For transcription initiation, σ54-RNA polymerase yields open promoter complexes through its remodelling by cognate AAA+ ATPase activators. Since activators can be bypassed in vitro, bypass transcription in vivo could be a source of emergent gene expression along evolutionary pathways yielding new control networks and transcription patterns. At a single test promoter in vivo bypass transcription was not observed. We now use genome-wide transcription profiling, genome-wide mutagenesis and gene over-expression strategies in Escherichia coli, to (i) scope the range of bypass transcription in vivo and (ii) identify genes which might alter bypass transcription in vivo. We find little evidence for pervasive bypass transcription in vivo with only a small subset of σ54 promoters functioning without activators. Results also suggest no one gene limits bypass transcription in vivo, arguing bypass transcription is strongly kept in check. Promoter sequences subject to repression by σ54 were evident, indicating loss of rpoN (encoding σ54) rather than creating rpoN bypass alleles would be one evolutionary route for new gene expression patterns. Finally, cold-shock promoters showed unusual σ54-dependence in vivo not readily correlated with conventional σ54 binding-sites.Enhancer-dependent transcription involving the promoter specificity factor σ This silenced state of RNAP may then have become a target for regulation. Intriguingly, to date no bacteria have been found where the gene encoding σ54 (rpoN) is present in the absence of any cognate AAA+ activator (54 functioning without activators are uncommon. Yet variants of σ54 bypassing activator requirements for making open promoter complexes exist and have been characterized extensively in vitro (54 can yield activator bypass forms. Quite why σ54 has seemingly not (or rarely) evolved to activator independence is rather unclear. Here we have investigated the in vivo global actions of wild-type σ54 in Escherichia coli bacteria and one bypass variant to explore potential restrictions on evolving bypass gene regulation systems.Amongst the strategies used to regulate transcription initiation in bacteria, the emergence of the enhancer-dependent system that uses the σnly seen ,2. Ratheomplexes ,4. Potenin vitro ,7. SinglE. coli increased activator bypass transcription suggesting multifactorial global constraints prevent unregulated transcription by the σ54 containing RNAP holoenzyme (σ54RNAP). Intriguingly, some genes with σ54 binding sites were found to be repressed in a σ54-dependent manner. In some cases the putative σ54 binding site overlapped that of a putative σ70 binding site suggesting a competition for promoter sites, which we confirmed by in vitro experiments. In other cases, σ54 and σ70 RNAP appear to act antagonistically through e.g. convergent transcription events.We found that only a very limited number of knock-outs (KOs) of non-essential genes in glnAp2 strains (Supplementary Table S1) P1vir transduction was used to insert rpoN208::Tn10 (K1471 donor strain) into the native chromosomal rpoN locus of BW25113 (recipient). Subsequently, transformation was used to transfer the pSB4A3 plasmid harbouring ΦPglnAp2-gfp  and pBAD18 cm . For the construction of the pspA strains (Supplementary Table S1) P1vir transduction was used to insert rpoN208::Tn10 (K1471 donor strain) into the native chromosomal rpoN locus of BW25113 (recipient). Next the ΦPpspA-lacZ  into the BW25113 rpoN208::Tn10 ΦPpspA-lacZ. The F3K3 plasmid containing the filamentous phage pIV secretin was also transformed into this strain. To transfer the single-gene KOs from the KEIO collection (vir lysates were produced from the pooled strains. These were then used as a donor for P1vir transduction of the KOs into the pspA-SABRS recipient strain (Supplementary Table S1).For the construction of the llection  and smalin vivo, strains were grown on LB indicator  plates and in minimal Gutnick liquid media supplemented with L-glutamine (54 function (since the nitrogen assimilation pathways in E. coli rely on σ54 for expressing appropriate levels of glutamine synthetase). The antibiotic concentrations used were 30 μg/ml kanamycin, 50 μg/ml chloramphenicol and 50–100 μg/ml ampicillin.For gene expression assays lutamine  as the npspA-lacZ in vivo, β-Galactosidase assays were carried out as described  and green fluorescence  were measured simultaneously using a BMG FLUOstar Omega microplate reader. Promoter activity was expressed as fluorescence emission at 520 nm per OD600 measured in triplicate.To measure activity of ΦPescribed . To deteNcoI, a 6-base cutter. Re-ligation of the resulting fragments into circular DNA was done in a volume of 100 μl. This DNA was used as a template for PCR, using kanamycin cassette-specific forward and reverse primers (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to align sequenced regions to the E. coli chromosome and to determine the identity of the deleted gene.Inverse polymerase chain reaction (PCR) was used to identify selected KO mutants (Supplementary Table S2). Chromosomal DNA from strains that were positive for β-galactosidase activity on indicator plates and in liquid culture (Miller assay) was purified and digested with  primers . Product® Genomics suite 6.6, including a QA/QC step to gauge the sequencing quality. Each sample yielded close to equivalent total reads with 100% of reads aligned to the E. coli K-12 reference genome (NC_000913) (Supplementary Table S4). For each gene, reads were normalized for both the number of reads and length of RNA and expressed as RPKM (reads per kilobase per million)  for further processing. Total RNA was isolated from the cell pellets using a bead mill and the mirVana RNA isolation kit (Ambion) including DNase treatment. Ribosomal RNA molecules were depleted from the total RNA preparations using the RiboZero rRNA Removal Kit (Bacteria) (Epicentre). The rRNA depleted RNAs were fragmented with RNase III. Then, the RNA fragments were poly(A)-tailed using poly(A) polymerase and the 5′ PPP structures were removed using RNA 5′ polyphosphatase (Epicentre). An RNA adapter was ligated to the 5′-phosphate of the RNA fragments. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). The cDNA pools were size fractionated in the size range of 200–450 bp using a differential clean-up with the Agencourt AMPure kit. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina and sequenced on a Illumina HiSeq 2000 system using 50 bp read length. Near equimolar amounts of cDNA were used for sequencing. The cDNA reads were analysed via the RNA-seq workflow within Parteke NC_0009  PEG 8000) at 37°C for 10 min. For the σ70/σ54 competition assays, 0.4 μM σ54 were either added before or with σ70 for binding competition. The reactions were quenched by formamide stop dye and run on a 20% sequencing gel. Signals were detected by Fuji PhosphorImager and quantified using the AIDA Image Analyzer software (raytest).Small primed RNAs (spRNAs) were synthesized in 10 μl volumes containing: 0.125 μM σ54 lacking the repressive Region I, σ54ΔRI, can bypass activator requirement for transcription in vitro  in single copy integrated into the att site of the E. coli chromosome we sought bypass activity from the σ54-dependent pspA promoter in vivo. Reporter strains lacked rpoN and were complemented with either the wild-type or the ΔRI form of σ54 expressed from pBAD18 cm . While rpoN and ΔRIrpoN were under control of the araBAD arabinose inducible promoter, leaky expression (without arabinose) yielded sufficient levels of σ54 to enable pIV-dependent transcription from the pspA promoter . Thus unless stated otherwise (Supplementary Table S2) the experiments were performed in the absence of arabinose. Under standard growth conditions, no clear bypass transcription was observable with the σ54ΔRI form  or on lactose as a carbon source for increased expression of PpspAlacZ. Screens were carried out at 37 and 42°C, and in the absence and presence of 0.001% arabinose (Supplementary Table S2). Colony PCR was used to confirm all necessary genetic elements  were intact in candidate upregulated clones. Coverage was three to four-fold of the chromosome assuming all KOs were represented in the library.To seek genetic determinants potentially restricting pspA promoter based on an increased β-galactosidase activity on indicator plates (Supplementary Table S2). Of these, six KOs  which were found in a screen at 37°C also show increased β-galactosidase activity in liquid culture. These KOs were directly identified by inverse PCR and sequencing. Five KOs were discounted as being direct restrictive determinants of in vivo levels of bypass transcription on the basis of (i) just causing changes to the cell's envelope which led to enhanced β-galactosidase activity, potentially a permeability issue with the ONPG substrate or (ii) reconstitution of the native chromosomal rpoN locus. The ttdR gene however encodes a nucleoid binding protein and its loss seems to genuinely cause increased bypass transcription .We considered that the amount of a gene product might be limiting, rather than inhibitory, for supporting bypass transcription by σ54 and compared outcomes to those from cells with wild-type σ54 and to cells lacking σ54. Cells were cultured at 42°C, which enhances bypass in vitro .We conducted RNAseq of cells containing the ΔRI bypass form of σin vitro , to favo54 compared to cells with σ54ΔRI or no σ54 revealed no major global bypass transcription  from class I and II for binding repression by σ54in vitro  using initiating dinucleotides (−1/+1 or +1/+2). The experiments were performed under two conditions. In the first, σ54 and σ70 were co-incubated with the RNAP prior to addition of promoter DNA probes. Here σ54 was in three-fold molar excess to σ70 and RNAP. This competition from excess σ54 reduced the amount of spRNA synthesised by 10–60% . Since the binding affinity and dissociation constant of σ54 and σ70 for RNAP are very similar  despite lacking any clear consensus −12/−24 sequences . However, in vitro transcription from cspA  has been shown to be essential for mounting a cold shock response through the transcription of σ54-dependent cold shock transcriptional regulators BkdR and YplP (cspA and cspH promoters contain putative heat shock sigma factor (σ32) binding motifs , and may influence DNA structure to normally repress bypass transcription.The σen cycle . Bioinfofunction . Recent proteins . Studieds of RNA ,29–33. Orcoiling ,32. The glnAp2 promoter) bypass transcription could be elevated by altering the TGCA sequence around the −12 GC element  correlation between low-level bypass transcription with a less conserved C −12 nucleotide and (ii) an apparent lack of in trans encoded repressors of bypass transcription.Earlier work from the Gralla lab established that with single promoter studies (the  element . Where wwith DNA ,35. We pin vitro ,21,36. O54 (54 can act to repress gene transcription by other forms of RNA polymerase through a binding competition for promoter sites. Our analysis of any repressive capacity is unlikely to be comprehensive, since a single growth condition was studied. Given more σ54 binding sites than conventional activator dependent σ54 promoters have been identified using a range of genomic methods, we propose that the repressive function is pervasive. Indeed, the σ54 factor is designed to inhibit RNAP by penetrating and blocking DNA entry routes (54 or some of its domains was to globally repress transcription. The unusual behaviours of the cold-shock promoters with respect to σ54 dependence is intriguing, and suggests some non-canonical functions of σ54 and or its holoenzyme remain to be discovered. These may involve factors outside of the well-recognized AAA+ activators which reorganize σ54 at its classical promoter sites.Overlapping promoters and competition between sigma factors are recognized as being important regulatory features of a range of genes –41, alth54 . Here, wy routes ,42,43, aSupplementary Data are available at NAR Online.


