In particular, we show that the process was successfully used to realize and characterize a simple, optically transparent graphene-based wire-grid polarizer at microwave frequencies (X band). The availability of graphene operating in a  Up to now, graphene properties in the microwave range have been studied by means of theoretical models68lossy-dielectric region0/2), which strongly affects the performance of any microwave device that requires metallic components.Chemical Vapour Deposition (CVD) graphene has been indicated as the ideal conducting material for future 2D technologies. However, the most current and lowest reported experimental values of its sheet resistance around 1000 Ω/sq) literally invalidate any such prospects000 Ω/sq 2 V−1 s−1) is approximately one order of magnitude lower than the carrier mobility of the mechanically exfoliated graphene, if measured inside a large single CVD graphene grainIn this regard, the transport properties of CVD graphene are determined by both intrinsic and extrinsic factors such as (i) its polycrystalline nature and (ii) the lack of reproducible methods that would allow its transfer to substrates of technological interest without generating holes, cracks and ripples, especially on large areas. These defects, as well as grain boundaries, act as barriers to charge transport and result in sheet resistance higher than that of mechanically exfoliated micrometer grapheneIn theory, the use of multiple layers of CVD graphene acting as parallel resistances should amount to a simple method to lower the sheet resistance. However, experiments suggest that adding parallel CVD graphene layers does not necessarily decrease sheet resistance, since the partial contact or the presence of additional impurities  contribute contact/series resistances between graphene layers12) has been successfully used for hole-doping of graphene2 consists of the exploitation of the intrinsic defects in CVD graphene as enhanced reactive centers for the attachment of dopant functionalities. This provides the stability of covalent chemical doping without detrimental effects on material mobility due to the creation of new C-sp3 acting as charge scattering centers in the C-sp2 lattice.Chemical doping is an effective way to enhance graphene sheet conductivity via the attachment of electron donors or acceptors. For example, the treatments with thionyl chloride . In contrast, chemically doped graphene can overcome the low threshold limit with only two layers. The minimum sheet resistance value measured for chemically doped graphene is 27 Ω/sq, and was achieved with five-layer graphene samples. If additional graphene layers are added, the effect of series resistances between adjacent graphene layers becomes predominant, resulting in higher Rs.The effect of the combined use of SOCl2 treatment.The optical transmittance of chemically doped graphene from one to five layers is reported in quasi-metallic graphene) allowed us to test the electromagnetic response of hole-doped graphene at microwave frequencies. In particular, we measured reflectance, transmittance, and absorbance of samples with different sheet-resistances. The measurements were carried out by means of a microwave setup consisting of a klystron connected to a WR90 rectangular waveguide (the waveguide supports only the TE10 mode). A slotted-line acquired the electromagnetic power (square law) with a spatial resolution of 1 mm in the rectangular waveguide at 9 GHz . Further details on the measurement setup are reported in the The possibility to operate with very-low sheet resistance samples . The comparison between the analytical model and the experimental results shows very good agreement.quasi-metallic region acts as a metal by efficiently reflecting the impinging electromagnetic field. For example, when Rs = 27 Ω/sq the reflectance is larger than 80%. At the same time, the doped graphene sheet conductivity is independent over a wide frequency range  in the stripes over an area of about 50 × 55 mm2 , while the electric field with vertical polarization (EV) is reflected /T90 of the polarizer Tpol with respect to transmission at 90° (T90). We adopted this normalization to emphasize the difference at 0° . In fact, the graphene-based wire-grid polarizer (red curve) shows a near-unity transmission at 90°.We compared the graphene-based wire-grid polarizer with a wire-grid polarizer made with copper stripes of identical dimensions (5 mm width) and identical foot-print with respect to the graphene-based polarizer. The comparison reveals that the two polarizers only show a constant 3 dB difference over the entire angular range while their angular behavior is identical. This result is quite remarkable, considering that the copper layer is 35 μm thick, while graphene thickness is about 1 nm.In conclusion, we have detailed a chemical protocol for the optimization of graphene in terms of (i) doping stability, (ii) scalability to large areas, and (iii) reduction of the series resistances between graphene layers in multilayer graphene, thus achieving multilayer graphene with sheet resistance values below 30 Ω/sq.lossy-dielectric to that of a quasi-metal.This protocol allowed us to experimentally verify the optimized graphene behavior in terms of reflectance, transmittance, and absorbance of hole-doped graphene when the sheet resistance ranges from that of a Finally, the optimized graphene was exploited for the realization of an optically transparent graphene-based polarizer. The comparison between the graphene-based polarizer and its copper-based counterpart revealed that the two polarizers show only a constant 3 dB difference over the entire angular range while their angular behavior remains identical.quasi-metallic region with very-low sheet resistance graphene may allow the realization of microwave devices (e.g. microstrip patch antennas) and the dynamical tuning of their S-parameters over a wide-range. In this regard, graphene-based antennas with low-sheet resistance graphene may help overcome the limitation of low gain23The possibility to operate in the 4/H2 as precursors. The graphene was transferred onto corning glass substrate (treated by O2 plasma to improve graphene adhesion) by the thermal tape method. The copper was etched in a solution of ammonium persulfate. Multilayer graphene samples were fabricated by transferring of additional graphene layers onto graphene/glass substrates.Graphene was grown by CVD on a 25 μm thick copper foils in a typical quartz tube CVD reactor at 1000 °C using CH2 treatments were performed in a dry chamber by placing graphene/glass substrate and 1 mL of liquid SOCl2 (avoiding direct contact) at 105 °C for 60 min. Doping of multilayer samples was performed by repeating SOCl2 treatment after transferring and stacking each graphene layer.SOCl2 in air and at room temperature  media (impinging from medium 1 to medium 2), that introduces a finite sheet conductivity ση1 and η2 are the wave impedances in the two media, respectively, and σD2 ≈ σDC = 1/Rs. Moreover, the sheet conductivity is independent over a wide frequency range, as found in references 6 at microwave frequencies (X-band). If we apply the boundary conditions for the electric and magnetic fields at the interface z = 0 we obtain:where where Γ and T are the reflection and transmission coefficients, respectively.Solving the Equation GR and transmittance TGR, GR may be evaluated as AGR = 1 – RGR – TGR. If we assume η1 = η2 = η0 (where η0 corresponds to the impedance of free space) it is possible to define two different regions for a single graphene sheet: the lossy-dielectric region quasi-metallic region where we set How to cite this article: Grande, M. et al. Optically Transparent Microwave Polarizer Based On Quasi-Metallic Graphene. Sci. Rep.5, 17083; doi: 10.1038/srep17083 (2015).


These new findings, together with archaeozoological and human skeletal collagen bulk stable carbon isotope proxies, unequivocally confirm rejection of marine resources by early farmers coinciding with the adoption of intensive dairy farming. This pattern of Neolithization contrasts markedly to that occurring contemporaneously in the Baltic, suggesting that geographically distinct ecological and cultural influences dictated the evolution of subsistence practices at this critical phase of European prehistory.The appearance of farming, from its inception in the Near East around 12 000 years ago, finally reached the northwestern extremes of Europe by the fourth millennium BC or shortly thereafter. Various models have been invoked to explain the Neolithization of northern Europe; however, resolving these different scenarios has proved problematic due to poor faunal preservation and the lack of specificity achievable for commonly applied proxies. Here, we present new multi-proxy evidence, which qualitatively and quantitatively maps subsistence change in the northeast Atlantic archipelagos from the Late Mesolithic into the Neolithic and beyond. A model involving significant retention of hunter–gatherer–fisher influences was tested against one of the dominant adoptions of farming using a novel suite of lipid biomarkers, including dihydroxy fatty acids, ω-( The temperature programme comprised an isothermal hold at 70° for 2 min, ramping to 220° at 10° min−1, followed by the second ramp at 4° min−1 to 300°, with a 10 min isothermal hold.Aliquots of the solvent and alkaline extracts were filtered through a silica column and treated with 40 µl (d)3). Fatty acids were methylated using 100 µl BF3/MeOH  and extracted (3 × 2 ml CHCl3). Fatty acid methyl esters were analysed using a GC/MS fitted with a polar column, with the MS operated in full scan (m/z 70–650) and SIM  modes.Aliquots of the total lipid extract were hydrolysed . The neutral fraction was removed (3 × 3 ml hexane) followed by acidification to pH 3 using 1 M aqueous HCl and the extraction of free fatty acids . Analyses were performed using a Varian 3400 GC coupled to a Finnigan MAT Delta-S IRMS with a modified Finnegan MAT interface, Cu and Pt wires (0.1 mm o.d.) in an alumina reactor (0.5 mm i.d.). Samples were injected via an SPI injector onto a non-polar column . The temperature programme consisted of a 2 min isothermal hold at 50° and then ramped at 10° min−1 to 300°C followed by a 10 min isothermal hold. Results were calibrated against reference CO2, which was injected directly into the source three times at the beginning and end of the run. All samples were run in duplicate with external standards every four runs; any runs of unacceptable integrity were discarded and repeated. The δ13C values were derived according to the following expression and are relative to the international standard vPDB: δ13C‰ = ((Rsample – Rstandard)/Rstandard) × 1000, where R = 13C/12C. The δ13C values were corrected for the carbon atoms added during methylation using a mass balance equation [The δequation .3.(a)n-alkanoic acids, which reflect a predominantly ruminant origin for the majority of fats , with the incidence of a distinctive series of mid-chain ketones (e.g. approx. 