particular concern due to this population's greater susceptibility. Meteorological factors  can influence both biological and chemical components of air and might induce negative effects on respiratory allergic diseases. With climate change, ambient humidity is likely to impact asthma, hay fever and other allergic diseases via its influence on the concentration and distribution of pollens, mould spores, house dust mites, and other aeroallergens However, in this review, some studies also report a negative association between ambient humidity and pediatric allergic diseases As mentioned above, different studies may present inconsistencies in the direction and magnitude of the effects of ambient humidity, with other studies even concluding that the occurrence of relevant climate-sensitive diseases appears to be unrelated to humidity This systematic review begins to address the current knowledge gap in the relationship between ambient humidity and child health. This is the first attempt to synthesize, using a systematic approach, the existing evidence of humidity-related health outcomes, across different countries, among children between 0–18 years of age. According to Despite targeting the reporting quality of observational studies, many items of the STROBE checklist were no doubt selected due to evidence of association with susceptibility to bias. Thus experts have suggested that the STROBE statement provides a suitable starting point for development of a quality assessment tool However, regarding the results of this systematic review, some limitations should be acknowledged. First, this review is limited by its emphasis on papers published in English in peer-reviewed journal databases, so we may have missed potentially useful studies. Second, despite the highly sensitive and comprehensive nature of the search strategy employed, it is unlikely that all existing studies have been identified, and publication bias cannot be entirely excluded. Third, the adapted version of the STROBE checklist was initially implemented to describe the reporting quality of weather-related studies, so the validity and reliability of the checklist for our purposes have not been completely tested and formally evaluated. However, in this review, the tool was used only to describe the reporting quality, and the quality scores were not included as part of the relevance criteria. Fourth, it has been reported that temperature and relative humidity are not independent of one another, so the effects of relative humidity may be confounded by temperature. However, in our review, the majority of studies adjusted for potential confounding factors (e.g. temperature) when analyzing the impact of humidity on child health, and studies that conducted no adjustments were given lower quality ratings. Additionally, relative humidity is a popular indicator that has been widely used in current research and meteorological monitoring. Therefore, it is helpful and of practical significance to choose relative humidity rather than absolute humidity or water vapor pressure as the exposure variable. Finally, the data extraction and quality assessment were not performed independently by more than one reviewer, which may have led to subjective errors and introduced bias. However, in the present review, due to the explicit inclusion criteria, specially pre-designed data extraction form, definite exposure variable and health outcome, and the adapted quality assessment tool, the authors were able to ensure the integrity of the review, and to be unbiased during the conduction of assessment.In summary, this study demonstrates that due to various inherent and unique characteristics, children are often susceptible to ambient humidity. We find that with climate change, there is a significant impact of humidity on child health, especially for climate-sensitive infectious diseases, diarrhoeal diseases, respiratory system diseases, and pediatric allergic diseases. However, the humidity-related effects on child health are various and complex. Ambient humidity continues to impact children unequally and in different ways, with impacts ranging from direct and indirect, and negative and positive. In this review we find that the influence of humidity on child health involves heterogeneous and even converse effects among different study regions, research designs, health outcomes, adjustments of potential confounding factors , and subgroups of children, which suggests that further studies focusing on the association between ambient humidity and child health are needed to deal with the aforementioned problems.Current increasing interest in the relationship between climate change and child health may provide an ideal opportunity to employ various epidemiological and statistical methods to further explore the impact of ambient humidity on child health. Such research and epidemiologic evidence can inform the local health officials and policy-makers to make and implement necessary preparations (mitigation and adaptation), ultimately contributing to better health outcomes for a humidity-impacted population.S1 ChecklistPRISMA checklist.(DOCX)Click here for additional data file.