We find that the transition gate voltage can be reduced arbitrarily by alloying, for example, for MoxWx1−Te2 monolayers. Our findings identify a new physical mechanism, not existing in bulk materials, to dynamically control structural phase transitions in two-dimensional materials, enabling potential applications in phase-change electronic devices.Dynamic control of conductivity and optical properties via atomic structure changes is of technological importance in information storage. Energy consumption considerations provide a driving force towards employing thin materials in devices. Monolayer transition metal dichalcogenides are nearly atomically thin materials that can exist in multiple crystal structures, each with distinct electrical properties. By developing new density functional-based methods, we discover that electrostatic gating device configurations have the potential to drive structural semiconductor-to-semimetal phase transitions in some monolayer transition metal dichalcogenides. Here we show that the semiconductor-to-semimetal phase transition in monolayer MoTe Control of conductivity and optical properties via atomic structure changes is of technological importance in information storage. Here, Li et al. show that electrostatic gating has the potential to drive structural semiconductor-to-semimetal phase transitions in some monolayer transition metal dichalcogenides. Recently, group VI TMDs have attracted increasing attention because they can exist in a semiconducting phase212223Another group of materials that can undergo phase transitions are layered transition metal dichalcogenides (TMDs), which have received recent attention as single- and few-layer materials, although research on bulk TMDs dates back decades11132, MoTe2, TaSe2 and the alloy MoxWx1−Te2. We consider MoS2 because it has received considerable attention as an exceptionally stable semiconductor, and MoTe2 because DFT calculations indicate that its energy difference between semiconducting and semimetallic phases is exceptionally small among Mo- and W-TMDse or greater than 0.09 e per formula unit is required to observe the semiconductor-to-semimetal phase transition in undoped monolayer MoTe2 under constant-stress conditions (e is the elementary electric charge) and a much larger value of approximately −0.29 e or 0.35 e per formula unit is required in the undoped monolayer MoS2 case. The charge densities discussed in this work refer to excess charge density and should not be misinterpreted as the electron or hole density in a charge-neutral material that one might obtain from chemical doping. We also study the potential of phase control in monolayer MoTe2 and TaSe2 through electrostatic gating using a capacitor structure. We discover that a gate voltage as small as a few volts for some choices of gate dielectric can be applied to drive the phase transition in monolayer MoTe2 using a capacitor structure. While the required field magnitudes are large and may be challenging to achieve, we find that the transition gate voltage may be reduced to 0.3–1 V and potentially lower by substituting a specific fraction of W atoms within MoTe2 monolayers to yield the alloy MoxWx1−Te2. To accomplish these calculations, we have developed a DFT-based model of the electrostatically gated structure  to determine the phase boundaries of single-layer MoStructure . This ap2, where M is a transition metal atom and X is a chalcogen atom. Each monolayer is composed of a metal layer sandwiched between two chalcogenide layers, forming a X–M–X structure162025TMDs are a class of layered materials with the formula MXet al.2 transistors of high mobility, large current on/off ratios and low standby power dissipation. Unlike group IV and group V TMDs , which have been observed in the metallic 1T crystal structure2 under ambient conditions162 at high temperature2 , suggesting the potential for a transition that is exceptionally close to ambient conditions. Therefore, we choose to focus on determining the phase boundary of monolayer MoTe2. While the computed energy difference between 2H and 1T′ is considerably larger for MoS2 (548 meV per formula unit), we also compute phase boundaries for this monolayer at constant charge because it has received more attention in the laboratory to date. Among 2D group VI TMDs, monolayer MoS2 has attracted the most experimental attention for its stability and relative ease of exfoliation and synthesis. Monolayer MoTe2 has also been exfoliated31The relative energies of Mo- and W-based TMDs monolayer crystals shown in d and capacitance C is sandwiched between monolayer TMDs and plate II. This dielectric medium can be vacuum. Distance sI is the separation between the centre of monolayer TMDs I and the right surface of the dielectric medium, while sII is the separation between the surface atoms of plate II and the left surface of the dielectric medium.  is the sum of three parts: energy stored in the dielectric medium (Ec), energy of the plate II (EII) and energy of the charged monolayer TMDs (EI), as shown in When the charge C is the capacitance of the dielectric medium. EI is the ground-state energy of the electrically neutral monolayer TMDs and EI−EI is the energy required to move electrons Q from the Fermi level of the monolayer TMDs to the dielectric surface. EII is defined analogously. We take the monolayers to be undoped in this work.where EI, is calculated using DFT for each phase of the monolayer TMDs to yield a E(Q) for each monolayer phase  becomes the relevant thermodynamic energy defined as:When the voltage is fixed rather than the charge, the grand potential ΦE(Q) is computed using QV term in this expression represents external energy supplied to the system when the charge Q flows through an externally applied voltage V. The equilibrium charge Qeq can be calculated through minimization of the grand potential at a given gate voltage V.where Qeq(V) to Applying the computed Hereafter, we omit the superscript ‘eq' for the equilibrium grand potential In addition to the electrical constraint, the nature of the mechanical constraint on the monolayer is also expected to play a role in the phase boundary, discussed in The distinction between the constant charge and voltage cases is most important when a phase transformation occurs. We discover that the transition between semiconducting 2H-TMDs and semimetallic 1T′-TMDs can be driven by excess electric charge (positive or negative) in the monolayer. A constant charge condition exists when the charge on the monolayer remains constant during the phase transition as if it is electrically isolated. An approximate condition of constant charge could exist when adsorbed atoms or molecules donate charge to the monolayer.d away from the electron reservoir . Because the dielectric medium is vacuum in this schematic, EI and Ec in EII in When computing the energy difference between a system where the monolayer is in the 1T′ phase and another system where the monolayer is in the 2H phase sI+d rather than sI and d independently. Variation of the results of sI+d (chosen to be 15 Å in The blue lines are constant-stress (stress-free) cases, in which both phases exhibit minimum energy lattice constants and atomic positions. This condition is expected to hold when the monolayer is freely suspended or is not constrained by friction on a substrate. The red lines represent constant-area cases, where the monolayer is clamped to its 2H lattice constants. This condition might be expected to hold when there is a strong frictional interaction between the monolayer and substrate preventing the monolayer from relaxing freely.2 has lower free energy and is the equilibrium state when the monolayer is electrically neutral or minimally charged. For the stress-free case (blue line), when the charge density is between −0.04 e and 0.09 e per formula unit, 2H-MoTe2 is the thermodynamically stable phase. These charge densities correspond to −3.7 × 1013 and 8.2 × 1013 e cm−2, respectively. Outside this range, semimetallic 1T′-MoTe2 will become the equilibrium phase and a transition from the semiconducting 2H phase to the semimetallic 1T′ phase will occur.In the constant-area case (red line) in 2 requires much larger charge density than the MoTe2 case. If the negative charge density is >0.29 e per MoS2 formula unit, semimetallic 1T′-MoS2 will have lower free energy and be more stable. For negative charge densities <0.29 e per formula unit, semiconducting 2H-MoS2 will be energetically favourable. This is consistent with previous experimental reports that adsorbed species donating negative charge to monolayer MoS2 can trigger a trigonal prismatic to octahedral structure transformation242 single layer is reported to adopt a distorted octahedral structure when bulk MoS2 is first intercalated with lithium to form LixMoS2 with x≈1.0 and then exfoliated by immersion in distilled watere per MoS2 may trigger the phase transition from 2H phase to 1T′ phase MoS2. See Another relevant type of electrical constraint is fixed voltage or electron chemical potential. This constraint is most applicable when the monolayer is in an electrostatic gating structure similar to field-effect transistors made using monolayers. Such a device structure enables a dynamical approach to achieve semiconductor/semimetal phase control in monolayer TMDs, suggesting intriguing applications for ultrathin flexible 2D electronic devices including phase-change memory.2 is deposited on top of a dielectric layer of thickness d, which we take to be HfO2 with a large dielectric constant of 25  with gate voltages as large as −50 V. For monolayer MoTe2 (rather than few layers), our model predicts that a gate voltage >200 V is required to drive the phase transition for this device configuration. Both the increase of dielectric thickness (from 5 to 270 nm) and the decrease of dielectric constant (from 25 for HfO2 to 3.9 for SiO2 (ref. Field-effect transistors based on few-layered MoTeiO2 ref. ) will re2-WTe2 monolayers. This is because in monolayer MoTe2, the 2H phase is energetically favourable by 31 meV per formula unit relative to the 1T′ phase, whereas in monolayer WTe2, the energy of the 1T′ phase is 123 meV per formula lower than the 2H phase, as shown in Monolayer alloys present the possibility for reducing the required gate voltage by varying the chemical composition. Recently, monolayer alloys of Mo- and W-dichalcogenides have attracted increasing attention for their tunable properties40414243xWx1−Te2  to 0.4 V in the constant-area case of Mo0.67W0.33Te2 monolayer.For the alloy configuration we employed in this work (assumed at constant area), the 2H phase is a semiconductor with a semilocal quasiparticle Kohn–Sham bandgap of ∼0.9 eV, and its free energy is 15 meV lower than the 1T′ phase at constant monolayer area, which is metallic or semimetallic. One might expect that the transition gate voltage in monolayers can be tuned and reduced potentially arbitrarily by controlling the chemical composition of this and other potentially alloys. To enable a structural phase transition driven by a small gate voltage, elements should be selected for alloying so that the energy difference between charge-neutral 2H and 1T′ phases can be tuned through zero with alloy composition. Alternative mechanical constraints placed on the alloy monolayer  can also be expected to shift the phase boundary and transition gate voltage.2 and TaS2, have been reported in experiments using a STM tip172 in the capacitor gating structure shown in 2 has a phase boundary at only positive gate voltage, qualitatively different from MoTe2 and MoS2. We find that this difference results from the metallic nature of both 2H- and 1T-TaSe2, further discussed in Electrically induced structural phase changes between 2H and 1T phases in the Ta-based TMDs, TaSeA qualitative difference between these calculations and the STM experimentThe electrical dynamical control of structural phase in monolayer TMDs has exciting potential applications in ultrathin flexible 2D electronic devices. If the kinetics of the transformation are suitable, nonvolatile phase-change memory2, MoS2 and TaSe2. We discover that changing carrier density or electron chemical potential in the monolayer can induce a semiconductor-to-semimetal phase transition in monolayer TMDs. We find that a surface charge density less than −0.04 e or greater than 0.09 e per formula unit is required to observe the semiconductor-to-semimetal phase transition in monolayer MoTe2 under constant-stress conditions, and a significantly larger value of approximately −0.29 e or 0.35 e per formula unit is required in the monolayer MoS2 case. A capacitor structure can be employed to dynamically control the semiconductor-to-semimetal phase transition in monolayer MoTe2 with a gate voltage ∼2–4 V for MoTe2. These transition charges and voltages are expected to vary considerably with the nature of the mechanical constraint of the monolayer and also potentially the presence of dopants or Fermi level pinning. While the gate voltages required to observe the transition in MoTe2 are likely near breakdown and could be challenging to realize in the lab, we find that the voltage magnitudes can be reduced arbitrarily by alloying Mo atoms with substitutional W atoms to create the alloy MoxWx1−Te2.To summarize, we have identified a new mechanism, electrostatic gating, to induce a structural semiconductor-to-semimetal phase transition in monolayer TMDs. We have computed phase boundaries for monolayer MoTe−8 eV per MX2 formula unit and 0.5 × 10−7 eV per MX2 formula unit, respectively. A Gaussian smearing of 50 meV was used. The computational cell length is 36 Å along the c axis. Spin–orbit coupling is employed in all DFT calculations. The ionic relaxations were performed using conjugate gradient algorithm.All periodic DFT calculations were performed within the Vienna Ab Initio Simulation PackageAll calculations in this work were performed at zero ionic temperature, omitting the vibrational component of the free energy. How to cite this article: Li, Y. et al. Structural semiconductor-to-semimetal phase transition in two-dimensional materials induced by electrostatic gating. Nat. Commun. 7:10671 doi: 10.1038/ncomms10671 (2016).Supplementary Figures 1-10, Supplementary Table 1, Supplementary Notes 1-7 and Supplementary References.