30% of Early Neolithic residues) indicating that the fats extracted were regularly reaching temperatures in excess of 270°C ,18. Noned,e). By the Viking and Norse period, from which nearly 100 sherds were investigated, marine biomarkers are considerably more widely detected in pottery 12–C14) fatty acids in milk fats compared with adipose fats. The stable carbon isotope signatures confirmed an overwhelming predominance of dairy products associated with Neolithic pottery throughout the northeast archipelago . During the earliest Neolithic, dairy fats comprised some 80% of lipid residues identified in pottery vessels (figures 1i and 3d). Contributions from wild terrestrial fauna (notably deer) can be discounted, since they typically make up less than 10% of faunal assemblages on the Western Isles of Scotland [While aquatic commodities were rarely identified in the lipid residues of Neolithic pottery of the northeast Atlantic archipelagos, the intact triacylglycerols often exhibited a wide acyl carbon number distribution. This distribution is characteristic of dairy-derived fats, arising from the higher abundance of lower carbon number U-test (SPSS v. 21) was run to determine whether there was any significant difference between the δ13C values of Neolithic milk (Δ13C ≤ −3.4‰) fatty acids from island compared with mainland coastal locations. Analysis of milk residues only was chosen in order to remove any possibility of different types of fat affecting the analysis. Distributions of δ13C values from island and mainland locations were similar, as assessed by visual inspection, for both C16:0 and C18:0 fatty acids, and the dataset did not deviate significantly from the assumptions required for a Mann–Whitney U-test. Interestingly, an offset is observed between the range of compound-specific δ13C values from the smaller and larger island masses. The median δ13C value was statistically significantly higher for both fatty acids from island locations  compared with mainland locations .The non-parametric Mann–Whitney 13C signatures in the herbivore fats in Hebridean, Shetlandic and Orcadian pottery.These differences between the absolute values of fatty acids from milk fats can probably be explained by the greater relative expanse of coastline. More saline conditions, which are encountered at shoreline locations, are known to cause stomatal closure and hence higher stable carbon isotope signatures in the terrestrial non-halophytic plants ,33. This(d)13C values are only observed in Late Iron Age and Viking/Norse residues. When viewed together with the archaeozoological evidence and human collagen δ13C values from these later periods , and (iii) collagen stable carbon isotope signatures of Mesolithic and Neolithic humans from Britain showing a terrestrial-based diet of coastal-dwellers in the Neolithic (figure 3a) [To summarize, our findings provide unequivocal evidence that marine products were of little overall importance to the Neolithic farmers of the northeast Atlantic archipelagos, as evidenced by (i) almost non-existent evidence for aquatic product processing in pottery, (ii) low presence of aquatic species in faunal assemblages from southern Britain  and the figure 3gure 3a) ,5,21. Whgure 3a)  and our -13910*T allele, and would have been enhanced by the practices introduced by the milk-producing dairy farming populations identified herein [This contrasts markedly with recent findings from Late Mesolithic and Neolithic human bone stable isotopes and organic residues from pottery from the Baltic region –39, whicd herein –43; and d herein . Given t


Aldoketoreductase 1B10 gene (AKR1B10) encodes one of the enzymes belonging to the family of aldoketoreductases and may be involved in detoxification of nutrients during digestion. Further, AKR1B10 mRNA (messenger ribonucleic acid) expression was diminished in brain regions potentially involved in the regulation of eating behavior in rats which are more sensitive to cocaine and alcohol. We hypothesized that the human AKR1B10 gene may also play a role in the regulation of human eating behavior.The human AKR1B10 on human eating behavior among 548 subjects from a German self-contained population, the Sorbs, and in 350 subjects from another independent German cohort. Among the Sorbs, we observed nominal associations with disinhibition at the 5′ untranslated region (5′ UTR) variant rs10232478 and the intragenic variants rs1834150 and rs782881 . Further, we detected a relationship of rs1834150 and rs782881 with waist, smoking consumption (rs782881) and coffee consumption (rs1834150) . Albeit non-significant, replication analyses revealed similar effect directions for disinhibition at rs1834150 (combined P = 0.0096). Moreover, in the replication cohort we found rs1834150 related to increased restraint scores with a similar direction as in the Sorbs (combined P = 0.0072).We investigated the effects of 5 genetic variants of AKR1B10 locus may influence human eating behavior.Our data suggest that genetic variants in the The online version of this article (doi:10.1186/s12863-015-0189-9) contains supplementary material, which is available to authorized users. Akr1b10 expression was shown to be lower in brain of Lewis rats (LEW) that are more sensitive to cocaine or ethanol compared to a control group of rats (Fischer 344 (F344)) [Akr1b10 in nucleus accumbens and the frontal cortex, areas suggested to be involved in eating behavior. Taken together, (i) the important role of AKR1B10 in detoxifying aldehydes and several other deleterious compounds during digestion and (ii) its putative central effects in brain regions potentially involved in eating behavior allowed us to hypothesize that AKR1B10 may play a role in human eating behavior.The human aldoketoreductase 1B10 (AKR1B10) was identified in human hepatocellular cancer as a member of the AKR superfamily . It redu (F344)) . Higuera (F344))  observedfat mass and obesity associated gene (FTO) were not only associated with higher body mass index (BMI) but also with loss of control over eating and selection of food higher in calories and fat [glutamate decarboxylase gene (GAD2) were significantly associated with increased disinhibition and hunger in women from the Quebec Family Study [GAD2 associated with lower birth weight and subsequently higher BMI Z-Scores in French children [leptin gene (LEP) and cholecystokinin gene (CCK) were shown to be associated with “snacking behavior” [AKR1B10 gene and in the upstream untranslated regulatory region. We evaluated its genetic association with human eating behavior, a variety of metabolic and anthropometric phenotypes, and consumption of consumer goods such as alcohol intake, smoking behavior and coffee consumption. To the best of our knowledge this is the first study investigating AKR1B10 variants and their potential relationship with anthropometric and metabolic parameters as well as eating behavior traits.There is a good evidence for the role of genetics in human eating behavior -19 or in and fat -23. Singly Study  while a ly Study . Meyre echildren . Furtherehavior”  and biggehavior” . Dotson ehavior” ,29 foundIn the present study we used a self-contained population, the Sorbs from Germany, comprising 1046 individuals as described earlier  . To estimate coffee and alcohol intake, subjects were asked for average intake but not for a certain time period  none; 2) occasionally at special events; 3) 2–3 glasses of wine or bottle of beer per week; 4) 1 glass of wine or bottle of beer per day; 5) 2 glasses of wine or bottles of beer per day; 6) more than 3 glasses of wine or bottles of beer per day; coffee: number of cups per day) ,37. We dFor replication purposes we used another independent German cohort as described elsewhere  includinAll human studies were approved by the ethics committee of the University of Leipzig and all subjects gave written informed consent.AKR1B10 SNPs  were selected for the analysis. We selected tagging SNPs for our analysis which are in linkage disequilibrium (LD) with several other SNPs within and around the AKR1B10 locus to enlarge the probability to find associating variants in the gene without the need to analyze all existing SNPs. The genotype information for rs10232478 was extracted from genome wide Affymetrix SNP data [2 > 0.8, minor allele frequency (MAF) < 0.05) and genotyped using the KASPar SNP genotyping system  according to manufacturer’s protocol (TD PCR (touch down polymerase chain reaction) with following conditions: 94°C for 15 sec, 94°C for 20 sec, 61°C for 60 sec, for 10 cycles, drop down 0.6°C per cycle, 94°C for 10 sec, 55°C for 60 sec, for 26 cycles). Genomic deoxyribonucleic acid (DNA) for genotyping was extracted from blood with QIAmp DNA Blood Midi Kit  according to the manufacturer’s protocol. Fluorescence was detected using ABI PRISM 7500 Sequence Detecting System. To guarantee genotyping reproducibility, a random ~5% of the samples were re-genotyped in all SNPs; all genotypes matched initial designated genotypes. The average genotyping success rate for all analyzed SNPs was 96.38%.Five SNP data  for the 10-transformed to approximate normal distribution prior to analysis. All variants were in Hardy-Weinberg-Equilibrium. Genetic association with eating behavior traits, anthropometric and metabolic phenotypes were calculated using linear regression models adjusted for age, gender and lnBMI (except for BMI). Cronbach’s alpha coefficients were calculated to measure the internal reliability of the three eating behavior subscales. Coffee consumption was defined as cups per day, smoking behavior as number of cigarettes per day and alcohol intake as number of glasses per week. We did not account for different contents of alcohol per glass/bottle or caffeine content per cup. P-values for smoking, alcohol intake and coffee consumption were corrected for age and gender. Additive, dominant and recessive models of inheritance were tested. To correct for multiple testing we lowered the significance threshold to (0.05/(315 tests) = 1.6×10−4). All P-values > 1.6×10−4 but ≤ 0.05 were considered to be of nominal statistical significance. All P-values are provided uncorrected for multiple testing. Meta-analyses were performed using METAL [Statistical analyses were performed using the SPSS statistics software version 20.0 . Non-normally distributed parameters were logng METAL . All anaWe observed nominally significant associations with disinhibition at the variant rs10232478 located in the 5′ untranslated region and the intronic SNPs rs1834150 and rs782881 , we did not detect evidence for any significant associations .Additional analyses were performed to test the hypothesis that variants within N = 350). Despite failing to reach statistical significance our replication analyses revealed similar trends for higher disinhibition scores in homozygous or heterozygous minor allele carriers  and the 5′ UTR variant rs10232478  Although we present a second independent replication cohort the sample size of our study is rather small which may lead to false positive and false negative results. (ii) There is a large discrepancy in the male/female ratio and in age between the study cohorts which may have, in terms of different eating behavior patterns between men and women, influenced our results as demonstrated by others -50. HoweAKR1B10 may be a potential contributor to human eating behavior. However, our data need to be cautiously interpreted and larger studies are definitely warranted to replicate and verify the observed effects.In conclusion, our data in regard to eating behavior factors support our hypothesis that AKR1B10 with eating behavior. The established role of AKR1B10 in cancers via influencing important signaling pathways like the retinoid acid metabolism [To the best of our knowledge this is the first evidence for an association of tabolism ,8 togeth


Several modified and new rigidly foldable tessellations are presented.Rigidly foldable origami allows for motion where all deflection occurs at the crease lines and facilitates the application of origami in materials other than paper. In this paper, we use a recently discovered method for determining rigid foldability to identify existing flat-foldable rigidly foldable tessellations, which are also categorized. We introduce rigidly foldable origami  An origami tessellation is rigidly foldable if all sectors remain rigid and deflection only occurs at the crease lines. Because many materials used in engineering are much stiffer than paper, non-rigidly foldable tessellations (those that require deflection of the paper sectors) may have restricted movement when constructed out of these rigid materials. However, rigidly foldable tessellations may be constructed using stiff materials, leading to potential applications such as architecture and deployable arrays.The study of origami has inspired engineering applications in recent years, including deployable structures such as solar panels ,2 and stet al. [et al. [Origami research has focused on several related areas. Hull developed theorems which govern flat-foldable origami patterns  and evalet al.  characte [et al.  showed tResearch on rigidly foldable origami has addressed the issue in several different ways. Huffman derived basic relationships between the various dihedral angles in a rigidly foldable degree-4 polyhedral vertex using spherical trigonometry . Wu usedgadgets, which are tools in the modification and creation of rigidly foldable tessellations. These gadgets are used in two different ways. First, they are used to replace portions of existing rigidly foldable tessellations to create slightly modified tessellations. Second, the gadgets are tessellated to make new rigidly foldable patterns.A method for analysing the rigid foldability of origami patterns composed entirely from flat-foldable, degree-4 vertices has been developed previously by the authors . While m2.An origami pattern is said to be rigidly foldable if all panels remain rigid