The enhanced doping effect caused by graphitic nitrogen in the GG produced an asymmetry electron–hole transport characteristic, indicating that the long–range scattering (ionized impurities) plays an important role in determining the carrier transport behavior. Analysis of temperature dependent conductance showed that the carrier transport mechanism in the GG was thermal excitation, whereas that in the PG, was a combination of thermal excitation and variable range hopping.Single–layer and mono–component doped graphene is a crucial platform for a better understanding of the relationship between its intrinsic electronic properties and atomic bonding configurations. Large–scale doped graphene films dominated with graphitic nitrogen (GG) or pyrrolic nitrogen (PG) were synthesized on Cu foils via a free radical reaction at growth temperatures of 230–300 °C and 400–600 °C, respectively. The bonding configurations of N atoms in the graphene lattices were controlled through reaction temperature, and characterized using Raman spectroscopy, X–ray photoelectron spectroscopy and scanning tunneling microscope. The GG exhibited a strong n–type doping behavior, whereas the PG showed a weak n–type doping behavior. Electron mobilities of the GG and PG were in the range of 80.1–340 cm Graphene, a two–dimensional crystal with carbon (C) atoms packed in a hexagonal lattice, has attracted extensive research attention in the past decade, owing to its extraordinary properties123891011121516171991011124/NH3 as precursors, graphitic N dominated graphene was synthesized on Cu foils at 800–1000 °C92H4/NH3 as the precursors23Chemical vapor deposition (CVD) is a powerful method among the synthesis techniques of heteroatom doped graphene films910Herein, we proposed a temperature controlled method for N–doped graphene film growth on Cu foils via a free radical reaction. The graphene was predominantly doped with graphitic and pyrrolic N atoms at different temperatures of 230–300 °C and 400–600 °C, respectively. The bonding configurations of N atoms in the graphene lattice were characterized using Raman spectroscopy, X–ray photoelectron spectroscopy (XPS) and scanning tunneling microscope (STM). The graphitic N doped graphene exhibited a strong n–type transport behavior, whereas the pyrrolic N doped graphene showed a weak n–type transport behavior with a symmetric electron–hole transport. Analysis of temperature dependent conductance showed that the carrier transport mechanism was a thermal excitation process for the graphitic N dominated graphene, whereas it was a combination of thermal excitation and variable range hopping for the pyrrolic N dominated graphene. As–grown mono–component doped graphene film is an important platform for the understanding of the relationship between the bonding configurations and its intrinsic physics of N–doped graphene, as well as development of graphene–based devices.Growth of the N–doped graphene film on Cu foils via the free radical reaction was previously reported by our groupTo reveal the influence of temperature on the growth of N–doped graphene via the free radical reaction, the Cu foil substrates were kept at various temperatures between 230 °C and 600 °C for growth. The morphology of the resultant film was characterized using SEM  and opti126−1 and ~2680 cm−1 are assigned to the G band and 2D band, respectively, which confirm the presence of graphitic carbon−1 activated by the defects via an intervalley double–resonance process is assigned to D bandD/IG) reveals the degree of defects and in–plane crystallite size (LD). Herein, the ratio (ID/IG) decreases as the temperature is increased in the range of 230–400 °C . The downshift of position agrees well with the electron doping feature in the graphene. Otherwise, the change of the fwhm(2D) appeared to be irregular with a larger bandwidth, which can’t be applied alone to identify the doping effectThe position and shape of Raman peaks can also provide doping information in the graphene. Herein, the position of G band was upshifted to the range of 1588.1–1589.0 cmr the GG  comparedsp2 hybridized C–C bond (C1). The peaks at 285.4 ± 0.2 eV and 286.4 ± 0.1 eV correspond to the trigonal phase with a sp2 C (C2) and the tetrahedral phase with a sp3 C (C3), respectively922sp2 C bond (C2). It may be come from pyridinic N or graphitic N atoms in graphene framework as well as the nitro– group connected to graphene framework, considering the spatial and electronic structure after incorporated these N atoms16sp3 C bond (C3). This bonding is originated from new type of incorporation N atoms other than pyridinic N or graphitic N atoms. Meanwhile, this bonding will increase the structural distortion