The ionic conductivity is higher by one order of magnitude than plain Sm-doped CeO2 films. By using scanning probe microscopy, we show that the fast ion-conducting channels are not exclusively restricted to the interface but also are localized at the Sm-doped CeO2 nanopillars. This work offers a pathway to realize spatially localized fast ion transport in oxides of micrometre thickness.Enhancement of oxygen ion conductivity in oxides is important for low-temperature (<500 °C) operation of solid oxide fuel cells, sensors and other ionotronic devices. While huge ion conductivity has been demonstrated in planar heterostructure films, there has been considerable debate over the origin of the conductivity enhancement, in part because of the difficulties of probing buried ion transport channels. Here we create a practical geometry for device miniaturization, consisting of highly crystalline micrometre-thick vertical nanocolumns of Sm-doped CeO Vertical nanocomposite films can exhibit a significant enhancement in oxygen ion conductivity, which is useful for oxide-based electrochemical devices such as fuel cells. Here, the authors directly probe this effect in high crystalline nanopillars using scanning probe microscopy. Fast oxygen ion transport at reduced temperatures is highly desired in many oxide-based electrochemical devices, including solid oxide fuel cells (SOFCs)1234591011123453101112132O3-stabilized ZrO2 (YSZ) sandwiched by SrTiO3 (STO) layers, which were reported to exhibit eight orders of magnitude higher than the oxygen ion conductivity of bulk YSZ1516171819et al.et al.et al. reported density functional calculations and electron energy loss spectroscopy study, supporting that the nature of conduction is ionic18Tremendous efforts have been made to increase oxygen ion conductivity in oxides by designing artificial oxide heterostructures245In spite of the above issues, strain engineering and interface effects in oxide heterostructures have attracted considerable attention as a means to enhance oxygen ion conductivity. Recently, vertically aligned heterostructure films (referred to as nanoscaffold films here) have emerged as a very promising type of nanostructured systems for tuning the strain states of materials among other coupling effects212223242526272122232425262729302 (SDC)–STO films is explored using SPM. We apply electrochemical strain microscopy (ESM)32333436Here, oxygen ion transport in vertical heteroepitaxial micrometre-thick Sm-doped CeOθ−2θ scan for an ∼1-μm-thick nanoscaffold SDC–STO film. In the 2θ range of 15°–125°, only SDC (00l) reflections are observed at 2θ=33°, 69° and 117° without any additional peaks indicative of no intermixing phases and solid solution phases. To compare the crystallinity of the nanoscaffold film, we grew a plain SDC film of the same thickness on a Nb:STO substrate. Unlike the nanoscaffold film, in the SDC film (the blue line in l)-oriented SDC thick films are not easy to grow on (001) STOThe SDC nanocolumns are (001)-oriented on the Nb:STO substrate. The red line in φ-scan (qz axis (∼5.34–5.51 Å), that is, a wide spread of lattice parameters. The combination of the poor lattice match with the substrate and the strain relaxation in the films leads to the presence of the aforementioned polycrystalline phase with little strain in the film. On the other hand, the nanoscaffold SDC–STO film exhibits a very narrow qz axis (5.42−5.44 Å), which is almost identical to the lattice constant of cubic SDC phase in the bulk. The results are consistent with the lower FWHM values of the (002) SDC peak measured for the nanocomposite film from above. Together, these results indicate that the SDC nanocolumn lattice is epitaxially controlled along the vertical direction by the surrounding STO matrix, an effect observed in other nanoscaffold film systems22232425It is well known that nanoscaffold films are a powerful platform for preserving the strain state of a film with less thickness limitation, leading to achievement of high crystallinity2223242526φ-scan . Figure f)-dependent ac conductivity σac, widely used for measuring ionic conductivity in disordered solids240f-dependent real part of σac  of the nanoscaffold SDC–STO film in the f-range of 40–4 × 106 Hz. The f-dependent σac′ in the ionic conductors connected with ion-blocking electrodes exhibits the characteristic electrical response, as shown in σac′ decreases with decreasing f at high frequency because σac′ is governed by the short time ion dynamics (which is characterized by back-and-forth motion over the limited ranges) in the bulkσac′ is not a strong function of f, as displayed in the existence of plateau. It is well known that this distinct plateau is due to long-range ion transport, ‘diffusive' dynamics, providing the dc conductivity of ions2σac′ shift up with an increase in temperature, as represented by the solid circles in σac′ decreases again with decreasing f since oxygen ions are blocked by the Pt top electrodes and accumulate there, leading to the formation of space-charge layers and rapid voltage drop in such layers, the so-called ‘electrode polarization' effect  in the entire temperature measurement range. This result shows that the electronic contribution to the ac impedance measurements can be considered to be negligible and, thus, that the measured ac transport is dominated by oxygen ions, not by electrons or holes . For comparison, we grew individual SDC, YSZ and STO films (see Methods) and measured their σ values by means of the same procedure as the nanoscaffold film. As shown in the guidelines, the temperature dependences of σ follow the well-known Arrhenius behaviour in all samples. The estimated σ values of SDC, YSZ and STO were ∼10−3, 10−4 and 10−6 Ω−1 cm−1 at 400 °C, respectively. Namely, the oxygen ion conductivity in the nanoscaffold film is much higher than that in other comparison films by one to four orders of magnitude, indicative of considerable enhancement of ionic conductivity in the nanoscaffold films. In addition, our nanoscaffold SDC–STO films have higher ionic conductivity than other CeO2-based ionic conductors and heterostructures reported in the literature (solid lines in 0.1Ce0.9Ox (GDC)10.075Nd0.075Ce0.85Oδ2−  of a ∼230-nm-thick nanoscaffold film top surface. Bright circles  and dark surrounding region indicate SDC nanopillars and STO matrix, respectively. In the FORC-ESM measurements, the amplitude of a triangular wave was linearly increased until the maximum value of 10 V was reached (2). Further experimental details are described in Methods. fcr, which is one of the key pieces of information obtained from the measurement. The fcr value in the STO matrix is relatively high and uniform, whereas that in SDC nanocolumns is low and inhomogeneous. Since fcr is very sensitive to the changes in mechanical properties of the tip–surface junction, the variation of fcr within SDC nanocolumns is attributed to chemical inhomogeneity of the SDC phase  because of the doping of them by Sm and surface hydroxylationfcr values in the STO region. Moreover, we found clear radial direction-dependent fcr values in the SDC nanocolumns  were probed directly at the nanoscale. We first performed FORC-ESM measurements at room temperature, enabling nanoscale mapping of the electrochemically active regions34 reached . The ESMumns see ; the pilWe observed that SDC nanopillars have higher ORR/OER rates than the STO matrix. 342) of the nanoscaffold film top surface. Owing to asymmetric current behaviour depending on the polarity of dc bias, that is, high (low) current at negative (positive) bias  showed an initial non-hysteretic curve that gradually transformed into a hysteretic one above −6 V 36ve) bias , we carrove −6 V .fcr gradient along the radial direction, which was observed in the FORC-ESM measurement, the results indicated that the SDC column core has more mobile oxygen vacancies. I–V curves at the pixels corresponding to the core of SDC nanocolumn and STO region, respectively. The core of the SDC column exhibits high current and clear loop opening, whereas the STO region shows nearly zero current. Therefore, it can be concluded that oxygen anionic current is well localized to the SDC nanocolumns in the nanoscaffold film. In addition, it can be confirmed again that the electronic contribution of the STO component of the film to the enhancement of ionic conductivity is negligible, since the STO matrix does not exhibit any conduction (either ionic or electronic).Contrary to the average behaviour, the local current exhibits high spatial variability. 