while all deflection occurs at the crease lines during deployment. We previously presented a method for determining if an origami pattern composed of degree-4 vertices is rigidly foldable . Becausesector and the angle between adjacent creases is called a sector angle, designated α. The sum of all sector angles around a vertex is 2π. The angle of the fold itself is the dihedral angle or fold angle, denoted by γ, which is the angle between the surface normals of the two incident sectors. A crease may be a valley fold (γ>0), a mountain fold (γ<0) or unfolded (γ=0). We will indicate mountain folds by solid lines and valley folds by dashed lines. We index the sector angles αi and dihedral angles γi so that sector αi lies between folds γi and γi+1, as illustrated in In this paper, we focus on patterns composed of degree-4 vertices. As illustrated in 2.1π in the secondary flat position of a degree-4 vertex. Likewise, an origami pattern composed entirely of degree-4 vertices is considered flat foldable if there exists a configuration where all dihedral angles in the pattern are equal to ±π. The conditions for flat-foldability are well known ;— the largest angled sector, if unique, is incident to folds of the same parity (‘iso’); andπ;— opposite sector angles sum to A flat-foldable vertex begins in an initial flat position and can be folded to achieve a secondary flat position. All dihedral angles are equal to ±wn (e.g. ); for dewhere a fold's ‘parity’ refers to its assignment as a mountain or a valley fold.minor creases and the two opposite creases with equal parity the major creases.If there exist two equally valued smallest angled sectors, at least one of these sectors must be anto and its opposite sector must be iso. We call the two opposite creases with opposite parity the These conditions give us the following relationship between sector angles in a degree-4 vertex :2.1α1+αThese are necessary conditions, not sufficient; an origami pattern composed entirely from flat-foldable vertices may self-intersect before the final flat state. Such a pattern is considered non-flat-foldable. Also, any origami tessellation containing one or more non-flat-foldable vertices cannot be flat-foldable.2.2We will use fold-angle multipliers to evaluate tessellations. Fold-angle multipliers, as defined in , define The following relationships between dihedral angles in a flat-foldable degree-4 origami vertex are given in :2.2γ3=−μi, for the ith sector is defined as follows:The fold angle multiplier, Using equations –2.4), t, t2.4), α2+α3=π), zero and infinite fold-angle multipliers are obtained. This occurs because the major crease lines must be completely folded before the minor crease lines begin folding. Because such vertices have more than 1 d.f., they are not considered in this paper.There is a special case to note: when the major crease fold lines are collinear .This method can be used to include additional information to crease patterns to demonstrate distinct fold angles in the tessellations. For each type of vertex, the fold angle multipliers ensure that certain pairs of fold angles are the same in the vertex, and we indicate sameness by adding a colour background to each fold line, choosing different colours for each distinct fold angle. Then it is clear to see that a given pattern is rigidly foldable by noting that the fold lines emanating from each vertex match in both fold direction and fold angle (magnitude) with the fold lines of each surrounding vertex. This not only demonstrates the rigid foldability of the tessellations, but is an example of the power of the fold angle multiplier method. One can both construct and prove rigid foldability by purely graphical techniques and inspection augmented by scalar algebra.In this paper, we first evaluate existing flat-foldable tessellation to identify those that are rigidly foldable. We then present gadgets that facilitate the creation of rigidly foldable tessellations and show some resulting tessellations.3.There are several origami tessellations which have been known to be rigidly foldable. 3.1a. Two of the sector angles are equal to π/2 and the other two are equal α and π−α. The tessellation is locally rigidly foldable for any α≠π/2. However, as seen in figure 3c, this tessellations folds into a cylindrical form and global self-intersection occurs before it reaches the final flat state.The first tessellation we consider was presented by David Huffman , which wfigure 3a have equal fold angle magnitude, as do all crease lines with a negative slope. As a result, this tessellation contains two unique dihedral angles.Equations –2.8) sh sh2.8) sfigure 3a shows the sector angles of this vertex and labels the dihedral angles γ1 through to γ4. The fold-angle multipliers associated with this vertex may be calculated using equations . F. Fa showquations 3.1μ1=sib. For any polygon, this procedure is repeated for all vertices of the polygon. Because the Huffman grid includes only one unique vertex, further calculations are not necessary.The other fold-angle multipliers are then calculated using equations –2.8). T. T2.8). figure 43.2α<π/2 although it folds into a cylindrical form and global self-intersection occurs before a second flat position is obtained, as is seen in figure 6c. This tessellation contains two unique dihedral angles; all valley creases have the same fold angle, as do all mountain creases.The chicken wire tessellation  is cons3.3α<π/2. The pattern stays in a planar form, as shown in figure 7b,c and is able to reach its final flat state without self-intersection. This tessellation contains two unique dihedral angles; all vertical moving chains are equal in fold angle magnitude, as are all horizontal chains.Paulo Barreto presented a tessellation, ‘Mars’, which included a single degree-4 vertex and its inversion  as shown3.4α<π/2. Figure 8a shows the fold pattern for the Miura-ori. The pattern stays in a planar form as shown in figure 8b,c and thus is able to reach its final flat state without self-intersection. The Miura-ori has two unique dihedral angles; all vertical creases are equal in angle, as are all other creases.The Miura-ori is an origami tessellation developed by Miura  which is3.5a shows the fold lines for the Yoshimura pattern. Figure 9b,c shows one of the possible fold paths for this tessellation. Although the pattern is locally rigidly foldable for α<π/2, except under specific circumstances, global self-intersection occurs before the second flat state can be reached with this fold path.Yoshimura developed an origami pattern  based onFigure 9As is noted in 3.6Tachi analysed quadrilateral mesh origami and presented conditions for rigid foldability . As the 3.7αi<π/2. The generalized Miura-ori may have many unique dihedral angles. All vertical creases have the same fold angle, while all chains of intersecting creases have another unique fold angle. By requiring a quadrilateral mesh origami to contain vertices with mirror symmetry, the generalized Miura-ori is obtained . The gen4.gadget as a localized section of crease pattern that can replace an existing patch to add functionality or otherwise modify the pattern [Author Lang defined an origami  pattern . As a gan>4 polygons in rigidly foldable tessellations.The average polygon degree in a repeatable tessellation containing only degree-4 vertices must be equal to 4 (see appendix A). As a result, any tessellation constructed using only degree-4 vertices and featuring polygons of more than fourth degree must also contain triangles. Because of this, of particular interest are gadgets which include triangles and allow for the use of 4.1β changes the size and shape of the depression, but does not change the fold angle multipliers between any of the exterior creases. It is required that β>α1. Replacing a vertex with a corner gadget adds one unique dihedral angle to the tessellation. A flat-foldable degree-4 vertex may be modified to create a rigidly foldable network of four degree-4 vertices with four outwardly extending creases in the same location as the original creases as shown in 4.1.1β. As with the original, it is also required that β>α.A special case of the corner gadget is one where the minor crease lines of the original vertex are collinear. As a result, the gadget created is symmetric about this crease line as seen in 4.2Any flat-foldable degree-4 origami vertex which does not contain two collinear crease lines and does not contain 90° sector angles may be modified to become a network of four vertices enclosing two isosceles triangles. This network contains six outwardly extending creases, three of which are parallel to one of the original minor creases and the other three are each coincident to each of the other three original creases . The folLabelling the opposite crease as crease number one and numbering counter-clockwise , the mulb. To form a set, the two gadgets must have equal values for α and β. A set of triple parallel gadgets may replace a combination of two vertices which are mirror-symmetric, as shown in figure 16a) where this third angle is entirely contained in the set of gadgets.Of particular interest is an arrangement where two of these gadgets are combined in such a way that the three parallel crease lines from one gadget meet the three parallel crease lines from another. We call this a set of triple parallel gadgets, shown in figure 164.3α1,α2) fully define the gadget. The gadget is flat foldable, therefore, all other angles may be calculated by recalling that opposite sector angles in a vertex sum to π. A chain of level shifters has three unique fold angles. Two angles intertwine along the direction of the chain and a third angle passes through the horizontal creases.A level shifter is an origami gadget that has found use in formal origami design and shows up often in origami in the form of a ‘spread sink’ . As its 4.3.1α1=α2. This creates a symmetric level shifter and we use α to designate the input angle. The dihedral angles for each of the horizontal crease lines in figure 18b are equal. Therefore, this gadget may be useful in creating large tessellations which do not self-intersect.A special case of the level shifter gadget occurs when 5.In this section, we explore modifications that can be made to existing rigidly foldable tessellations. These modifications use the gadgets presented previously to directly replace existing portions of the tessellations.5.1c and 19c. Also, when constructed using hinges with finite stiffness, this configuration is stiffer than the original Miura-ori because of the addition of springs in parallel.By replacing the vertices of a Miura-ori tessellation with corner gadgets, the tessellation shown in figures 85.2As with the Miura-ori, the ‘Mars’ tessellation may be modified by replacing vertices with corner gadgets. Such a resulting tessellation is shown in 6.We present several new rigidly foldable patterns in this section. Many of these patterns were constructed using the previously mentioned gadgets.6.1α≤β<90°. Note that the case where α=β results in the ‘Mars’ tessellation. This tessellation contains four unique dihedral angles. Every other set of mostly vertical chains have equal fold angle magnitudes, as do every other set of mostly horizontal chains.A special case of a generalized quadrilateral mesh origami occurs when only two different vertices are used. These vertices, along with their inversions are used to create the tessellation shown in 6.2This tessellation is created by attaching chains of level shifters in opposite directions. The chains are arranged so that adjacent chains are in alternate directions and are connected using hexagons . This te6.3This tessellation is created by attaching chains of level shifters in the same direction. These chains are attached using pentagons . This te6.4b. Likewise, level shifter chains with valley folds separating the triangles act as mountain-like folds during the intermediate positions as shown on the right and left in figure 26b. As the pattern approaches the final position, these level shifter chains become flat again as shown in figure 26c. The tessellation shown has four unique fold angles .Level shifter chains may be combined with Miura-ori patterns to construct new tessellations such as that shown in figure 266.5a provides one example where alternating chains of the Miura-ori are replaced with chains of back-to-back level shifters. The gadgets are arranged so that the three parallel folds from each gadget meet the three parallel folds from another gadget. This tessellation is rigidly foldable under the same conditions that the gadgets are rigidly foldable. However, as is shown in figure 27c, global self-intersection occurs before the final flat state. This tessellation includes three unique fold angles; the vertical crease chains are of equal angle, all folds stemming from these chains are of equal angle, and each set of triple parallel gadgets forms a third fold angle. As with the level shifter gadget, the triple parallel gadget may also be used in many different combinations with the Miura-ori pattern. Figure 277.gadgets have been designed which may facilitate the modification of creation of rigidly foldable origami tessellations. New flat-foldable and rigidly foldable origami tessellations involving these gadgets have been presented. These tessellations have final geometries that are more compact in one dimension than their counterparts. Because rigid foldability can be a critical feature for origami applications in materials other than paper, the identification of rigidly foldable tessellations, introduction of rigidly foldable gadgets and examples of how to create new tessellations by combining these concepts lays the foundation for future application.This paper has identified and categorized existing flat-foldable rigidly foldable origami tessellations. Using the method of fold-angle multipliers, several origami