by disrupting the plane framework of graphene. It would be assigned to pyrrolic N bonding configuration. The relative content of each component  is higher than that of C–N (305 kJ mol−1)These bonding configurations of N atoms are determined by bonding energies and their thermal stabilities. The coupling reaction could reduce the energy barrier for dechlorination and leave pyridinic ring radicals intact at low reaction temperatures , although the bonding energy of C–Cl  occurs, the conductance is linearly depended on Vg in both electron– and hole– transport regimes, indicating a long–range scattering (incorporated impurities) in the GG1946μ = (ΔIds·L/W)/(ΔVgs·Vds·Cox), where, Cox is the silica gate capacitance (1.15 × 10−8 F·cm−2 for a gate oxide thickness of 300 nm). As a result, the hole and electron mobilities are in the range of 59.5–429.8 cm2 V−1·s−1 and 80.1–340 cm2 V−1· s−1, respectively. The obtained high mobility of the carriers would be attributed to minimized defect formation upon direct substitution  at Vg = Vg,crossing and Vg,crossing (indicated by arrow). In our transport measurements, the gate voltage at Gcrossing is found to be equal to the Vg,min , implying the potential fluctuations induced by the randomly distributed charged impurities  localize in the graphene system with a homogenously spatial distributions5g,min and Vg,crossing observed in some nitrogen and potassium doped grapheneg regimes for both in the GG and PG  transport5050Incorporation of the pyrrolic nitrogen into graphene introduced not only defects but also a strong disorder resulting in localization of carriers, which is similar to the carriers in the rGO sheetWe have synthesized graphitic and pyrrolic N dominated graphene films by controlling the growth temperature via the free radical reaction. Raman spectra, XPS and STM results confirm the graphitic N and pyrrolic N dominated graphene films are grown at temperatures in the range of 230–300 °C and 400–600 °C, respectively. Furthermore, the graphitic N dominated graphene exhibits a strong n–type doping effect, whereas the pyrrolic N dominated graphene shows a weak n–type doping effect. The enhanced doping effect in graphitic N dominated graphene results in an asymmetry electron–hole transport which suggests that the scattering by ionized incorporated N impurities plays an important role in determining the carrier transport behavior. Temperature dependent conductance shows carriers transport mechanism in the GG was thermal excitation, whereas that in the PG was a combination of thermal excitation and variable range hopping.2 atmosphere (90 sccm) at 15 torr, followed by cooling down to the designated temperature (230–600 °C) and then the pentachloropyridine was heated to 130 °C and kept for various durations (0.5–5 min). After growth, the furnace was cooled down to room temperature. As–grown N–doped graphene was transferred onto the SiO2/Si substrates using a PMMA supported method for the characterization and device fabrications.Cu foils  and pentachloropyridine  were separately placed in a furnace with two–temperature zones. Prior to the growth of graphene, the Cu foils were annealed at 1000 °C for 20 min in H2/Si substrates without further annealing. The SEM images, STM images and XPS data were collected directly from all the doped graphene on Cu foils after growth.As–grown N–doped graphene was characterized using scanning electron microscopy , transmission electron microscopy , optical microscopy (Leica DM4500P), Raman spectroscopy , atomic force microscopy , X–ray photoelectron spectroscopy  radiation as a probe) and scanning tunneling microscope . Raman spectra, optical and AFM images of all the doped graphene were collected from the transferred doped graphene onto the SiO2/Si substrate with a 300 nm thermal oxide layer, and then patterned into micro–ribbonsL) and width (W) were measured to be 14 μm and 24.5 μm, respectively. To obtain a better contact, the devices were thermally annealed at 200 °C in H2/Ar (10/20 sccm) atmosphere for 20 min. Electrical measurements were carried out in a vacuum after pumping for 12 hours at room temperature using a semiconductor analyzer (Keithley 4200–SCS) combined with a Lakeshore probe station. Low temperature measurements were carried out from 5.2 K to 300 K in a vacuum of 1.58 × 10−6 torr.Electrical properties of the N–doped graphene films were evaluated based on a field effect transistor (FET) configuration. The N–doped graphene film was firstly transferred onto a SiOHow to cite this article: Zhang, J. et al. Tunable electronic properties of graphene through controlling bonding configurations of doped nitrogen atoms. Sci. Rep.6, 28330; doi: 10.1038/srep28330 (2016).