363–CoFe2O4 nanocomposite systems303–Sm2O3 nanocomposite systemSpatially resolved mapping of oxygen ionic conduction at the nanoscale reveals that only the SDC nanopillars have high interfacial ORR/OER rates as well as significant bulk oxygen ion conductivity. Especially, the highest ion conductivity is observed at the column centre, rather than at the SDC–STO interface. This contrasts with the common consensus that the interfaces in planar oxide heterostructure films act as the fastest ion transport pathwaysqz  SDC X-ray peak and much narrower RSM peak width of along qz . Hence, 2/BaF2 heterostructure filmsFinally, since the diameters of the SDC nanopillars are only ∼30 nm, we should consider the effect of space-charge regions at the SDC–STO interfaces and the possibility that they could overlap and therefore influence the oxygen ion conductivity. When two different phases meet, a highly ionic conductive path can be created along the interfaces as a result of ion redistribution in a space-charge region5253In conclusion, we have explored the oxygen ion transport in micrometre-thick vertical nanocomposite SDC–STO films. The macroscopic ionic conductivity in nanoscaffold films was higher than those in plain SDC, YSZ and STO thick films by up to four orders of magnitude. We have investigated interfacial ORR/OER process as well as bulk oxygen ion transport using SPM techniques because of the unique nanoscaffold geometry studied here where conduction channels can readily be probed, which is not the case for buried interfaces in standard planar films. Spatially resolved mapping of oxygen ion transport at the nanoscale revealed that only the SDC nanopillars have high oxygen ion conductivity, while the surrounding STO matrix showed negligible conduction. On the basis of these SPM results combined with complementary macroscopic measurement results, the high crystallinity of SDC nanopillars comparable to a single SDC phase is the primary origin of oxygen ion conductivity enhancement in nanoscaffold films. Note that such high crystalline quality of epitaxially grown SDC nanopillars without crystalline imperfections was achieved by interfacial strain coupling of the SDC to the surrounding STO matrix through the whole micrometre-thick film.This work highlights that the crystalline quality of bulk ionic conductors is very important for ionic conductivity enhancement in oxide heterostructures, a fact that is often overlooked. In particular, the conduction in nearly single-crystalline bulk phase materials can be more dominant in oxide heterostructures composed of heavily doped ionic conductors. In addition, direct spatially resolved mapping of oxygen ion conduction at the nanoscale is needed to verify the underlying mechanism of ionic conductivity enhancement. The vertical nanocomposite structures studied here provide unique structures that allow for easy probing of interface and bulk regions. They also represent a simple, self-assembled system for realizing micrometre-thick fast ionic conduction channels and are widely applicable for clean energy, multifunctional ionotronic and novel information devices.−2. During the deposition, we heated the substrate at 825 °C and flowed 200 mbar oxygen. The samples were post annealed at 700 °C for 1 h under 900 bar oxygen to assure proper oxygen stoichiometry and to minimize the superfluous oxygen vacancies inside films. We selected 0.5% Nb:STO (001) single crystal as the substrate since it has been considered as a SOFC anode material because of redox stability and electrochemical properties40The SDC–STO nanoscaffold films were grown to 0.5% Nb:STO (001) single crystal by a simple one-step process of pulsed laser deposition. We ablated a polycrystalline target containing SDC (20% wt. SDC) and STO (50:50 molar ratio) by a KrF excimer laser (248-nm wavelength) with a laser fluence of 1.5 J cmKα radiation (λ=1.5405 Å). To measure transport characteristics with temperature variation from 20 to 550 °C, we used a probe station equipped with a hot plate. The ac and dc measurements were performed by an HP 4294A Precision Impedance Analyser and Keithley 2,440 sourcemeter, respectively. For all transport measurements, we grounded the Nb:STO substrate and applied the voltage to the Pt electrodes.The microstructural characterization has been conducted using TEM (FEI Tecnai G2 F20) as well as aberration-corrected STEM (FEI Titan G2 80–200), both operated at 200 kV. STEM HAADF images were taken using an annular detector with a collection range of 60–160 mrad. Cross-sectional samples for TEM characterization were prepared by a standard grinding and thinning and a final ion-milling (Gatan PIPS 691 precision ion polishing system). The X-ray diffraction was carried out with a Panalytical Empyrean high-resolution X-ray diffractometer using Cu-ESM and FORC-IV measurements were performed using a commercial atomic force microscope  interfaced with National Instrument cards controlled by the Labview and Matlab software. The signal was applied to the cantilever with a conductive Pt/Cr coating . Image and data processing were performed using WSxM ref.  and cust36How to cite this article: Yang, S. M. et al. Strongly enhanced oxygen ion transport through samarium-doped CeO2 nanopillars in nanocomposite films. Nat. Commun. 6:8588 doi: 10.1038/ncomms9588 (2015).Supplementary Figure 1-11, Supplementary Note 1-3 and Supplementary References.


Supplemental Digital Content is available in the text Preeclampsia is a multisystem disease that significantly contributes to maternal and foetal morbidity and mortality. In this study, we used a nonbiased microarray approach to identify novel circulating miRNAs in maternal plasma that may be associated with preeclampsia.Plasma samples were obtained at 16 and 28 weeks of gestation from 18 women who later developed preeclampsia (cases) and 18 matched women with normotensive pregnancies (controls). We studied miRNA expression profiles in plasma and subsequently confirmed miRNA and target gene expression in placenta samples. Placental samples were obtained from an independent cohort of 19 women with preeclampsia matched with 19 women with normotensive pregnancies.IGF-1, a target gene of miR-206, was also found to be downregulated (0.41 ± 0.04) in placental tissue from women with preeclampsia. miR-206 expression was also detectable in myometrium tissue and trophoblast cell lines.From the microarray, we identified one miRNA that was significantly differentially expressed between cases and controls at 16 weeks of gestation and six miRNAs that were significantly differentially expressed at 28 weeks. Following qPCR validation, only one miR-206 was found to be significantly increased in 28-week samples in women who later developed preeclampsia (1.4-fold change ± 0.2). The trend for increase in miR-206 expression was mirrored within placental tissue from women with preeclampsia. In parallel, Our pilot study has identified miRNA-206 as a novel factor upregulated in preeclampsia within the maternal circulation and in placental tissue. Preeclampsia is a syndrome that occurs during the second half of pregnancy in approximately 1–5% of women in the developed world [MicroRNAs (miRNAs) are small noncoding RNAs that act at a posttranscriptional level to degrade target genes recognised by complementary base pairing in the 3’ untranslated region of the mRNA. miRNAs may provide a novel strategy for better understanding of preeclampsia and its diagnosis. miRNAs have been shown to play a functional role in physiological processes important in pregnancy such as placental development, trophoblast proliferation and migration and angiogenesis . miRNAs Microarray technology allows the expression of many miRNAs to be detected simultaneously creating a patient-specific profile. In this pilot study, we have used an OpenArray microarray to complete a nonbiased screen of circulating miRNA expression in plasma samples taken from 18 women who developed preeclampsia and 18 matched women with normotensive pregnancies at 16 and 28 weeks of gestation. We hypothesized that there are detectable changes in circulating miRNA expression at the preclinical stage of preeclampsia. Our aim was to identify these miRNAs and evaluate their relevance as an early biomarker and their potential pathological role in disease.2, family history of preeclampsia in mother or sister [For the microarray study, plasma samples from women recruited between 2007 and 2010 as part of the prospective ‘Proteomics in Preeclampsia’ (PIP) study were used. The study protocol has been described elsewhere [r sister ), at gesn = 3 before the onset of labour and n = 3 in labour) and preterm (defined as 24–36 weeks: n = 2 before the onset of labour). All of the women in the preterm group were delivered preterm due to preeclampsia. In the term group, caesarean section was due to breech position (n = 2) or prior elective caesarean section (n = 7). In both groups, individual characteristics were recorded at the time of tissue collection and delivery details were recorded from patient notes. Customized birth weight centiles were calculated using the Gestation NetworkCentile Calculator 5.4 (http://www.gestation.net/birthweight_centiles/centile_online.htm). All biopsies were flash frozen in liquid nitrogen and stored at −80°C until analysis.Placental biopsies were collected at delivery from a second cohort, independent from the PIP study, of 38 women, 19 of whom were diagnosed with preeclampsia according to ISSHP criteria and 19 normotensive individuals matched for age, BMI and parity. Myometrium specimens were obtained from a third independent cohort wherein all women were normotensive. Myometrium biopsies were taken from the upper margin of the lower segment uterine incision during caesarean section in women who delivered at term . Both media were supplemented with foetal calf serum (10%), penicillin/streptomycin (1%), 200 mmol/l l-glutamine (1%) and Fungizone (1%). C2C12 cells were