Anticipated synchronization (AS) is an anti-intuitive phenomenon that can occur in two coupled dynamical systems when there is a dominant connectivity between the elements. AS occurs when a dynamical system A dominantly connects to another system B and B synchronously pulses before A does. It has been recently shown ,2 that A


BRCA1 mutation. The exposure to ionizing radiation for diagnostic or therapeutic purposes of BRCA1 mutation carriers is counterintuitive, since BRCA1 is active in the DNA damage response pathway. The aim of this study was to investigate whether healthy BRCA1 mutations carriers demonstrate an increased radiosensitivity compared with healthy individuals.Breast cancer risk increases drastically in individuals carrying a germline 2 micronucleus assay, reflecting defects in DNA repair and G2 arrest capacity after exposure to doses of 2 or 4 Gy. We investigated if a correlation between the RIND score and nonsense-mediated decay (NMD) could be established.We defined a novel radiosensitivity indicator (RIND) based on two endpoints measured by the GBRCA1 mutation carriers compared with healthy controls. In addition, our analysis showed a significantly different distribution over the RIND scores  for healthy BRCA1 mutation carriers compared with non-carriers: 72 % of mutation carriers showed a radiosensitive phenotype (RIND score 1–4), whereas 72 % of the healthy volunteers showed no radiosensitivity (RIND score 0). Furthermore, 28 % of BRCA1 mutation carriers had a RIND score of 3 or 4 (not observed in control subjects). The radiosensitive phenotype was similar for relatives within several families, but not for unrelated individuals carrying the same mutation. The median RIND score was higher in patients with a mutation leading to a premature termination codon (PTC) located in the central part of the gene than in patients with a germline mutation in the 5′ end of the gene.We found significantly increased radiosensitivity in the cohort of healthy BRCA1 mutations are associated with a radiosensitive phenotype related to a compromised DNA repair and G2 arrest capacity after exposure to either 2 or 4 Gy. Our study confirms that haploinsufficiency is the mechanism involved in radiosensitivity in patients with a PTC allele, but it suggests that further research is needed to evaluate alternative mechanisms for mutations not subjected to NMD.We show that The online version of this article (doi:10.1186/s13058-016-0709-1) contains supplementary material, which is available to authorized users. Approximately 15 % of all patients with BC have at least one relative affected by BC. About 15 % of all familial BCs can be attributed to a mutation in the BRCA1 or BRCA2 gene [2 phases of the cell cycle [2/M checkpoint control, allowing the cell to repair DNA damage before proceeding to the next phase of the cell cycle [Breast cancer (BC) is the most common malignancy in the Western world (CA2 gene ). BRCA1 ll cycle –6. BRCA1ll cycle .BRCA1 mutation may show enhanced radiosensitivity associated with an increased carcinogenic risk after exposure to diagnostic or therapeutic ionizing radiation (IR). Several studies have shown that exposure to diagnostic X-rays may cause cancer in healthy BRCA1 mutation carriers [BRCA1/2 mutation carriers [BRCA1 and BRCA2 mutation carriers reported no univocal conclusion [Carriers of a heterozygous carriers –11, whercarriers –15. Alsonclusion . The conSince long-term studies of the effect of exposure to IR in mutation carriers are difficult to set up and are reputed to be unethical, it is clear that more empiric data are needed to determine in vitro the radiosensitivity of patients carrying a germline mutation. To date, research has yielded contradictory results –26.0 micronucleus assay performed on peripheral blood lymphocytes exposed to in vitro doses of 2 to 4 Gy is frequently used to assess chromosomal radiosensitivity. However, this assay is not optimized to detect defects in DSB repair activated during the G2 phase of the cell cycle or the G2/M checkpoint, two processes in which BRCA1 plays a major role because irradiation takes place in the G0 phase of the cell cycle.The G2 phase of the cell cycle. This assay efficiently detected increased radiosensitivity in a patient with a mild form of ataxia-telangiectasia (A-T) and in heterozygous relatives [We previously reported a modified micronucleus (MN) assay optimized to detect defects in the S or Gelatives .2 micronucleus assay to further elucidate whether healthy BRCA1 mutation carriers are characterized by an increased in vitro radiosensitivity. The endpoints of the study were (1) micronucleus yields and (2) G2/M checkpoint efficiency ratio. Both endpoints were assessed after irradiating phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes with doses of 2 Gy and 4 Gy. With this assay, we assessed the mean differences in radiosensitivity for heterozygous BRCA1 mutation carriers compared with healthy volunteers. We also scored the overall radiosensitivity in each mutation carrier using a radiosensitivity indicator (RIND) scoring system. In addition, as our BRCA1 population consisted of individuals carrying different BRCA1 mutations, we investigated if there was a link between radiosensitivity and the degree of nonsense-mediated decay (NMD) of the specific mutant allele.In the present study, we applied the G2 MN assay was performed on heparinized blood samples. In addition, we collected heparinized blood samples from healthy volunteers (n = 20) without a personal or family history of BC to determine the normal distribution of micronucleus yields in controls. Lymphocytes were isolated from the blood samples using Lymphoprep™  and were preserved in liquid nitrogen for analysis of NMD of the mutant allele. For a number of mutation carriers (n = 4) and healthy volunteers (n = 7), a second blood sample was taken to determine the reproducibility of the results obtained with the G2 MN assay at different time points.Blood samples were collected from individuals consulting the clinic of the Centre for Medical Genetics, Ghent University Hospital, Belgium (CMGG). Both ethylenediaminetetraacetic acid (EDTA) and heparin blood samples were collected. EDTA samples were used for mutation analysis at CMGG, whereas the Gn = 18) were counselled by clinical geneticists in the context of a predictive (n = 16) or diagnostic (n = 2) test for hereditary BC and signed an informed consent form. Two of the eighteen BRCA1 mutation carriers had developed BC, but their cancer treatment had finished more than 2 years ago; these women are both carriers of a substitution affecting the start codon (M01 and M02). The mean ages of the mutation carriers and healthy volunteers were 40.9 and 35.4 years, respectively .This study was approved by the ethics committee of the Ghent University Hospital (B670201111641 d.d. 20/09/2011). All study participants  after 3 days of incubation, when the blood culture is irradiated. On the contrary, in the G0 MN assay, blood is first irradiated and then cultured in the presence of PHA, resulting in the irradiation of T lymphocytes in G0 phase. The addition of cytochalasin B (cyto B), an agent that blocks cytokinesis, to the blood cultures allows the identification of first-division cells as binucleated (BN) cells. A non- or misrepaired DSB can result in an acentric chromosomal fragment, which is detected as a micronucleus in the cytoplasm of the BN cell [For the G BN cell , 28.2 MN assay, a 50-ml blood culture was set up using 5 ml of heparinized blood, 45 ml of complete RPMI (RPMI with 1 % l-glutamine and 0.5 % penicillin/streptomycin) containing 10 % foetal calf serum (FCS) and 1 ml of PHA  in a T75 culture flask [60Co gamma rays were performed. An overview of the experiments is shown in Fig. 2/M checkpoint, was added to determine the G2/M checkpoint efficiency ratio [2 phase   and thrice with methanol and acetic acid . Finally, the cell suspension was concentrated and spread on slides.More precisely, for the Gre flask . This lacy ratio , 30. On cy ratio  and new ase Fig. . The cul2/M checkpoint efficiency ratio, the quotient was determined for the MN yield obtained in the presence versus the absence of caffeine (MNCaf+/MNCaf−). The lower this ratio was, the more radiosensitive an individual was.After 4′,6-diamidino-2-phenylindole staining, the slides were scanned with a Metafer 4 . This automated image analysis system selects BN cells and determines the number of MN per BN cell see Fig. . For eac2/M checkpoint efficiency ratio assessed in the cohort of healthy volunteers served to determine cut-offs to define the radiosensitivity of individual BRCA1 mutation carriers. An individual value equal to or greater than the mean MN yields in controls + 1 SD scored 1 point (colour-coded orange). A value equal to or greater than the mean MN yields of controls + 2 SD scored 2 points (colour-coded red). An individual value less than the mean of controls + 1 SD was scored as naught (colour-coded white). For the G2/M checkpoint, a similar conversion of the data was performed, with a value less than the mean checkpoint ratio assessed in controls − 1 SD scored 1 point (colour-coded orange) and the mean − 2 SD scored 2 points (color-coded red). An individual value greater than the mean of controls − 1 SD was scored as naught (colour-coded white). The combined scores from MN yields  and checkpoint ratios  were then added to form a single RIND score (range 0–4).To assess the overall radiosensitive phenotype of each patient, a RIND score was calculated. The mean and standard deviation (SD) of the micronucleus yield and the G1 phase of the cell cycle at the moment of irradiation, whereas BrdU-negative binucleated cells were irradiated in G2 phase. We corrected for daughter nuclei incorporating BrdU after binucleation by adding BrdU 2 h before fixation to another subset of cultures. BrdU was visualized by fluorescence immunostaining with a monoclonal BrdU-specific antibody .BrdU was added to blood cultures of three individuals to analyse the phase of the cell cycle at the time of irradiation. BrdU  was added to the cultures immediately after irradiation. Binucleated cells that incorporated BrdU during synthesis were in S or G6 frozen lymphocytes were thawed and cultured in a mix of 1 ml of cRPMI, FCS (10 %), 2-mercaptoethanol  and sodium pyruvate . PHA (10 μl/ml) was added to stimulate cell division. At day 7, whole RNA was extracted using the QIAamp® RNeasy Mini Kit  according to the manufacturer’s instructions. Four hours before extraction, all cultures were split in two, and puromycin  was added to one part. Puromycin was added to avoid NMD, a pathway responsible for the degradation of aberrant mRNA [For this experiment, 2 × 10ant mRNA .cDNAp. Before this cDNA synthesis, samples were treated with DNase  to remove any possible remaining genomic DNA (gDNA).The total RNA and purity was measured using the DropSense96 reader . The RNA was converted into complementary DNA (cDNA) using the iScript™ cDNA Synthesis Kit . When RNA was extracted from cultures with puromycin, the converted cDNA is referred to as BRCA1 allele versus