We develop an orbital-dependent potential to describe electron-hole interaction in materials with structural 2D character, i.e. quasi-2D materials. The modulated orbital-dependent potentials are also constructed with non-local screening, multi-layer screening, and finite gap due to the coupling with substrates. We apply the excitonic Hamiltonian in coordinate-space with developed effective electron-hole interacting potentials to compute excitons’ binding strength at M (π band) and Γ (σ band) points in graphene and its associated multi-layer forms. The orbital-dependent potential provides a range-separated property for regulating both long- and short-range interactions. This accounts for the existence of the resonant π exciton in single- and bi-layer graphenes. The remarkable strong electron-hole interaction in σ orbitals plays a decisive role in the existence of σ exciton in graphene stack at room temperature. The interplay between gap-opening and screening from substrates shed a light on the weak dependence of σ exciton binding energy on the thickness of graphene stacks. Moreover, the analysis of non-hydrogenic exciton spectrum in quasi-2D systems clearly demonstrates the remarkable comparable contribution of orbital dependent potential with respect to non-local screening process. The understanding of orbital-dependent potential developed in this work is potentially applicable for a wide range of materials with low dimension. Thus, despite extensive studies in graphene’s optical properties, the aforementioned controversy invokes more heuristic analysis to gain better understanding of exciton in graphene, particularly by detail account for electron-hole (e-h) interaction in the context of quasi-2D dimension, which represents the 2D nature in structure together with inevitable spreading of wave functions perpendicular to the plane. In this work, to account for both π and σ excitons, we develop the orbital-dependent electron-hole interaction in graphene and its associated multi-layer structures. The unscreened orbital dependent interactions in graphene, with clear quasi-2D character, deviate from Coulomb k∙p effective mass method, we compute the binding strength of excitons from π bands at M point and σ bands at Γ point in graphene. Furthermore, we demonstrate the existence of σ exciton in thicker multi-layer graphene stack, and address the coupling between graphene and substrate to be important for clarifying the controversy between the measured little shift in σ exciton peak position of samples on the substrate and computed player-dependence peak shift of σ exciton in the suspended graphene structures. Finally, we perform systematic analysis to scrutinize the contributions of orbital dependent potential and non-local screening to the non-hydrogenic exciton spectrum which appears to be quite general for materials with 2D structures.The optical properties of low-dimensional spTo construct the coordinate-space excitonic Hamiltonian including both effective-mass kinetic term and effective potential, we represent the quasi-particle energy by the sum of k-dependent single-particle kinetic terms and the scissor operator approximation based constant band gap correction. The interaction kernel is approximated by the dominate Coulomb coupling term. Thus, a stationary exciton is the eigenstate of the following BSE in momentum spacek∙p effective mass approximationcgv, gc/gv being the conduction/valence bands degeneracy . The Fourier transform of Eq.Here ⊗ denotes the Kronecker product, 2with So for small k and k′, we haveIn 3D case, which reminds us of the Coulomb terms between sub-bands in quantum wells Veh, we first evaluate the function W(q) in matrix elements given by Eq. q increasesW(q) can be nicely approximated by an orbital-dependent exponential function WOD(q).To determineWOD(q) agrees with the integral W(q) to the first order of q, which is a good approximation at small q as demonstrated in where The above Eq. WOD(q) is a soft-core Coulomb potentialIf the dielectric function is local and q-independent VOD contains the clear physical origin with explicit orbital-dependence beyond the atomic limit, and a well-defined quantity, VOD approaches a finite value at small distance, resulting from the finite extension of wave function perpendicular to the 2D atomic plane. It differs from both bare Coulomb between two point charges, which diverges as r−1 at small distance, and screened Coulomb potential in 2D system which diverges logarithmically35VOD into the coordinate space, we obtained the e-h interaction between σ electrons and between π electrons, as presented in This form coincides with the approximated one observed by Ohno when evaluating two-centre Coulomb integrals between atomic orbitals in Pariser-Parr-Pople method132et al.VOD-NLS approaches a finite valueThe dielectric function has the non-local form D0 and stronger non-local polarizability α leads