maintained in Dulbecco's modified Eagle medium  supplemented with foetal calf serum (10%), 200 mmol/l l-glutamine (1%) and penicillin/streptomycin (1%). Confluent cultures were subject to a 1 : 3 subcultivation ratio using trypsin/ PBS.BeWo cells (Cat. No. CCL-98) and JAR cells (Cat. No. HTB-144) were maintained in Ham's F12K medium and modified RPMI-1640 medium, respectively, in 5% CORNA extraction from plasma, cells and tissues was performed using the miRNeasy mini kit  according to manufacturer's instructions. Samples were processed according to manufacturer's protocol. Each sample's total RNA concentration was determined by Nanodrop  and total RNA was stored at −80°C.The OpenArray real-time PCR system  was used to screen for miRNA expression levels in plasma. Total RNA from patient plasma samples was processed according to manufacturer's instructions, using the low input modifications for blood plasma samples. Briefly, reverse transcription of each sample was carried out with two separate primer pools in parallel, using 19.15 ng of total RNA per sample per reaction. Following reverse transcription, a preamplification step with 16 cycles was carried out and the resulting products diluted 1 : 20. The diluted samples, matched and grouped, were applied to OpenArray qPCR panels in a randomized fashion, for profiling of 754 miRNAs per sample. Before calculating the Ct values for each miRNA with the supplied software (Life Technologies), the default minimum fluorescence signal threshold was adjusted to 200 and the ‘subtract baseline’ method was applied. Prior to comparing miRNA expression levels, any miRNA probes on the array panels that did not meet the quality control criteria were excluded from analysis. Ct values from the microarray were not subject to normalization, as suitable stable miRNAs could not be found. Therefore, all statistical analysis was performed on raw Ct values presented in the text. miRNAs with a Ct of 35 or above were not included in analysis.Gene expression RT-PCR was performed using the Taqman Reverse Transcription Kit (Applied Biosystems) according to manufacturer's instructions with 400 ng RNA input. The reaction was run on a Multi Block System Satellite 0.2 Thermo Cooler (Thermo Fisher Scientific) on the following settings: 25°C 10 min, 48°C 30 min, 95°C 5 min. miRNA RT-PCR was performed using the Taqman miRNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer's instructions with 5 ng initial RNA input. The reaction was run on a Multi Block System Satellite 0.2 Thermo Cooler (Thermo Fisher Scientific) on the following settings: 16°C, 30 min; 42°C, 30 min; 85°C, 5 min.2O and relevant Taqman probe (Applied Biosystems). miRNA protocol was run on an ABI PRISM 7900HT PCR system at the following settings: 95°C, 10 min; followed by 40 cycles of 95°C, 15 s; 60°C, 1 min. Gene expression protocol was run on an ABI PRISM 7900HT PCR system at the following settings: 95°C, 15 min; followed by 40 cycles of 95°C, 15 s; 60°C, 1 min. miRNA results were normalized using syn-cel-miR 39 as a spiked-in reference miRNA. PPIA, TOP1 and GAPDH were chosen as reference genes for gene expression studies [PPIA, TOP1 and GAPDH were chosen as reference genes for gene expression studies. Ct values were analysed using the 2(-delta delta Ct) method, with dCt indicating normalization to the geometric mean of the three reference genes.Reactions were set up using the following reagents: Taqman Universal Mastermix (Applied Biosystems), dH studies . In orde studies . PPIA, Tt-test where ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.All data are represented ± standard error of the mean (SEM). Parameters measured in patient cohorts, miRNA changes identified from the microarray and all qPCR data were analysed using a Student's http://links.lww.com/HJH/A495). Following statistical analysis of the microarray data, six miRNAs were found to be significantly differentially expressed between the case and control groups at either time point  all had increased expression in cases at 28 weeks of gestation only. Following these findings, the six miRNAs identified by the microarray were subject to standard qPCR validation. There were no significant differences in expression between cases and controls in miR-23a∗ in plasma samples taken at 16 weeks . Of the differences in expression between cases and controls detected by the microarray at 28 weeks, only miR-206 was found to be significantly different between the groups in validation experiments . In keeping with the microarray data, miR-206 was found to be upregulated in women who went on to develop preeclampsia (1.4-fold change ± 0.2) compared with those with normotensive pregnancies.Plasma samples collected at 16 and 28 weeks gestation from 18 women who went on to develop preeclampsia and 18 matched normotensive women (Table n = 19) and matched women with normotensive pregnancies (n = 19) (Table P = 0.11) , insulin-like growth factor 1 (IGF-1) and neurogenic locus notch homolog 3 (NOTCH-3) was also measured in the same placental tissue . IGF-1 was found to be significantly downregulated in placental tissue from cases (0.41-fold change ± 0.04).Interrogation of the miRNA databases TARGETSCAN-VERT  and miRbsue Fig. b. There n = 2) and term myometrium (n = 9) from normotensive women . miR-206 expression was also detected in two trophoblast cell lines, BeWo and JAR .Further investigation of miR-206 in pregnancy related tissues showed that miR-206 was detectable in both preterm women with re-eclampsia  gene that expresses IL-17, a potent cytokine mediating pro-inflammatory responses [miR-206 is predominantly expressed in skeletal muscle and is part of the ‘myomiR’ family of miRNAs known to function in myogenesis and striated muscle growth and repair . It is wesponses . T-helpeesponses . In the esponses . WhetherVEGF[NOTCH-3[IGF-1[HIF-1α) [VEGF expression in response to hypoxia.Our findings show that miR-206 expression was observed to be higher in both the circulation and the placenta and is therefore a promising candidate for further study. Notably, it has experimentally validated interactions with a number of genes known to be directly involved in the pathology of preeclampsia, particularly at the placenta: VEGF,24, NOTC[NOTCH-3,26, IGF-[HIF-1α) . In our [HIF-1α)  and is tIGF-1 mRNA expression was downregulated in placental tissue in preeclampsia in the present study. miR-206 has been previously been shown to target and downregulate IGF-1 expression in other tissues, specifically rat myoblasts [IGF-1 has important growth effects on the foetus [IGF-1 in the first and second trimester of pregnancy in preeclampsia [Placental e foetus  and myome foetus , which aclampsia . It is nOne of the strengths of our study is the screening of samples from two gestational time points, 16 and 28 weeks, both before the clinical signs of preeclampsia were apparent, whereas other studies using large-scale approaches such as microarray or next generation sequencing have only looked at one gestational timepoint –35. Our VEGF causing a parallel increase in its negative regulator, miR-206. This then results in changes in expression of other placental genes regulated by miR-206 such as IGF-1. Notably, placental expression of miR-206 was upregulated to a similar degree but did not reach statistical significance. We suggest that there are two reasons for this within our study. Firstly, the placenta is a diverse tissue formed by the cooperation of many different cell types. Secondly, our placental samples were representative of the entire tissue and not specific regions. It has previously been shown that miRNA levels can vary according to the region of placenta that is sampled [http://links.lww.com/HJH/A495).It is unknown from our work whether the increase seen in miR-206 is a cause or effect of preeclampsia. Therefore, it can only be considered as a starting point in order to further investigate novel mechanisms of the disease. As the changes identified in our study were only seen at 28 weeks of gestation and not at an earlier time point, miR-206 upregulation may be a response to the established placental hypoxia characteristic of preeclampsia. We hypothesize that the hypoxic response leads to a compensatory increase in  sampled . TherefoIn conclusion, earlier diagnosis and better understanding of the disease of preeclampsia is at the forefront of preeclampsia research. Our study has identified miR-206 as a novel differentially regulated miRNA in preeclampsia that may give information regarding the pathology of the disease. As this is a pilot study, a large independent cohort is needed to determine whether the small increase in miR-206 expression seen is reproducible and, if so, provides further insight into the complex disease of preeclampsia.The study was funded by grants from the European Union  and the Scottish Government's Chief Scientist Office (reference ETM/196). H.Y.S is funded by a British Heart Foundation Student Fellowship. L.S. is funded by a grant from the Russian Ministry of Education and a grant from the Boehringer Ingelheim Fonds, and the work was according to the Russian Government Program of Competitive Growth of Kazan Federal University. R.F. is the recipient of an MRC studentship.There are no conflicts of interest.