the wild-type (WT) allele at the mRNA level, we performed polymerase chain reaction (PCR) amplification and Sanger sequencing of the amplicon harbouring the germline mutation and a heterozygous single-nucleotide polymorphism (SNP) (c.2311 T > C), if present. An overview of the primers used can be found in Additional file BRCA1 mutation.To determine the ratio of the mutant  (SNP) c.311 T > C2 phase (negative at BrdU staining) at the time of irradiation. In sham-irradiated samples, the percentage of BrdU-negative binucleated cells was much lower (56 ± 8 %). These observations suggest that synchronization of the cells in G2 phase took place because of radiation-induced G2 arrest. When caffeine, an agent that abrogates the G2/M checkpoint [2 phase decreased . The high percentage of cells irradiated in G2 phase indicates that the harvest of BN cells 8 h post-irradiation is optimal.On the basis of data reported in the literature, the cell cycle of proliferating lymphocytes takes between 18 and 22 h. The cell cycle is presented in Fig. eckpoint , was addBRCA1 mutation carriers compared with healthy volunteers, a significantly increased radiosensitivity was observed for both endpoints at both radiation doses of 2 Gy and 4 Gy. Our results also show that the 4 Gy dose point was the most discriminative  for both endpoints. We thus selected this dose to determine the individual overall radiosensitive phenotype. Results of the mutation analysis and individual results obtained for the two radiosensitivity endpoints upon 4 Gy irradiation are shown in Table The MN yield obtained for the sham-irradiated samples did not show a significantly different mean result between mutation carriers and healthy volunteers Table . The meaBRCA1 mutation carriers (median 2) and healthy volunteers (median 0) . Figure BRCA1 mutation carriers and healthy control subjects for the five different RIND scores. The distribution amongst the RIND scores between the two groups is significantly different . The significantly different median and the significantly different distribution over the RIND scores obtained for both groups point towards a difference in response to radiation.Significantly different RIND values were found for p > 0.05 for both MN yield and G2/M checkpoint efficiency; repeated-measures analysis of variance).Repeated assessments were performed on blood specimens taken on two different occasions from a random sample of seven healthy volunteers and four mutation carriers to exclude intraindividual variations. Although minor variations were observed, no significant differences were observed between repeated measurements , we found evidence for NMD. In general, 25–30 % residual truncated mRNA was detected in carriers of a premature termination codon (PTC) mutation. Ten of fifteen patients with a truncating allele showed a radiosensitive phenotype .n = 3), we have no evidence for NMD. The effect at the mRNA level for the c.212 + 3A > G splice-site mutation has previously been studied with quantitative PCR by our group. We showed that no full-length transcript is formed, but that it leads to a significantly increased expression of an alternative transcript , which is not subjected to NMD [2/M checkpoint efficiency. For M01, carrier of the c.1A > G start codon mutation, no decline of the mutant allele could be detected when analysing the SNP data . Moreover, 28 % of BRCA1 mutation carriers had RIND scores equal to 3 or 4, scores that were never observed in healthy volunteers. This simple scoring system can be valuable in assisting physicians in their decision-making in clinical follow-up and for refinement of radiotherapy at the individual level. Since our study is limited in sample size, a larger prospective study with blood samples of BRCA1 mutation carriers will be undertaken to confirm and prove the importance of an in vitro radiosensitivity scoring system to assist clinical management of BRCA1 mutation carriers.Since it was our aim to develop a radiosensitivity assay applicable in a clinical setting, we performed the Gdevelops , 43, 44.Haploinsufficiency has been suggested as the main mechanism for hereditary breast carcinogenesis . HoweverThe majority of patients included in this study are heterozygous for a mutation located in the central part of the gene leading to a PTC. These PTC-inducing mutations result in a truncated mRNA, which can be degraded by NMD. Generally, we observed that the mutated PTC allele was expressed at a ratio of 25–30 % of the WT allele at the mRNA level. Our results are consistent with the data of Anczuców et al. and Perrin-Vidoz et al. , 47. BotBRCA1 mutation carriers are compared as a group with non-carriers . For the patient with the c.212 + 3A > G mutation (M03), equal expression of a full-length transcript and a transcript lacking the last 22 nucleotides of exon 5 was observed in previous research by our group and confirmed by others , 36. A hs e.g., , 38, 48), 48BRCA1BRCA1 mutation carriers and patients with BC [Unique to this radiosensitivity study compared with others are that we had access to material from several individuals from one family and that we could also evaluate the radiosensitive phenotype from several unrelated individuals carrying the same mutation. For four unrelated carriers of a Belgian founder mutation c.2359dup (p.Glu787fs*3), a large variation for the radiosensitive phenotype based on the RIND score was observed (range 0–4) . All study participants signed an informed consent form.


The deposition and evolution of the FDTS-based seed layer is investigated by X-ray photoelectron spectroscopy, Auger electron spectroscopy, and transmission electron microscopy. Crystalline quality of graphene transferred from Cu is monitored during formation of the seed layer as well as the HfO2 growth using Raman spectroscopy. We demonstrate that FDTS-based seed layer significantly improves nucleation of HfO2 layers so that graphene can be coated in a conformal way with HfO2 layers as thin as 10 nm. Proof-of-concept experiments on 200 mm wafers presented here validate applicability of the proposed approach to wafer scale graphene device fabrication.We investigate the use of perfluorodecyltrichlorosilane-based self-assembled monolayer as seeding layer for chemical vapour deposition of HfO We adapt an established process used for surface energy modification in the fabrication of micro-electro-mechanical systems (MEMS) structures and their cavities2 on graphene. Wafer scale compatibility and maturity of the proposed pre-treatment can enable fast adoption for the fabrication of graphene-based devices on large-diameter wafer scale.In this work we investigate a pre-treatment of graphene with perfluorodecyltrichlorosilane (FDTS) prior to HfO2/Si(100) wafers before and after FDTS coating are shown in 20−1, FWHM(2D): 36 cm−1) proving that the FDTS coating process does not negatively affect the crystalline quality of graphene sheet. The observed red shift of about 8 cm−1 after SAM formation may be associated with a process induced electrostatic doping of graphene22The representative Raman spectra of a graphene layer transferred to 300 nm SiO2 substrate and weaker C signal due to the absence of graphene. The additional scans performed on the remaining area (regions 1 and 2) show reduced Si and O signals and an intense C peak due to the closed graphene layer. A fluorine signal of similar intensity is detected in all investigated regions proving that the FDTS SAM was coated homogeneously on the substrate surface. More detailed chemical analysis of the SiO2/graphene/FDTS interfaces was performed by X-ray photoelectron spectroscopy (XPS), as shown in The optical microscope image of a transferred graphene layer after the FDTS deposition process is shown 2 substrate which can be detected through the graphene layer. After the FDTS coating process, a clear F1s appears and the O1s peak and Si 2s and 2p peaks are attenuated. Based on these XPS results, we can also conclude, that no additional elements are incorporated during the FDTS deposition into the graphene layer  rises with temperature. As to be seen in the spectra, the characteristic oxidized carbon species (COOR) signals are also observed at a binding energy of 288 eV282/Si substrate is adsorbed, resulting in an increase of the signal of oxidized carbon species C-O and COOR and a peak shift of the C-O component28The intensity of the aromatic C-C signal is reduced after the annealing at 400 °C, as illustrated in As the decomposed FDTS chains could interact with graphene, a Raman analysis was performed after the annealing procedure in order to investigate the quality of the graphene layer. As illustrated in The following conclusions can be drawn from the discussions above: The FDTS seed layer covers graphene homogenously at room temperature and this process does not affect the crystalline quality of graphene. During thermal treatment, long fluorine chains partially decompose and some fluorine species desorb from the surface as it is evidenced by reduced F1s signal. After annealing the high crystalline quality of graphene remains unaffected as proved by Raman spectroscopy investigations.2 by CVD on pretreated graphene layers. As demonstrated by Lukosius et al.2 on transferred graphene is feasible without any initial pretreatment of the graphene surface with the restriction, that HfO2 films have to be thicker than 20 nm to be uniform and closed. Due to nucleation sites created by the FDTS based seed layer, the minimum HfO2 film thickness should be drastically reduced. In the first step, we chose a thickness of nominally 10 nm HfO2 to test the influence of the FDTS nucleation capability. Two graphene samples were transferred to pre-structured 75 nm TiN/Si substrates, which will be used for ongoing electrical characterization. After the transfer process one sample was coated with FDTS, while the other sample was left pristine. Finally, 10 nm HfO2 were deposited by CVD onto both samples.The next section is dedicated to the growth of HfO2 layer, transmission electron microscopy (TEM) was applied. The samples were firstly coated with an Al-layer to protect the sample and then a lamella was prepared. According to the high resolution TEM images, there is a huge difference between the two samples, as illustrated in 2 layer, the sample with FDTS coating  to remove the polymer. Target substrates in this study were 200 mm Si(100) wafers covered with either 300 nm thermal SiO2 or 75 nm CVD TiN layers.Commercially available graphene was transferred from Cu growth substrates by detaching graphene from Cu foils with poly(methyl methacrylate) (PMMA) assisted wet etching in Ammonium persulfate (80 mg/ml in water)3(CF2)7(CH2)2SiCl3) coating of graphene was carried out with a 200 mm AURIX tool . The standard FDTS coating process used in MEMS fabrication is performed at substrate temperatures of 30 °C and is divided into two parts: the first part cleans and activates the surface by exposure of the wafer to O2 plasma, the second part provides the N2 bubbled exposure to FDTS and DI water at a process pressure of 40 Torr and for a duration of 300 s. The flow rate of the two carrier gases are 20 sccm for FDTS and 100 sccm for DI water. As O2 plasma readily introduces defects in the crystalline lattice of graphene, this step was omitted in the process flow.The FDTS (CF2 growth was performed in an atomic vapor deposition tool with Hf(NMeEt)4 precursor and oxygen as the reactive gas2 deposition was set to 400 °C for a duration of 3 min. The deposited layers are polycrystalline. More details about the depositions of HfO2 by CVD have been reported elsewhereHfO−8 Torr and the spectra were measured with X-ray beam size of 100 × 100 μm. A PHI 670 Auger Nanoprobe (Perkin Elmer) was used for Auger electron spectroscopy. The spectra were measured with a beam voltage of 10 keV. Structural investigations and EDX-measurements were done by transmission electron microscopy using a FEI Tecnai Osiris instrument operated at 200 kV.A Renishaw InVia micro-Raman spectrometer equipped with a 514 nm wavelength laser, an 1800 lines/mm grating and 50x objective was used to acquire Raman spectra. XPS measurements were performed using PHI 5000 Versa Probe II (ULVAC-PHI) with monochromatic Al-Kα irradiation. The chamber pressure was ~3 × 10How to cite this article: Kitzmann, J. et al. Perfluorodecyltrichlorosilane-based seed-layer for improved chemical vapour deposition of ultrathin hafnium dioxide films on graphene. Sci. Rep.6, 29223; doi: 10.1038/srep29223 (2016).