Tbx5, a known modifier of CHD, to heart defects on a trisomic backgroun. Mice that were heterozygous for a Tbx5 null allele were crossed with Ts65Dn mice. Thoraxes of progeny were fixed in 10% formalin, embedded in paraffin, and sectioned for analysis of CHD. Gene expression in embryonic hearts was examined by quantitative PCR and in situ hybridization. A TBX5 DNA binding site was verified by luciferase assays.Nearly half of all individuals with Down Syndrome (DS) have some type of congenital heart defect (CHD), suggesting that DS sensitizes to CHD but does not cause it. We used a common mouse model of DS, the Ts65Dn mouse, to study the contribution of Mice that were heterozygous for a Tbx5 null allele were crossed with Ts65Dn mice. Thoraxes of progeny were fixed in 10 % formalin, embedded in paraffin, and sectioned for analysis of CHD. Gene expression in embryonic hearts was examined by quantitative PCR and in situ hybridization. A TBX5 DNA binding site was verified by luciferase assays.Tbx5 null allele with Ts65Dn mice. Mice that were trisomic and carried the Tbx5 mutation  had a significantly increased incidence of overriding aorta compared to their euploid littermates. Ts65Dn;Tbx5+/− mice also showed reduced expression of Pitx2, a molecular marker for the left atrium. Transcript levels of the trisomic Adamts1 gene were decreased in Tbx5+/− mice compared to their euploid littermates. Evidence of a valid binding site for TBX5 upstream of the trisomic Adamts1 locus was also shown.We crossed mice that were heterozygous for a Tbx5 and trisomy affects alignment of the aorta and this effect may stem from deviations from normal left-right patterning in the heart. We have unveiled a previously unknown interaction between the Tbx5 gene and trisomy, suggesting a connection between Tbx5 and trisomic genes important during heart development.Haploinsufficiency of The online version of this article (doi:10.1186/s12861-015-0080-y) contains supplementary material, which is available to authorized users. Congenital heart defects (CHD) comprise the most common congenital anomaly in live births . There iTbx5, a known contributor to heart development, as a modifier and assess its interaction with trisomy. Tbx5, a transcription factor, has well-described effects in cardiac and limb development [Tbx5 has a well-studied role in the morphogenesis of the four heart chambers. It is expressed in the left ventricle (LV) and both atria during chamber maturation and septation [Tbx5 in the left ventricles (LV) of mice suppresses formation of the ventricular septum, resulting in formation of a single ventricle [Tbx5 expression might affect heart development on the “sensitized” trisomic background.We consider the role of elopment , 11. Mutelopment . Tbx5 haeptation . Ectopicentricle . Its impCreld1 and Hey2 genes as disomic modifiers of septal development on this trisomic background [Tbx5 in heart development.Animal models provide important information for understanding the pathogenesis of CHD and the molecular mechanisms that give rise to these conditions. Orthologs of many genes on Hsa21 are found on mouse chromosome 16 (Mmu16), with smaller subsets on Mmu10 and Mmu17 . The mosckground . HaploinTbx5 has been described [Tbx5−/− embryos (Black Swiss/SvJ background) die by embryonic day 10.5 (E10.5) and lack cardiac looping and endocardial cushion formation, among other severe defects [Tbx5+/− mice are greatly influenced by genetic background. Bruneau et al. report a 10 % frequency of the Tbx5 null allele on a 129SvEv/129SvJ background at birth and 28 % on a Black Swiss/129SvJ background, instead of the expected Mendelian ratio of 50 %. Deviation from the expected frequency indicates that prenatal loss has occurred; the different frequencies in different mouse strains suggest that genetic background contributes to the penetrance and expressivity of heart phenotypes in this situation [Tbx5 dosage are susceptible to additional genetic modifiers. The molecular mechanisms by which Tbx5 influences heart development are incompletely described and possible