These PG-FETs allowed usto investigate behavior of revealed hydrogen intercalation and diffusion aspectswith time and spatial evolutions in nanowires. These results show that air nanogapscan operate as an electrochemical reaction field, even in a gaseous atmosphere, andoffer new directions to explore emerging functions for electronic and energy devicesin oxides.Reversible and drastic modulation of the transport properties in vanadium dioxide(VO In VO2 nano tomicrostructures567891011127892 causedrastic changes in the transport properties, equal to that caused by inducing theMIT1415161718191415171420212222O3 with a nanoporous structure) gatesincluding water212 nanowires byelectrochemical gating via air gap under humid condition and establishment of theion diffusion model, giving the hydrogen ion intercalation and diffusion aspects withtime and spatial evolutions in VO2.The transport characteristics of transition metal oxides are sensitive to redox reactionsbecause the valence numbers of the transition metal ions are easily changed by thereactions, which affect the carrier density and/or stabilization of the crystalstructure1232. Especially a narrowerchannel often enhances interface effect. An atomic force microscope image of adevice is shown in w) were systematically changed from3 μm to 400 nm  illustrated in 2 nanowire channel(w = 500 nm), found by applying apositive and negative VG at 300 K under a humidityof around 50%. The normalized resistance (R/R0), whereR and R0 are the resistance and the pristineresistance before applying a VG at 300 K,respectively, slowly continued to drop during the application ofVG = + 100 Vdown to the saturation line at roughlyR/R0 = 0.75. This state washeld after removal of the VG. Namely, it exhibited anon-volatile memory effect. In contrast, the resistance increased again atVG = −100 V.Regarding slow resistive drops by an electric field, it is suggested that theorigin would be related to mechanical relaxation or slow traps according to somereports252612 cm−2 electronsshould be accumulated atVG = 100 V and400 nm-gap distance. From Hall measurements in VO2 thinfilms on Al2O3 substrates18 cm−3,equivalent to the Debye length of ~2 nm. Givenmodulation of resistivity in both lateral sides in our device geometry, thecalculated modulation rate would be ideally 0.4%. Thus, it is considered that apure carrier accumulation acts at lateral sides in dry air condition though theefficiency on the electrostatic effects was not so high compared with the idealcase. From the presence of the steep resistive switching in 212427d-orbital sensitive systemsVG = 100 V)and non-intercalation(VG = 0 V) cases indetail) and/or color-switching properties known as electrochromism2 intercalated hydrogen ions, a strong H-O bondinduces electron transfer from hydrogen onto the oxygen atom, resulting inhigher 3d-orbital occupancy of vanadium fromV4+(3d1) toV3+(3d2)5. G dependence ofR/R0 20 minutes after applying theVG to investigate the magnitude of resistance changes withvariety of VG. The threshold voltage starting to the reduction(Vth) was approximately 20 V and the magnitudeof the resistance changes was enhanced with increasing VG. Theresistance fluctuation in over 20 V would be due to fluctuation forlevel of humidity. The resistance modulation is sensitive to the level ofhumidity. In addition, repetitive intercalation and desorption of hydrogen ionsmay slightly change crystallinity of VO2 channels, causing theresistive fluctuation. In response with the resistance changes, the currentbetween the gate and source electrodes (IGS) suddenlyincreased at around 20 V as seen in Vth in VG and the number of intercalated hydrogen ions inVO2 increases. Additionally, VG = 100 V, investigatedby ToF-SIMS (time-of-flight secondary ion mass spectrometer). It can beindicated that hydrogen content in VO2 channel is higher than that inother area in a device after applying the VG, though thehydrogen content is roughly averaged because spatial resolution in themeasurement is several hundred nanometer at most, whereas the hydrogen contentsremain unchanged in a pristine device in 2 unit cell inducesalmost one order of magnitude reduction in resistivity172 can be evaluatedby investigating the resistive behavior with a variety of VGvalues. G under a humidity of 60%. Theresistance began to decrease aboveVG = 22 V, approximatelycorresponding to the Vth. In the characteristic features, themagnitude of resistance reduction rate increases with increasingVG and the initial reduction speed is faster in the largerVG, while the reduction speed became slower with time.It is known that a 1% hydrogen intercalation per VOVG,theoretical investigations have been carried out with a combination of chemicalreaction kinetics at the interface and ion diffusion model under an electricfield2 at the interface. The reaction rate depends on the externalhydrogen ion concentration (nH+) generated by electrolysis ofthe absorbed water. The nH+ tends to increase with increasingcurrent between the gate and channel electrodes  can be written as a differential equation withrespect to time (t):To understand such transport behaviors in a variety of k1 and k2 are the forward and reversereaction rate constants, depending on the activation energy at the interface andtemperature. Next, we consider how the intercalated ions diffuse inVO2. Theoretically, for ion diffusion, the ionic fluxes likelyarise from the gradients of the ion concentration and the electric gradients insolid-state materialsnHVO2 is thehydrogen ion concentration in VO2, the hydrogen ion flux(JHVO2) can be described as: D is the diffusivity, μ is the mobilityand E is the internal electric field in VO2. The first andsecond terms represent ion diffusion by the ion concentration gradient and by anelectric field, respectively. The E resulting from VGis screened by mobile electrons in VO2 according toPoisson’s equation, given as a function of the distance (x)from the interface (x = 0), namely, e is the elementary charge,N0 is the carrier density in VO2, andεr and ε0 arethe relative permittivity of VO2 and the permittivity of a vacuum,respectively. x0 can be expressed as: VG, where d is the distancebetween the gate and channel. N0 andεr change with depending on the magnitude ofintercalated hydrogen ions, however,N0/εr can be treated as aconstant value because the change rate of εr isroughly proportional to that of N0x0 would be determined only by the magnitude ofVG, and E linearly decreases as a function ofx and become zero at x0, as shown in where ρIH) and non-intercalated(ρ0) parts in 2 unit cell inducesone order of magnitude reduction in the resistivity17R/R0 could be evaluated as the followingequation:Furthermore, using a parallel resister model for the resistivity in theintercalated , derived using the finite difference method in equation where k1,k2 and μ are fitting constants, thoughthe more perfect reproduction requires more consideration efforts in the settingparameters, for example, considering k1 andk2 parameters depending on VG, whichmake enhanced the reduction rate of resistivity with increasingVG, and more precise resistive simulation like a randomresistor network. Furthermore, this simulation enough includes the importantessence of ion diffusion behavior, time and spatial evolutions of the iondiffuse in VO2 are shown in VG = 27 and38 V, respectively, where x = 0indicates a channel edge. Hydrogen ions expand with time and the concentrationincrease with increasing VG. Within the frame work of thismodel, interestingly, hydrogen ions accumulate in an inner area, clearlyobserved at VG = 38 V inx. As a token of this scenario, following the removal of anyVG, this accumulation fades away in time due to theion-diffusion by concentration gradient and finally the concentration becomehomogeneous and equilibrium states. This diffusion behavior atVG = 0 is significantlyreproducible for the persisting resistance decrease even after removal of theVG in The experimental results in w) because the effect on theinterface diffusion due to the electrochemical gating from lateral sides becomemore prominent in narrower w. R/R0 with a variety of wire widths atVG = 100 V and300 K. The saturation values for R/R0 wereenhanced with decreasing w and the sharpness of the resistance deductionin the initial process differs among the three devices as seen in the left insetof Moreover, this model predict that the magnitude of resistance reduction rateenhance with decreasing the channel width  single crystal substrates by pulsed laserdeposition using an ArF excimer laser at 450 °C under anoxygen pressure of 1.0 Pa. Using X-ray diffraction measurements, itwas confirmed that the films were b-axis-oriented without any impurityphases.35-nm-thick VO2 andSF6 gases. As advantages of this method, we can easily obtain200 nm to 400 nm-air gaps between VO2 gateelectrodes and VO2 channels, and fabricate many PG-FETs at oneprocess. Also, since the resistivity of VO2 is roughly 1 ohm cm evenin insulating region at room temperature, VO2 gate should enough workas gate electrodes in electrostatic effect. Pt/Cr electrodes were deposited byradio-frequency sputtering. Ohmic contacts between the VO2 films andelectrodes were confirmed.The films were patterned into nanowire channels with planer-type field gates bynanoimprint lithography and reactive ion etching using OVG was applied using a Keithley 236 and thecurrents between the gate and source electrodes were monitored simultaneously.The temperature of the device was controlled by a Peltier-based temperaturestage . The gaseous conditions, from dry air to 80% humidity werecontrolled in a glovebox. The humidity fluctuation waswithin ±2%.The transport characteristics were measured using a two terminal method with aKeithley 2635A. How to cite this article: Sasaki, T. et al. Electrochemicalgating-induced reversible and drastic resistance switching in VO2nanowires. Sci. Rep.5, 17080; doi: 10.1038/srep17080 (2015).


Moniliophthora perniciosa is responsible for considerable economic losses for cacao producers. One of the ways to combat WBD is to plant resistant cultivars. Resistance may be governed by a few genetic factors, mainly found in wild germplasm.Witches’ broom disease (WBD) caused by the fungus We developed a dense genetic linkage map with a length of 852.8 cM that contains 3,526 SNPs and is based on the MP01 mapping population, which counts 459 trees from a cross between the resistant ‘TSH 1188’ and the tolerant ‘CCN 51’ at the Mars Center for Cocoa Science in Barro Preto, Bahia, Brazil. Seven quantitative trait loci (QTL) that are associated with WBD were identified on five different chromosomes using a multi-trait QTL analysis for outbreeders. Phasing of the haplotypes at the major QTL region on chromosome IX on a diversity panel of genotypes clearly indicates that the major resistance locus comes from a well-known source of WBD resistance, the clone ‘SCAVINA 6’. Various potential candidate genes identified within all QTL may be involved in different steps leading to disease resistance. Preliminary expression data indicate that at least three of these candidate genes may play a role during the first 12 h after infection, with clear differences between ‘CCN 51’ and ‘TSH 1188’.We combined the information from a large mapping population with very distinct parents that segregate for WBD, a dense set of mapped markers, rigorous phenotyping capabilities and the availability of a sequenced genome to identify several genomic regions that are involved in WBD resistance. We also identified a novel source of resistance that most likely comes from the ‘CCN 51’ parent. Thanks to the large population size of the MP01 population, we were able to pick up QTL and markers with relatively small effects that can contribute to the creation and selection of more tolerant/resistant plant material.The online version of this article (doi:10.1186/s12864-016-2415-x) contains supplementary material, which is available to authorized users. Theobroma cacao L. (cacao), which is native to the Amazonian basin of South America [sensu lato [ America , 2, is ansu lato  and has nsu lato . Recentlnsu lato  whereas nsu lato .Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is a basidiomycete and produces basidiocarps, which emerge from dry brooms in the form of mushroom-like structures. Direct production losses are due to infected mature pods and flower cushions causing parthenocarpic fruits, thereby reducing significantly the potential production [Witches’ broom disease (WBD), which causes significant losses, is only present in South America and in the Caribbean  and was oduction .2 mapping population of ‘SCA 6’ × ‘ICS 1’. Two other QTL were identified on chromosomes IV and VIII [A major source of resistance was found in the clones ‘SCAVINA 6’ (‘SCA 6’) and ‘SCAVINA 12’ (‘SCA 12’)  and in tand VIII .2 mapping population resulting from selfing a single tree of the cross ‘SCA 6’ × ‘ICS 1’. This map contains 1,262 co-segregating markers and has a length of 733.6 cM [One of the main requirements of an efficient marker-assisted selection (MAS) program is to have reliable markers associated with traits of interest. The availability of dense genetic linkage maps is one of the prerequisites. The first linkage map in cacao, which contained 202 markers, was published in 1995 . This reHere, we describe the development of a dense genetic linkage map using single nucleotide polymorphisms (SNP) based on 459 trees of a cross between the WBD-resistant ‘TSH 1188’ and the tolerant ‘CCN 51’ at the Mars Center for Cocoa Science (MCCS) in Barro Preto, Bahia, Brazil. Furthermore, we identified seven QTL on five different chromosomes that are associated with WBD using a multi-trait QTL analysis for outbreeders. Various potential candidate genes that were identified within the QTL may be involved in different steps in disease resistance. Potential SNP markers that can be applied in the marker-assisted selection of WBD-resistant material are proposed.For this study, a segregating population ‘MP01’ consisting of 459 offspring from a cross between ‘TSH 1188’ and ‘CCN 51’ was used. Both parents are very different for various traits of interest and the offspring segregates for pod color , self-coTheobroma cacao Matina 1–6 leaf transcriptome. After alignment, a variant report was generated for all of the identified SNPs [DNA for genotyping was isolated from leaf samples according to the protocol described by Motamayor et al. . The SNPied SNPs . Furtheried SNPs . Of the ied SNPs .JoinMap®4.1  was usedVB and cushion brooms (CB) were counted once or twice a year over a period of 4 years  and removed after they had been counted. The data from the 459 genotyped trees were recorded as the number of VB per year and the number of CB per year., being the main feature of the phenotypic variation, was used for further analysis. To get an indication of the genetic component of variation for the number of VB and CB over time, determination coefficients were calculated from the variance components for genotypes and the genotype-by-year interaction using the REML facilities of GenStat 15 [The distributions of the numbers of VB and CB per year were extremely skewed with many zeros. Therefore, the presence/absence of broomsnStat 15 .10(P)) are based on the additive effect of ‘TSH 1188’, the additive effect of ‘CCN 51’, and the interaction effect. The initial selection of QTL was performed by simple interval mapping using a threshold of three for the -log10(P), and the final selection of QTL was performed by backward elimination using the default parameters provided by GenStat.The number of years in which each tree carried VB and the number of years in which it carried CB were used for further analysis; the values for each of these traits varied between zero and four. To remove the effect of possible spatial patterns of infestation by WBD, the data was adjusted for row and column effects using the REML facilities of GenStat 15 . The adjTo obtain a better sense of the precision of the QTL positions and their usefulness for cacao breeding, a simulation study was carried out in which random samples of 200 individuals were drawn from the original 459 individuals. For each sample, a QTL analysis was carried out as described above. The sample size of 200 was chosen as a reasonable size compared to the population size of many QTL experiments. For each sample, the marker positions that were selected as QTL were recorded as ‘hit’. The results of 500 random samples were stored, and a graphical display of the results was obtained using the procedure DQSQTLSCAN of GenStat 15.Haplotypes of the offspring and the parental haplotypes surrounding the SNP markers on each chromosome with a significant association with WBD resistance in the QTL analysis were identified using version 1.1 of the Matina 1–6 genome and iXora . To testT. cacao structural groups was run with fastPHASE [The phasing of 1162 individuals including three mapping populations and a set of diversity panel members [astPHASE . Sixty-tastPHASE .Using the location of the identified SNP markers with the highest association in the QTL analysis and the gene annotations in the version 1.1 Matina 1–6 genome [For the construction of the MP01 map, 3,564 segregating SNPs could be scored: 1,198 SNPs were classified as segregating in ‘TSH 1188’ (segregation type: ‘ab × aa’), 1,105 SNPs as segregating in ‘CCN 51’ (‘aa × ab’) and 1,261 as segregating in both parents (‘ab × ab’). Eleven SNPs were discarded because they had more than 5 % missing data. Nine SNPs that were classified as segregating in both parents were discarded because for these markers, the offspring individuals were scored in only two classes instead of three, leaving 3,544 SNPs for further analysis. Using a recombination frequency threshold of 0.2, ten major linkage groups were formed. One marker from chromosome III (according to its location on the assembled genome) was discarded because it was unlinked from other markers of the same segregation type. Additionally, 17 markers were removed after map inspection because of Nearest Neighbor Stress (N.N. Stress) values greater than 2.0 cM . An overview of the map construction process is given in Table , Additional file Compared to the most recent map , the MP01‘TSH 1188’ is considered resistant to WBD, whereas ‘CCN 51’ is considered moderately resistant. A recent study in MP01 focused on the number of cushion brooms showed that ‘TSH 1188’ had about 3 % infected flower cushions, whereas ‘CCN 51’ had close to 30 % infected flower cushions . NowadayFor each genotype, the total numbers of VB and CB per year were used . All seven positions exhibited QTL effects that differ in the two traits under study. A summary of the results after backward elimination is shown in Table The multi-trait QTL analysis identified various QTL, but after backward selection on VB and CB  seven positions on five different chromosomes were retained as potential QTL Fig. . All sev2 population obtained by selfing an individual of a cross between the WBD-resistant ‘SCA 6’ and the WBD-susceptible ‘ICS 1’ [2 mapping population [M. perniciosa. A simple regression analysis indicated that in addition to the known markers on chromosome IX, there were also markers that showed an association with resistance on chromosomes I, III and IV. The markers on chromosomes III and IX were from the ‘TSH 1188’ parent, whereas the markers on chromosomes I and IV were from the ‘CCN 51’ parent, most likely indicating an additional source of resistance [The major QTL (for both VB and CB) on chromosome IX most likely corresponds to the QTL identified in the F ‘ICS 1’ –14. This ‘ICS 1’ , 33; ‘TS ‘ICS 1’ , and ‘TS ‘ICS 1’ . The sec ‘ICS 1’ . ‘IMC 67 ‘ICS 1’ , 38, whe ‘ICS 1’ , 40. At  ‘ICS 1’  used 18 pulation –14) on 1sistance . In the sistance , 42–44. −22 (Table T = ‘TSH 1188’ and C = ‘CCN 51’), the highest associations were found for QTL9.1_T2-C1   Table . The freP = 1.33 × 10−9), followed by QTL9.1_T2-C2 with QTL6.1_T2-C2 (P = 5.79 × 10−8).To identify the best marker combinations of two QTL for the selection of resistant trees, each haplotype combination of QTL9.1 was combined with each haplotype combination for the other QTL. All the associated P-values are presented in Additional file P = 0.000057), followed by the combination of QTL9.1_T2-C2 with QTL9.2_T2-C2 and QTL6.1_T2-C2 (P = 0.000074). The percentage of resistant trees for the top five of each QTL combination varied between 76.9 and 95.0 % depending on the number of trees that were available with that specific combination  were combined with the more-significant haplotype combinations for two other QTL. All of the associated P-values are presented in Additional file Furthermore, we used the trees that showed a recombination event between the different haplotypes within a small region around the most significant marker and extended this region by adding flanking SNP markers. This extension was designed to help us to localize more precisely the most likely candidate genes, but we only managed to do this successfully for the most significant QTL9.1 on chromosome IX  and its neighbor (Tcm009s08051015), all of the trees that have the T2 haplotype, i.e., the G allele or the T allele, respectively, had ten or fewer total VB over the 4 years and can be considered resistant  surrounding the most significant marker for each QTL were investigated. The number of genes within each QTL ranged from 23 in QTL9.1 to 90 in QTL6.1, and the number of candidate genes that were potentially involved in disease resistance ranged from 6 in QTL9.1 to 29 in QTL6.1 -leucine-rich repeat (LRR) receptors by direct or indirect mechanisms . On QTL6Arabidopsis thaliana seems to be involved in defense response to bacteria and in the hydrogen peroxide biosynthetic process [The next stages of the plant immune response include a rapid influx of calcium ions, an oxidative burst of ROS and the activation of MAPKs –52. Four process .A. thaliana, one of the genes involved in this cascade is MAPK kinase 5, which is also one of the genes (Thecc1EG016564) that we identified in QTL3.1 [A. thaliana to both bacterial and fungal pathogens [The MAPK signaling cascade is involved in the biosynthesis and signaling of plant stress hormones, ROS generation, stomatal closure, defense gene activation, phytoalexin biosynthesis, cell wall strengthening and programmed cell death (PCD) . In A. tn QTL3.1 , 48, 51.n QTL3.1 . NDR1  by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and SA, and elevated resistance to biotrophic pathogens, as well as increased susceptibility to necrotrophic pathogens [A. thaliana may interact with a TIR-NBS-LRR class disease resistance protein. Among the other candidate genes, two were annotated as BURP domain-containing proteins. These proteins are involved in abiotic and biotic stresses in Brassica [Another important pathway in disease response and PCD is the ubiquitin/26S proteasome-mediated pathway, which is involved in the selective degradation of proteins in cells of eukaryotic organisms –61. At lathogens . Tcm009sathogens ), uveal athogens . This paathogens , 70, andathogens . Tcm009sathogens , this prBrassica .2+ and MAPKs also control the synthesis and/or signaling of the hormones that are involved in the later steps of defense gene activation and PCD. The SNP marker Tcm004s00110232 that is associated with QTL4.1 is located within Thecc1EG016777 encoding a UDP-glucose pyrophosphorylase 2. Overexpression of this enzyme in poplar showed extremely high quantities of the glycoside of salicylic acid (SAG). SA accumulation, and therefore SAG, could catalyze the increase in other plant defense metabolites [A. thaliana [Caabolites . Also inthaliana .Of the 11 candidate genes selected for differential gene expression . They identified 1,269 upregulated and 698 downregulated differentially expressed genes in cacao. Of those differentially expressed genes 26 were located within our identified QTL. Three of those genes were pathogen-related (CDG0008741 in QTL6.2) or receptor-like kinases involved in signal transduction (CDG00002586 and CDG00002587 in QTL6.1). The later one showed a positive fold change of 25.37 compared to the untreated control. This gene is also located between our best SNP marker on QTL6.1 (Tcm006s19715703) and the closest neighboring SNP (Tcm006s19387431). Three other genes, CGD0026033 in QTL6.2, CDG0031650 and CDG0031682 in QTL9.2, are involved in photosynthetic, carbon and nitrogen metabolism, more specifically in sugar transport, beta-oxidation of fatty acids and amino acid metabolism, respectively [A study performed by Teixeira et al.  showed aectively . It is iectively  during tOne of the main requirements of an efficient MAS program in cacao is to have reliable markers associated with the trait(s) of interest. These markers and ultimate knowledge of the underlying genes must be identified first. This knowledge demands a combination of large mapping populations with very distinct parents that segregate for the trait(s) of interest, a dense set of mapped markers, and rigorous phenotyping capabilities. The availability of a sequenced genome helps in the identification and annotation of potential candidate genes, although it is important to mention here that annotations are based on the Matina 1–6 genome, which is susceptible to WBD. It is therefore not unlikely that one or more genes responsible for the resistance phenotype occur specifically in the resistant cultivars, which might render the use of the Matina 1–6 genome inappropriate. We combined these requirements to identify several genomic regions that are associated with WBD resistance, and a novel source of resistance that most likely comes from the ‘CCN 51’ parent. Thanks to the large population size of the MP01 population, we were able to pick up QTL and markers with relatively small effects that can contribute to the creation and selection of more tolerant/resistant plant material.