Patients suffering from migraine with aura can have either pure visual auras or complex auras with sensory disturbances and dysphasia, or both. Few studies have searched for possible pathophysiological differences between these two subgroups of patients.N = 27) and another with complex neurological auras . We recorded pattern-reversal visual evoked potentials  and measured amplitude and habituation (slope of the linear regression line of amplitude changes from the 1st to 6th block of 100 sweeps) for the N1-P1 and P1-N2 components in patients and, for comparison, in 30 healthy volunteers (HV) of similar age and gender distribution.Methods - Forty-seven migraine with aura patients were subdivided in a subgroup with exclusively visual auras  and MA+  patients. However, VEP N1-P1 amplitudes across blocks were normal in MA patients, while they were significantly greater in MA+ patients than in HVs.VEP N1-P1 habituation, i.e. amplitude decrement between 1st and 6th block, which was obvious in most HV (mean slope −0.50), was deficient in both MA (slope +0.01, Our findings suggest that in migraine with aura patients different aura phenotypes may be underpinned by different pathophysiological mechanisms. Pre-activation cortical excitability could be higher in patients with complex neurological auras than in those having pure visual auras or in healthy volunteers. Migraine with aura (MA) is defined as attacks of neurological symptoms that last no more than 60 min and may be followed or accompanied by headache . The most common aura symptoms are visual (e.g. scintillating scotoma), while sensory and aphasic auras are present in a smaller proportion of patients [The most likely cause of the migraine aura, Leão’s cortical spreading depression (CSD), consists of a brief neuronal depolarisation followed by a long-lasting wave of neuronal depression that often spreads postero-anteriorly in the occipital cortex and can reach the parietal and/or temporal lobes , 5. IndiDuring the last decade various research groups have demonstrated significant changes of bioelectrical activity in the visual cortex of migraine patients over the migraine cycle. In particular, cortical visual evoked potentials (VEPs) are used to infer the mass activity of visual cortical neurons. Most, though not all , VEP, reIt was suggested that migraine with aura is a condition with a spectrum of clinical subtypes that likely differ in pathophysiological mechanisms . DistincN = 27) and those who had in addition paraesthesia and/or dysphasia . Auras usually developed gradually and were followed by headache. None had hemiplegic or brainstem auras or persistent aura without infarction. All patients had a varying combination of attacks with or without aura. We took information on various clinical characteristics by collecting up to two-month headache diaries at the time of the screening visit and the day of the recording session. Patients had to indicate duration of migraine history (years), attack frequency (n/month), attack duration (hours), and number of days elapsed since the last migraine attack (in 35 out of 47 patients)  who attended our headache clinic. We discarded recordings of 10 patients who did not fulfil our primary inclusion criteria (see below), and of one patient because he was an outlier. The final analysis set comprises therefore 47 patients . Patients were subdivided into those who experienced pure visual aura  generated on a TV monitor; the reversal rate was 1.55 Hz ). At the viewing distance of 114 cm, the single checks subtended a visual angle of 15 min, while the checkerboard subtended 23°. Recordings were done with the best corrected visual acuity of > 8/10 at the viewing distance. Subjects were instructed to fixate with their right eye a red dot in the middle of the screen with the contralateral eye covered by a patch to maintain stable fixation. VEP were recorded from the scalp through silver cup electrodes positioned at Oz (active electrode) and at Fz . A ground electrode was placed on the right forearm. Signals were amplified by Digitimer™ D360 pre-amplifiers  and recorded with a CED™ power 1401 device . A total of 600 consecutive sweeps each lasting 200 ms were collected and sampled at 4000 Hz.Subjects were sitting in an acoustically isolated room with dimmed lights in front of a TV monitor surrounded by a uniform luminance field of 5 cd/mAfter applying off-line a 35Hz low-pass digital filter, cortical responses were partitioned in 6 sequential blocks of 100, consisting of at least 95 artifact-free sweeps. Responses in each block were averaged off-line (“block averages”) using the Signal™ software package version 4.10 (CED Ltd). Artefacts were automatically rejected using the Signal™ artefact rejection tool if the signal amplitude exceeded 90 % of analog-to-digital converter (ADC) range and was controlled by visual inspection. Through this approach, we made sure to exclude all severe artefacts but not to remove any signal systematically because background EEG amplitudes vary between subjects. The EP-signal was corrected off-line for DC-drifts, eye movements and blinks.VEP components were identified according to their latencies: N1 was defined as the most negative peak between 60 and 90 ms, P1 as the most positive peak following N1 between 80 and 120 ms and N2 as the most negative peak following P1 at between 125 and 150 ms .We used the Statistica for Windows (StatSoft Inc.), version 8.0 for all analyses. Preliminary descriptive analysis showed that the VEP N1–P1 and P1–N2 peak-to-peak amplitudes of the six blocks and the habituation slopes had a non-normal distribution. After log transformation, all data reached normal distribution ; the within-subject factor was “blocks”. Two models of repeated measures ANOVA (rm-ANOVA) followed by univariate ANOVAs were employed to investigate the interaction effect. Univariate results were analyzed only if Wilks’ Lambda multivariate significance criterion was achieved. The sphericity of the covariance matrix was verified with the Mauchly Sphericity Test; in the case of violation of the sphericity assumption, Greenhouse-Geisser (G-G) epsilon (ε) adjustment was used. In rm-ANOVA and ANOVA models, partial eta2A General Linear Model approach was used to analyze the “between-factor” × “within-factors” interaction effect. The between-subject factor was “group” (HV vs. MAtot or HV vs. MA subgroups), using Tukey test for post hoc analysis. Also for ANOVA partial eta2 and op was used. Statistical significance was set at p < 0.05.A regression analysis was used to disclose linear trends in VEP amplitude across blocks (slope) in each group. For slope, we employed ANOVA with group factor “group” .Recordings from 47 participants who fulfilled the inclusion criteria yielded analysable VEP data. The two patient subgroups MA and MA+ did not differ in other clinical features Table .p > 0.05)  (Table p = 0.104), univariate rm-ANOVAs for N1–P1 peak-peak amplitude confirmed the significant interaction factor effect  observed (see above) at the multivariate test. Post-hoc analysis showed that VEP amplitudes differed between groups only in the last block . This was not so in MAtot, where this comparison did not reach the significance level (p = 0.994).In the rm-ANOVA model with N1–P1 peak-peak amplitude as dependent variable, multivariate test was significant for the “7) Table . After cgroup” × “blocks” interaction effect  . There were no other significant correlation between neurophysiological and clinical data.In the MAtot group the N1–P1 amplitude slope correlated positively with the number of days elapsed since the last migraine attack  . After checking that the sphericity assumption was not violated (Mauchley Test: p = 0.126), univariate rm-ANOVAs for N1-P1 peak-peak amplitude confirmed the significant interaction factor effect  observed (see above) at the multivariate test. On post-hoc analysis there was a significant increase of N1-P1 VEP amplitude from the 2nd to the 6th block in MA+ compared with MA, and from the 4th to the 6th block in MA+ compared with HV .In the rm-ANOVA model with N1–P1 peak-peak amplitude as dependent variable, multivariate test was significant for the “group” × “blocks” interaction effect .In the rm-ANOVA model with P1–N2 peak-peak amplitude as dependent variable, multivariate test was not significant for the “group” × “blocks” interaction effect .The linear regression N1–P1 slope of VEP amplitudes over all blocks differed significantly between the three groups  and positively with days elapsed since last attack , correlations that were not found in MA patients  patients without phenotype distinction. In some reports the grand-average of VEP N1–P1 and/or P1–N2 amplitudes was found greater in MwA patients than in controls –35 and/oFrom a pathophysiological point of view, it is interesting to compare MA+ and chronic migraine that is also thought to be associated with true cortical hyperexcitability. The evidence in chronic migraine comes from studies of somatosensory evoked potentials  and magn+/K+ ATPase pump [The pathophysiological determinants of different aura phenotypes and related differences in interictal visual evoked potential profiles remain speculative. Cortical spreading depression (CSD) is thought to be the pathophysiological substrate of the migraine aura. CSD is an electrochemical wave that usually starts in the posterior regions of the brain and spreads anteriorly at approx. 3 mm/min, accompanied by biphasic cerebral blood flow changes . In sevease pump . The disThe neurovascular tone is modulated by local factors such as oxygen availability or lactate concentrations, and by subcortical monoaminergic projections , 53. DurSeveral studies suggest that the abnormalities of energy metabolism could be more pronounced in migraine with complex neurological aura. The phosphocreatine/phosphate (PCr/Pi) ratio, a marker of the brain’s energy reserve, differed significantly between patients with different aura phenotypes and was lowest in those with more complex auras . In a 1HThat genetic load can influence CSD patterns and severity is evidenced by the studies of the “knock-in” mice wearing CACNA1A  or ATP1AOne can only speculate on the possible relation between the ictal phenomena, i.e. CSD and its spreading, and the VEP abnormalities found interictally. We know of only one study in photosensitive subjects with a photo-paroxysmal response to intermittent photic stimulation where increased VEP amplitude was correlated with spread of the paroxysmal EEG activity to more anterior brain areas . In photWhatever the possible relation between ictal CSD and interictal VEP might be, the pathophysiological mechanisms underlying VEP habituation are not permanently influenced by the ictal phenomena, even in MA+ patients. In the latter, indeed, like in MwA patients overall and in migraine without aura , the VEPAs other neurophysiological studies, ours has some methodological shortcomings. For instance, the investigators were blinded during off-line analyses of VEP data, as applied in previous studies by independent groups , 73, butThough we refer herein to habituation as the common feature of responses to any type of repeated sensory stimuli and to its classical definition of “a behavioral response decrement that results from repeated stimulation and that does not involve sensory adaptation/sensory fatigue or motor fatigue” , 79, we We also are aware that our samples are relatively small and that clinical correlations are retrospective. Further studies are needed to repeat the analysis in a larger clinical sample with various migraine phenotypes and with a longitudinal, prospective follow-up of patients, allowing to record them during attacks as well as at different time points between attacks.To summarize, this study shows that the clinical heterogeneity of migraine with aura is reflected in distinct visual evoked potential profiles. Patients with complex neurological auras differ from those with strictly visual auras by an interictal increase of VEP amplitudes suggestive of an underlying genuine persistent visual cortical hyperexcitability. Whether this is related to CSD features, their effect on cortex and thalamus or to a common neurobiological or genetic denominator between CSD and VEP profile determinants remains to be determined.