to a weaker e-h potential, esp. at small distance. Hence, the modified orbital-dependent parameter in Eq. Deff, increases as both Do and α increase.Note that both a larger s = 2 being spin degeneracyConsider a perfect 2D system, its static dielectric function is The electronic contributions to x, 2py, 2s) orbitals. We notice that in bulk graphite, the states with higher energy difference contribute to the dielectric function in RPA aswhere γ = 6.46 eV · Å is band parameter of grapheneLc is the lattice constant of graphite perpendicular to atomic plane. For graphite, the experimental reflection spectrum measurement provided where x being the thin film thickness. In another work by Berkelbach et al.ab initio calculation. These non-local terms arise from the same origin: inter-band polarizability from gapped band pairs. For conduction-valence band pair q2, and the energy difference is a finite value. Therefore, the leading term of 2D polarizability given by Eq. (10)From Eq. αD2. The Poisson equation for electrostatic potential Next, we derive the effective e-h interaction to address the thickness dependence of exciton binding strength in graphene stack. In multi-layer graphene, it is well known that the band structure depends on the stacking sequences of graphene layers. As reported previously40434420zn = (n−1)d is the z coordinate of nth layer. The solution of Eq. Supplementary Discussion C for details)Here D2, we first take To determine the appropriate αIn Eq. γ = 6.46 eV · Å, interlayer hopping Supplementary Discussion C for details)with band parameter The leading term of dielectric function has Thomas-Fermi screening form For small r (large q) limit, the exponential term εbgTo corroborate the asymptotic behavior at both long- and short-range, we choose the following form to describe the orbital dependent e-h interaction with the presence of multi-layer graphene screening as where the parameters z and px/y orbitals along z axis since the wave functions of π bands are combination of pz orbitals while those of σ bands are px/y orbitals. Interestingly, the effective potential energy of σ exciton is much stronger than that for k∙p effective mass description of bands in graphene (See %Supplementary Discussion B for details) from ab initio calculation (See %Supplementary Method for details) as mo being the free electron mass. Finally, we solve the excitonic Schrödinger equation in the coordinate space numerically to investigate the characteristics of π exciton at M point and σ exciton at Γ point of graphene and its associated multi-layer forms.We employ tight-binding wave functions constructed from carbon p orbitals with effective nuclear chargeSince M point is a saddle point, as shown in The first equation describes a 1D bound state problem while the second leads to a delocalized scattering state, with a total wave function n for first three excited states with n being the number of nodes. The binding energies and parities are shown in ab initio GW-BSE calculation (0.60 eV)5051121411and obtained the binding energy ED which controls the effective potential. Only when the short range part of the interaction is regularized and no longer diverges as 1/r at small r, the existence of such a nodeless state could be found. For instance, if we only include the non-local screening effect, without the orbital-size regularization, the computed binding energy obtained for SLG is 1.66 eV which is almost three times that of experimentalThe solution of 1D soft-core Coulomb problem has been studied numerically recently by Gebremedhin and WeatherfordIn this work we mainly focus on the binding strength of exciton, while the absorption intensity by these excitons can be numerically calculated as the imaginary part of frequency dependent dielectric function in the linear response formulism56To study σ exciton at Γ point, we start with a variational trial function to find the ground excitonic state for σ bands. Examining the Hamiltonian l there is a radial Schrodinger equationand for each quantum number Ai is the prefactor and Bi is the inverse of effective Bohr radius for each component. For each l the energy of the lowest state has an upper boundThe radial part of normalized 2D hydrogenic orbitals l, and the approximated values of some El0, are given in l = ± 1 and l = ± 3 which have 2p-like character. Such exciton spectra significantly deviate from standard 2D hydrogenic spectrum1s state has nine times the energy of 2s/2p state. Such deviation from 2D hydrogenic model has been found in other layered materials in recent years, and will be studied in details in next section. Including the layer-dependent screening effect, we computed the binding energies of excitonic states in multi-layer graphene as shown in 5260et al.so γ = 6.46 eV · Å is the band parameter of single-layer grapheneHere By including the high energy bands effects discussed in Eq. r1 ≈ 74 Å for SLG, r1 ≈ 633 Å for