To evaluate the role of in vivo dosimetry with radiochromic films for dose verification in cutaneous radiation therapy (RT).Five patients with 8 cutaneous or sub-cutaneous malignancies of the face, neck, trunk and extremity receiving RT were included. Orthovoltage, megavoltage photon therapies were applied based on anatomic location. The delivered dose for each target was measured with GAFCHROMIC EBT3TM film. The differences between the prescribed and measured doses in each target were analyzed based on the RT characteristics, target location and custom patient set up. The accuracy of EBT3TM film measurement was verified by measurements in a solid water phantom.The mean measured dose was -3.2%  lower than prescribed over 23 measurements. A wide range of under dose was detected in orthovoltage therapy when a gap existed between skin and a closed-ended applicator surface. The magnitude of the under dosage was correlated with the degree of the gap (P=0.01). The phantom study confirmed the accuracy of GAFCHROMIC EBT3TM film measurement and found that the low measured dose in orthovoltage therapy was caused by the deviation from the inverse square law (ISL) of the beam output at extended source surface distance (SSD) for closed-ended applicators.A significantly low delivered dose for extended SSD orthovoltage therapy was demonstrated during cutaneous RT. The dose fall-off with distance is not completely compensated by the ISL standoff correction for orthovoltage therapy. GAFCHROMIC EBT3™ film is a useful and accurate tool for quality assurance of patients receiving a curative intended cutaneous RT. Primary cutaneous malignancies mainly include basal cell carcinoma (BBC) and squamous cell carcinoma (SCC). BCC is usually slow growing, rarely metastasizes and may be cured with effective local therapy including surgical resection, radiation therapy (RT) or both -3. RT foOrthovoltage X-rays or megavoltage (MV) energy electrons are the main cutaneous radiotherapy tools ,7,8. MV Accurate dose delivery is important to achieve and report control rates during curative-intent cancer therapy. It is possible that delivered dose may be different from the estimated dose due to unanticipated or under-appreciated skin gaps, tissue folds, or set-up and positioning variation.in vivo dosimeter for quality assurance during cutaneous radiotherapy. The manufacturer of GAFCHROMIC EBT3™ film did not provide any funding or have any input into the study design, conduct, analysis or reporting.GAFCHROMIC EBT3™ film has an active layer of radiation-sensitive organic microcrystal monomers on thin polyester located between two identical polyester layers. The film is near tissue equivalent. EBT3™ film is highly sensitive to ionizing radiation. These features make it potentially suitable as a pragmatic clinical dosimeter. The aim of the current study was to examine the clinical value of GAFCHROMIC EBT3™ film as an Five consecutive patients including one male and four females received RT to 8 cutaneous or sub-cutaneous lesions of the face, neck, trunk and extremity by a single radiation oncologist. The median age was 86 years (range 49–91 years). Skin lesions included primary BCC, SCC and metastatic melanoma. This investigation was considered a quality assurance study by the institutional research ethics board but individual consent was obtained from each patient prior to the film dosimetry measurements.2 field using 6 MV photons. A mould of the patient’s hand and wrist was made out of Polyethylene Terephthalate Glycol. The mould was mounted to a 10 × 22 × 30 cm3 plastic box filled with LiquiBlock™ to provide sufficient backscattering and build-up for a 6 MV photon beam LiquiBlock™ is a non-toxic absorbent polymer salt that when mixed with water forms a jelly-like substance used for bolus. The jelly was distributed so that 1–1.5 cm of build-up covered the target area. A 101 cm source to skin distance (SSD) was set to the skin surface. Table The RT dose was measured by placing film on the target surface for patients who received orthovoltage X-ray therapy. Additionally, one patient with MV photon therapy was included for comparison. Patients with lesions of the face, neck or chest wall skin were treated with orthovoltage photons using an Xstrahl-300 X-ray therapy unit  with treatment energies of 150 and 200 kVp and half-value layers of 6 mm Al and 1 mm of Cu, respectively. Treatment was delivered with either open-ended circular applicators (diameters of 1.5 or 3 cm) at 30 cm focus to skin surface distance (FSD) for small lesions or closed-ended square applicators at 50 cm FSD for larger lesions. Custom Pb cut-outs were manufactured for large irregularly shaped lesions treated with orthovoltage. One patient with a SCC of the left wrist had a positive margin after amputation and received adjuvant RT with a single 6 × 7 cmISL = [FSDN/(FSDN + S)]2 = SOF, where FSDN is the nominal FSD (30 cm for open-ended and 50 cm for closed-ended applicators), and S is the amount of standoff in cm. MUs for the MV photon treatment were calculated using standard tissue-maximum ratios.RT doses were prescribed, and film measurements were taken at the skin surface. Monitor units (MUs) for orthovoltage treatment were calculated based on the prescribed daily dose modified by a back scatter factor (BSF), ISL if there was standoff from the skin surface to the applicator, and the machine output factor. The ISL correction factor or standoff correction (SOF) factor was defined as: I 2 and the largest film was 6 × 6 cm2. Differences between prescribed and measured doses were calculated as: (DoseM –Dosep)/Dosep × 100%, where Dosep, and DoseM were the planned and measured doses, respectively.The delivered dose for each target was measured with GAFCHROMIC EBT3™ film  for a minimum of three fractions per lesion (except one lesion had two). EBT3™ film was placed on the skin surface. The goal was to measure a point dose at the centre of the lesion. For each case, the film was cut to conform to the lesion size and location. If there was an irregular surface, the film was cut small enough to be placed directly on the surface at the center of the field. The smallest film was at least 1 × 1 cmA single batch (Lot# A05151203) of EBT3™ film was used for all measurements because the film response and calibration curves can be batch specific . Films w2 pieces. Due to the energy dependence of EBT3 films from the kilovoltage range to the MV range [60Co unit  was used to calibration for the MV beam. The Xtrahl orthovoltage treatment beam was calibrated in absolute dose following the American Association of Physicists in Medicine (AAPM) TG61 guideline [60Co unit was calibrated following the AAPM TG51 guideline [The calibration films were cut into 4 × 4 cmMV range , separatuideline , and theuideline . A minimuideline .2 square, closed-ended applicator with no standoff (i.e. 0 cm) was delivered at 0, 1, 2, 3, and 5 cm standoff distances. The same experiment was repeated with a Markus plane parallel ionization chamber  in air for verification. A detailed description of the ion chamber measurements has been reported previously [A systematic difference between measured and prescribed dose was noticed for patients treated on orthovoltage in whom a standoff correction had been calculated Figures  and 2. Teviously . To direeviously .Figure 1The mean differences between film-measured and prescribed doses were compared using a two-tail Student’s paired t-test with a cut-off p-value < 0.05 to indicate statistical significance. ANOVA was used to analyze the differences between group means and their associated variation.We evaluated over 23 measurements for 8 lesions. On average the film measurements were 3.2% lower  than the prescribed doses Figure . For theThe observed correlation between standoff and measured under dosage was investigated using water phantom measurements. After factoring out the impact of back scatter, the film measurements compared those measured with an ion chamber were well within experimental uncertainty. The dose fall-off with gap distance measured by the EBT3™ film was greater than predicted by the ISL standoff correction as shown in Figure 2 open-ended or closed-ended applicators. The clinical decision to use a particular applicator is based on tumor size, lesion depth and location. When an air gap exists between the skin surface and the end of the applicator, an ISL correction is used to adjust the number of MUs delivered to compensate for beam divergence. When the standoff gap was larger than 1 cm, the dose measured with the EBT3™ film was significantly lower than the prescribed dose and the extent of under dosage was directly proportional to the size of the air gap between the skin surface and the end of the treatment unit applicator (Figure In the current study, film-measured dose during orthovoltage therapy displayed large deviations from calculated doses in particular, when there was standoff of 1 cm or greater. The Xstrahl-300 orthovoltage unit was commissioned for clinical beams of 100, 150, and 200 kVp using a range treatment delivery device from 1.5 cm circles to 20 × 20 cmA 1–2 cm air gap is most likely to occur when a closed-ended applicator is applied to an irregular surface Figure . For larPreviously , we quanThe film measurements from 6 MV photon revealed non-significant dose difference between estimated and delivered. Therefore, no more case studies were further performed. This was in line with our initial study objectives of any systematic uncertainty greater than 5% warranted further investigation. The concordance of doses during the MV photon treatment in this study could be considered an additional verification of the systematic variation noted between the film-measured and prescribed doses when using orthovoltage energy with closed end applicators with increasing applicator to skin gaps.in vivo dosimeter should be part of treatment planning quality assurance during curative intent cutaneous RT. At the very least it should be used for initial treatments on a new orthovoltage unit, and when there is measurable standoff between the applicator end plate and the skin surface.Only small dose variations were observed between fractions. This is attributed to variations in patient set-up, reproducibility of skin lesion positions and uncertainties in placement of the film dosimeter between fractions. The current results suggest that a simple, in vivo dosimetry should be used as a quality assurance tool that can help to assess dose delivery. Minimization of large skin gaps is crucial to avoid unacceptable under dosage.Cutaneous cancers occurring on irregular skin surfaces such as the face, ear or neck are commonly seen in the clinic. Orthovoltage RT provides a simple, non-invasive alternative to surgical excision and can achieve high rates of complete response and local control. For small lesions encompassed in open-ended cones without air gaps, calculated doses are reliable estimates of the delivered dose. For larger lesions and lesions located on irregular surfaces treated with closed-ended applicators, a 1 cm or larger gap requires a larger correction than estimated using the ISL correction. For such cases, EBT3™ film-based in vivo dosimetry demonstrated that delivered doses were significantly lower than calculated doses for extended SSD orthovoltage techniques using closed-ended applicators. Dose fall-off due to air-gaps as frequently occur when treating irregular surfaces were not completely compensated by the inverse-square law corrections. This is commonly neglected in clinical practice. In vivo film measurements can be used to improve quality assurance during curative intent cutaneous radiotherapy.EBT3™ film-based