The cephalochordate Amphioxus naturally co-expresses fluorescent proteins (FPs) with different brightness, which thus offers the rare opportunity to identify FP molecular feature/s that are associated with greater/lower intensity of fluorescence. Here, we describe the spectral and structural characteristics of green FP (bfloGFPa1) with perfect (100%) quantum efficiency yielding to unprecedentedly-high brightness, and compare them to those of co-expressed bfloGFPc1 showing extremely-dim brightness due to low (0.1%) quantum efficiency. This direct comparison of structure-function relationship indicated that in the bright bfloGFPa1, a Tyrosine (Tyr159) promotes a ring flipping of a Tryptophan (Trp157) that in turn allows a cis-trans transformation of a Proline (Pro55). Consequently, the FP chromophore is pushed up, which comes with a slight tilt and increased stability. FPs are continuously engineered for improved biochemical and/or photonic properties, and this study provides new insight to the challenge of establishing a clear mechanistic understanding between chromophore structural environment and brightness level. Aequorea victorea2First discovered in 1961 by Shimomura and colleagues in the cnidarian jellyfish GFP gene acquires fluorescence. This genetically encoded autonomy and self-assembly makes the GFP gene a powerful biological tag for the in vivo visualization of a wide array of cellular structures and processes29Critical steps in the wide-spread use of GFP included the elucidation of the three dimensional structure of the wild-type GFP67Aequorea victorea have been deposited into the Protein Data Bank (PDB), and are described in many publications (www.rcsb.org)AvGFP and the ability to decipher the effect of random mutations and directed amino acid substitutions on the proteins' optical characteristics1213Since the publication of the landmark crystal structures from the Remington and Phillips groups, respectively4Aequorea jellyfish, in order to identify whether Nature could have “engineered” GFPs with other structural backbones, or fluorescent proteins with different structure/s, and hopefully with different (and attractive) biophysical and photonic properties. As a result, an increasing number of GFP-like crystal structures from many different species of cnidarians initially, but more recently also from crustaceans, have been deposited in the PBD. In particular, a red fluorescing GFP-like protein, DsRED, was identified in the cnidarian stony coral Discosoma1617212226Some research efforts were also developed towards finding fluorescent proteins from other organisms than the −1 (ZsGreen1)−1]: 37 for TurboGFP, 40 for ZsGreen1, as low as 26 for T-Sapphire, and as high as 41 for Azami GreenQuantum yield (QY), also referred to as the quantum efficiency (QE), is the probability that an excitation of the electronic dipole of the chromophore leads to the emission of a photon instead of a heat dissipating transition as relaxation back to the ground state occursThe need for GFPs with improved brightness becomes apparent as researchers push the limits of signal detection. GFPs are widely used to address protein-protein interactions based on single molecule fluorescence detection, or fluorescence shifts from Förster Resonance Energy Transfer (FRET)2930Branchiostoma floridae35We recently identified a family of GFP proteins (bfloGFP) in the cephalochordate, E. coli with thrombin cleavable N-terminal hexahistidine tags, and purified via Ni2+-NTA affinity and size exclusion chromatography (SEC). SEC confirmed the monomeric character of eGFP while both bfloGFPa1 and bfloGFPc1 behaved as dimers  bfloGFPa1 = 497 ± 45 nm and bfloGFPc1 = 493 ± 55 nm for absorbance, and bfloGFPa1 = 512 ± 62 nm and bfloGFPc1 = 521 ± 58 nm for fluorescence emission. The excitation spectrum of bfloGFPa1 peaked at 500 nm (FWHM = 38 nm) for emission fixed at 512 nm , and retained their color throughout the duration of the data collection at 100 K. bfloGFPa1 crystals diffracted x-rays to 1.35 Å resolution while bfloGFPc1 crystals yielded measurable diffraction to 1.9 Å resolution. Phasing of bfloGFPc1 (8 molecules per asymmetric unit) via molecular replacement afforded high quality electron density maps for monomers A–D while the electron density for monomers E–G were of a lower quality. In the case of bfloGFPa1 (2 molecules per asymmetric unit), electron density maps for both monomers were of a high quality. Model building and refinement yielded final GFP structures exhibiting electron density for all amino acid residues except residues 1 and 182–187 (bfloGFPa1 monomer A), residues 1 and 219 (bfloGFPa1 monomer B), and residues 1–2 (bfloGFPc1a) .−1 cm−1 and 98,800 M−1 cm−1, respectively; both coefficients were significantly larger than the extinction coefficients of copGFP −1 cm−1−1 cm−1) (929Renilla reniformis (sea pansy) GFP with a value of 133,000 M−1 cm-13940Molar extinction coefficients measured for bfloGFPa1 and bfloGFPc1 were 120,900 M−1 cm−1) . bfloGFPAmphioxus GFPs compared to other GFPs; bfloGFPc1 has a very low QE of 0.0015, while bfloGFPa1 exhibited the maximum possible QE of 1.04 ± 0.05 −1  * cm)−1 . To the a could not be determined for the bfloGFPc1 fluorescence due to the barely detectable fluorescence well below the detection limit for conventional spectroscopic instrumentation. As for bfloGFPa1, it exhibited a pKa of 3.0 for its fluorescence, exhibiting high intensity for pH values ranging from 3.5 to 11 . The crustacean copepod GFP shares up to 35% amino acid sequence identity with bfloGFPs (compared to <19% between cnidarian GFPs and bfloGFPs), and is in fact also the closest GFP-containing evolutionary relative to AmphioxusDespite biochemical characteristics that were clearly different from one another (see above), we found that bfloGFPa1 and bfloGFPc1 shared essentially identical global tertiary and quaternary structures with a root mean square deviation (rmsd) for backbone atoms of 0.9 Å; for this reason, the results described in this section will use “bfloGFPs” to refer to both bfloGFPa1 and bfloGFPc1. bfloGFPs possess the classic 11-stranded β-barrel structure observed for all GFPs crystallized to date. In bfloGFPs, the barrel is capped by a combination of inter-strand loops at the bottom, and inter-strand loops and helices at the top. Following the central chromophore bearing helix (α1) there is a short helix at the top of the barrel (α2), common to all GFP structures, juxtaposed next to a third short helix (α3) that appears unique to While globally the architectures of bfloGFPa1 and copGFP displayed a high degree of structural similarity, the local environments of the chromophores in both cases were highly divergent, with only 7 out of 19 chromophore-contacting residues strictly conserved . Arg 195Amphioxus GFPs.When bfloGFPa1 is compared to bfloGFPc1, the degree of conservation between chromophore sites was 37% with only 7 out of 19 residues retained. However, only 4 out of 7 residues for each set were common to all three proteins . Interes3 vs. 386 Å3). Interestingly, the chromophore cavities of bfloGFPa1 and copGFP were similar in volume (265 Å3 vs. 280 Å3)Likewise, the indole ring of Trp 157, abutting the phenolic oxygen moiety of the chromophore, flips its relative orientation for bfloGFPa1 compared to bfloGFPc1. In bfloGFPc1, the Nε1 nitrogen of the Trp 157 forms a hydrogen bond with the phenolic oxygen while in bfloGFPa1 the ring flip negates a similar interaction . These d2, which is much smaller than 39.2 Å2 for bfloGFPc1 or 26.8 Å2 for copGFP. While only partially indicative of thermal motion associated with any particular atom or group of atoms, the low B-factors for the atoms making up the chromophore in the bfloGFPa1 crystal suggests the chromophore possesses particularly low thermal motion with energy dissipation occurring primarily through fluorescence in bfloGFPa1.The x-ray crystal structure of the brightly fluorescent bfloGFPa1 exhibited two unique features that were different from the structures of the dimmer bfloGFPc1 and copGFP. The first property relates to the atomic displacement factors (ADP) or B-factors, which partially represent the thermal motion and disorder of a particular atom averaged across one or more crystals used for the dataset employed during coordinate and ADP refinementThe second unique feature relates to the hydrogen-bonding network of the chromophores' phenolic hydroxyl groups. In the case of bfloGFPc1, copGFP, and many other GFPs, the phenolic hydroxyl moiety forms hydrogen bonds with one water molecule and two residues from the protein cavity, usually involving a Thr-Arg (copGFP), Ser-Trp (bfloGFPc1), or His-Thr (eGFP). In the case of brightly fluorescent bfloGFPa1, a Cys residue, Cys 139, resides proximal to and within hydrogen bonding distance of the chromophore's phenolic hydroxyl moiety. The remaining two hydrogen bonds form with water molecules anchored in the chromophore cavity , which eAmphioxus.Despite more than a decade of GFP engineering directed at developing FPs with varied photonic properties, an understanding of the factors modulating “brightness” remain uncertain. Given the remarkably high degree of brightness associated with bfloGFPa1 and the unexpectedly low degree of brightness for its evolutionary cousin, bfloGFPc1, we investigated the atomic resolution architecture of both proteins with the aim of establishing a structural basis for this surprising difference in GFP brightness within a single organism, the cephalochordate Amphioxus (bfloGFPs) with the structure of the evolutionarily related copepod GFP (copGFP). Two logical mechanisms for tuning brightness and QE emerged from this comparative analysis. Specifically, the mechanisms relate to the extreme conformational rigidity of the chromophore in the exceptionally bright Amphioxus GFP, bfloGFPa1, and its alteration of the hydrogen-bonding environment surrounding the chromophore's phenolic hydroxyl moiety.In this study, we address the structural basis of brightness in GFPs by comparing two contrasting GFP structures from per se, indicating that high fluorescence QE is not necessarily associated with planar chromophoric ringsCyphastrea microphthalma is the only other native GFP reported so far with 100% QE−1 cm−1−1 cm−1 for bfloGFPa1. Crystallography data from VFP are currently not available to address similarities or differences with structural features of bfloGFPa1.QE positively correlates with increased rigidity of chromophores and in GFPs, this rigidity is provided by its protective β-barrel structure144611482 (2CD1) and 21.1 Å2 (2DUG) , while being <10 Å2 for bfloGFPa1. The low