BLG. Since the band structure of AB-stacked tri-layer graphene is equivalent to a bi-layer with the inter-layer hopping parameter r1 ≈ 2749 Å. Since the screening parameter r1 is pretty large here, we need to consider its effect on the long range potential. Considering that r1 is much greater than Deff and where 0 is the Struve function while Y0 is Bessel function of the second kind. Since r−1 and r−3 terms in approximation. The dielectric function for large q, which is dominated by higher energy dispersions, should be least affected since the gap only affects the low-energy band structure. So, the asymptotic form of e-h interaction for large q is similar to Eq. Here HHere r1q, which is quite weak for small q. However, with the enhanced background dielectric constant induced by substrate effect, the total dielectric response at small increases which leads to the reduction of binding energy in SLG as compared to the suspended SLG graphene. Considering the TF like screening in BLG and TLG, the substrate-induced finite gap further regularizes the screening at small q from TF type, which decays as r−3, to insulator-like for both BLG and TLG. Thus, exciton binding strength is enhanced accordingly. Therefore, since the leading terms of the effective e-h potential in Eq. With these effective potentials, the calculated binding energies of σ excitons are 186 meV for SLG, 154 meV for BLG, and 135 meV for TLG, respectively, which are plotted in 1s and 2p excitons with respect to the standard 2D hydrogenic spectrum3 hydrogenated graphane by Cudazzo et al.2 carbon layer graphyne by Huang et al.et al.As discussed in previous session, a non-hydrogenic feature is identified for σ exciton at M point in graphene by examining the energy gap between VOD given Eq. VOD-NLS given by Eq. et al.r−1 divergence of Coulomb potential to a logarithmic divergence, and the orbital-size effect further weaken it to a finite value at r = 0. From r−3 at large distance, therefore, very effective on reducing the binding strength of excitons with small principal quantum number n, like 1s state, due to its tight spatial distribution. And among those states with the same n, states with larger angular quantum number l are more diffusive and have less density at small distance. Then the binding energy reduction of, for example, 2s state is greater than the reduction of 2p state, so the 2p state invokes less excitation energy than 2s one. For a given non-local screening, the non-hydrogenic feature becomes more evident as D increases since the orbital size information is nicely included in D. . Thus, it is interesting to compare the exciton 2s-2p splitting observed in WS2 and graphane. With two-photon luminescence measurement and GW-BSE calculation Ye et al.2 significantly deviate from 2D hydrogenic model. By matching the ratio between 2s-2p splitting and 1s binding energy with our model, we observed that WS2 has greater 2s-2p splitting than graphane as shown in 2. This also supports the important role of orbital-size dependence. Finally, we address the possibility of observing the 2s-2p splitting in graphene. The strong screening of π state imposes the formidable challenge in forming spectroscopically resolved series π exciton states. Motivated by the desirably stable σ exciton in graphene stacks, we estimate 2s-2p splitting of σ excitons in a suspended graphene to be in the order of 10 meV.To further understand the role of orbital-size effect and non-local screening, we solve two-body Schrödinger equations using parameters from σ bands. The 2p and 2s excitonic states for such model system, with bare Coulomb potential, In summary, we construct an orbital-dependent effective potential describing e-h interaction in graphene to unravel its quasi-2D characteristics originated in the orbitals’ spatial distribution. The modified e-h potentials are also developed by including effects of non-local screening, multi-layer screening, and the coupling with substrates. We demonstrate the inclusion of orbital dependence to be crucial for correctly accounting for the binding energy of π exciton at M point. We highlight the stronger e-h interaction in σ orbitals than that of π ones, which facilitates the formation of stable σ exciton in graphene stacks even at room temperature. The coupling with substrates opens a gap in graphene stacks. The synergized contributions between gap-opening and screening from substrates result in the fairly weak dependence of σ exciton binding energy on the thickness of graphene stacks. The orbital dependent potential plays a comparable role as non-local screening in scrutinizing the non-hydrogenic exciton spectrumin in quasi-2D materials.How to cite this article: Deng, T. and Su, H. Orbital-dependent Electron-Hole Interaction in Graphene and Associated Multi-Layer Structures. Sci. Rep. 5, 17337; doi: 10.1038/srep17337 (2015).


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