B-factor for bfloGFPa1 clearly indicates that the chromophore and its surrounding have limited structural flexibility (hence high stiffness), which is known widely in the literature to be associated with greater QE  is a partial measure of this rigidity, using as a proxy the level of flexibility induced by thermal energy. The B-factor is therefore resolution dependent, and when comparing structures resolved at the same crystallographic resolution, the lower the B-factors of a particular groups of atoms, the more rigid the associated structure. We screened the PDB for GFP structures refined to a similar resolution as bfloGFPa1 and found 15 of them  also stu2 (2DUG) . All the QE e.g.,4652.2, which is indicative of high stiffness of the chromophore and thus high QE. This suggests that there is another factor that affects QE, which could be the chromophore pocket. Indeed, we believe that the conformational rigidity of the bfloGFPa1 chromophore may be in part the result of the smaller volume of its chromophore pocket relative to its very dim cousin bfloGFPc1. Nevertheless, the volume of the bfloGFPa1 chromophore pocket is approximately 65% smaller than the same pocket calculated for the static x-ray crystal structure of eGFP, and roughly equivalent to the one found in an evolutionarily related GFP, namely copGFP, yet both of these latter GFPs are 5–10 times dimmer than bfloGFPa1. This observation indicates that volume of the chromophore pocket alone cannot be the only factor modulating QE and ultimately brightness. A restricted pocket size would likely contribute to maintaining the chromophore in a strained, high-energy conformation, thus contributing to an increased efficiency for photon transfer and fluorescence, while also increasing protection of the chromophore from quenching agents53If the B-factor clearly plays an important role driving the QE in GFPs, it is also likely not the only one, and several factors might have to act in synergy for reaching maximal QE. This might be indicated for example by the Glycine-containing mutant 3CB9, which corresponds to a redox sensitive GFP with insertion of an extra amino acid, in this case arginineAmphioxus GFP structures elucidated and described here, it is clear that the chromophore packs against a conserved Pro, Pro 55, that assumes distinct backbone conformational states in each GFP; in the intensely bright GFP, bfloGFPa1, the peptide bond, Ile 54 – Pro 55, adopts a cis orientation while the same peptide, Ile 54 – Pro 55 adopts a trans orientation in the very dim bfloGFPc1. These divergent backbone conformations shift the positions of the encapsulated chromophore helical segment, resulting in the formation of a single hydrogen bond between the imidazolinone nitrogen and the backbone carbonyl oxygen of the Pro 55 in bfloGFPc1. Such an alteration of the hydrogen bonding patterns of the β-barrel with the chromophore would expectedly lead to change(s) in the chromophores' emission efficiencies and the brightness of the resultant fluorescenceThrough comparisons of GFP structures possessing widely varying spectral properties but isolated from the same organism, we were able to clearly define other structural features not obvious from sequence alignments alone that likely contribute to the modulation of QE and brightness in these naturally evolved FPs. In the An additional set of changes in the chromophore environments when comparing bfloGFPa1 and bfloGFPc1 centers around the rotameric state of the chromophore contacting Trp 157. In bfloGFPa1, this rotation disrupts a hydrogen bond between the indole NH of Trp 157 and the hydroxyl moiety of Tyr 159 that occurs in bfloGFPc1. This structural distortion results in the phenyl portion of Trp 157 abutting the phenolic end of the chromophore, thus increasing the hydrophobicity of the bfloGFPa1 chromophore environment compared to that of bfloGFPc1. This rotation appears to be stabilized by the edge-to-face interaction between the aromatic moieties of Trp 157 and Tyr 159. Without an actual experimentally determined three-dimensional structure, this type of second tier interaction can be challenging to identify unequivocally.Amphioxus bfloGFPa1 appears to be an ideal candidate for future applications and engineering efforts. This has been materialized already by the use of bfloGFPa1 in bioassays where increased brightness and biochemical stability were key in providing signal not otherwise available using conventional FPs56Branchiostoma lanceolatum, the amphioxus species from Europe, is now commercially available (under the name of lanGFP) and showing about 4x-increased brightness compared to eGFPCurrently, genetic engineering of GFPs is focusing more and more on improving the QEs but remains an ongoing challenge compared to modifying the biochemical and/or biophotonic properties of FPs313We used conventional techniques of mutagenesis of critical residues in a first attempt to demonstrate the role of key residues from the chromophore pocket that are associated with high versus low brightness or quantum efficiency. We performed site directed amino acid substitutions on bfloGFPa1 based on differences with bfloGFPc1 and copGFP. bfloGFP mutants were generated using the QuikChange  PCR-based method. Mutant enzymes were expressed and purified as described in this study for wild-type bfloGFP. In addition to wtGFP controls, we completed five different single mutations chosen to provide further understanding of the relationship amongst the diverse GFPs within amphioxusThe five bfloGFPs mutants were successfully expressed and exhibiting strong fluorescence, except for C139S, F155L and R195A that showed relatively lower fluorescence intensity, yet always with a spectrum similar to that of bfloGFPa1. The absorbance spectrum was also measurable for each mutant, and remained similar amongst all bfloGFPs, yet showing relatively greater values for F155L. Complete and detailed biochemical and spectroscopic characterization of the mutants could however not be performed because all mutants were unstable, constantly precipitating out of solution, which was otherwise not observed for the wtGFP control, or when maintained in darkness. Consequently, determining accurate protein concentration necessary for calculation of the extinction coefficient, for performing biochemical experiments, and for interpretation of the spectroscopic measurements from the bfloGFPa1 mutants was not possible using the current protocols. However, these data suggests that the five mutations we performed in the chromophore pocket do not seem to qualitatively affect the spectral characteristics of bfloGFPs, while clearly indicating that each of the substituted residues  plays a key role in contributing to energetic stability of the whole protein.Amphioxus contains 16 bfloGFPs organized in six clades12Amphioxus bfloGFPa1 and bfloGFPc1 were cloned and purified as previously reportedAmphioxus GFPs−1 cm−1 at 446 nm)Protein concentrations were calculated using the extinction coefficient of the chromophore after denaturation in 0.1 N NaOH  of bfloGFPa1 and bfloGFPc1 was measured from six independent replicate expression batches, with and without His-tag, using the method of relative actinometry. Emission of the GFPs was then expressed relative to a known standard with the same absorption at the excitation wavelength of 450 nm, keeping all instrumental conditions identical2, 50 mM Tris-HCl, pH 8, and 1 mM DTT). The SpectraMax M2 plate reader equipped with a 495 nm excitation filter was used to follow refolding and re-oxidation, using as a proxy the fluorescence intensity of the GFPs. Samples were excited at 475 nm and fluorescence emission recorded at 508 nm (eGFP) or 516 nm (bfloGFPa1) every second for 3,000 sec (50 min). Both refolding and re-oxidation curves were modeled employing a two-phase exponential equation using Prizm 4.00 (GraphPad). The equation was as follow: Y = P + CF*exp(−KF*X) + CS*exp(−KS*X), where “P” is the Plateau value reached at infinite time for “X”, “CF” and “CS” are the two time constants for the Fast and Slow half-life, respectively, and “KF” and “KS” are the rate constants for the Fast and Slow half-life, respectively. The Fast and Slow half-lives were then computed as ln(2)/KF and Ln(2)/KS, respectively. Parameters of the equations were estimated by iteration from the raw values of fluorescence, following the nonlinear estimation method of Quasi-Newton, while the resulting best fit model is indicated by the R corrected for non-linear systemsRefolding and re-oxidation experiments were performed as previously describedAmphioxus GFP fluorescence at various pH values was evaluated using citric acid - sodium citrate (pH 3–5), sodium phosphate (pH 5–6), Tris-HCl - Tris-Base (pH 6–9), and glycine - NaOH (pH 9–12). Each sample consisted of concentrated GFP in a weakly buffered solution (~5 mM) diluted approximately 200-fold  into 50 mM buffer at a given pH. The SpectraMax M2 plate reader with a 495 nm cut-off filter was used to record fluorescence emission at 508 (eGFP) and 516 (bfloGFPa1) upon excitation at 475 nm. Absorbance spectra of each sample were subsequently recorded and used as internal controls for calibration of protein concentrations.bfloGFPc1 crystals were grown overnight by vapor diffusion at 4°C in 2.0 μL drops, consisting of 1 μL crystallization reservoir , and 1 μL protein solution [419 μM bfloGFPc1]. Diffraction data were collected at the Berkeley Advanced Light Source (ALS) synchrotron beamline 8.2.2 on a Quantum Q315 CCD detector. bfloGFPc1 crystallized in spacegroup C2, a = 158.76 Å, b = 130.46 Å, c = 106.33 Å, a = g = 90°, b = 128.39° with eight monomers in the asymmetric unit. Data were indexed, integrated, and scaled to 1.95 Å with HKL2000Phase determination of bfloGFPc1 was accomplished via molecular replacement (MR) using the program PhaserPhase determination of bfloGFPa1 was accomplished via molecular replacement (MR) using the program Phaser. The MR search model was the refined structure of bfloGFPc1. MR phases were used for manual model building of the bfloGFPa1 tertiary structure in CootD.D.D. designed research; E.K.B. and J.E.H. performed research; J.P.N. provided support for crystallization and x-ray data collection; E.K.B. and D.D.D. wrote the paper; all authors read and edited the manuscript. The authors declare no conflict of interest.Crystal structures deposition Structures of bfloGFPa1 and bfloGFPc1 were deposited at the RCSB Protein data bank under “Crystal structure of amphioxus green fluorescent protein, GFPa1, andGFPc1”, with assigned RCSB IDcode rcsb061402 and rcsb061411, respectively.Suppl Tables and Figures