interactions between Tbx5 and genes on Hsa21 are unknown. We hypothesize that Tbx5 acts as a genetic modifier to alter CHD in Ts65Dn mice. Here we provide evidence of an interaction between Tbx5 and trisomy and the effects of that interaction on trisomic gene expression and left-right patterning of the heart.A mouse model with a null allele for escribed . Homozyg defects . The viaituation . TherefoTbx5+/− male mice and B6C3.Ts65Dn females were established to examine the role of Tbx5 as a modifier of CHD. Genetic background of the Tbx5 mice affected viability  background  background. There is some loss of Ts65Dn fetuses during late gestation and the observed frequency of 42 % trisomic pups at P0 . ASD and Gerbode’s defect, an abnormal communication between the right atrium and left ventricle . Three of these animals were affected, with an AVSD, ASD and VSD, respectively  in ~58 % of Ts65Dn;Tbx5ce Table . The pencle Fig. , were alTbx5+/− mice is an interaction between Tbx5 and a trisomic gene(s) that is regulated by this transcription factor. We considered 109 genes that are trisomic in Ts65Dn mice [Adamts1, Dyrk1a, Rcan1, Ripply3, Sh3bgr and Wrb). We used the Transcription Element Search System program  to identify potential TBEs in the first exons and/or promoter regions of those genes [Adamts1 for further investigation.The simplest explanation for the increased incidence of overriding aorta in Ts65Dn;5Dn mice  and gene5Dn mice . Howeverse genes  (see MetAdamts1 encodes an extracellular matrix protease. Transcript levels were increased in Tbx5+/− mice (p = 0.04)  [et al. [Adamts1 transcription start site, was amplified by PCR and cloned into the pGL3 luciferase vector  Fig. , suggestGGTGTVR) . He et a [et al.  found thTbx5  . The putative TBE was mutated to inhibit TBX5 binding and luciferase levels decreased compared to the construct with the intact TBE (p = 1.51 × 10−4).We transfected the construct into cells stably over expressing −4) Fig. . IntroduTbx5 is generally considered to be an inducer of transcription, consistent with the increase in luciferase signal when TBX5 binds the upstream Adamts1 region included in the construct. However, Adamts1 expression is increased when Tbx5 is reduced in Tbx5+/− mice . The expected expression pattern was observed in other organs . Thus, atrial isomerism likely contributes to the significant increase in overriding aorta in the Ts65Dn;Tbx5+/− animals.Overriding aorta and Gerbode’s defect are seen more often in Ts65Dn;somerism . To detents Fig. . These rnts Fig. , where PTbx5 dosage and trisomy resulted in a significant increase in defects of aortic alignment and revealed a potential effect on left-right patterning of the heart. The action of genetic modifiers in heart development is evident from the differences in penetrance and patterns of heart phenotypes in DS, in DS mouse models and when the Tbx5 null allele is bred onto different mouse genetic backgrounds. In addition, Holt-Oram patients who inherit the same TBX5 mutation have variable heart phenotypes, indicating that additional factors affect development [TBX5 displaying OA and a number of additional defects have been described [Tbx5 expression and trisomy with increased OA as a primary outcome.The interaction between elopment . Previouelopment , 9. For escribed . Isolateescribed . Our resPitx2 knockout mice [Pitx2 expression is sufficient to cause septal and valve defects [Pitx2 and Cited2 genes which are involved in left-right patterning exhibit phenotypes similar to the ones reported, including Gerbode’s defect and overriding aorta [Pitx2 is an important determinant of left-right asymmetry of the heart and gut [Pitx2 expression [Pitx2 regulates Tbx5 expression in the abdominal wall during development [Tbx5+/− embryos have reduced Pitx2 expression, which can contribute to atrial isomerism and aortic defects.Disruptions in the left-right patterning pathway frequently lead to the kinds of heart defects seen here. OA is considered to be a milder form of double outlet right ventricle (DORV), the most common defect seen in out mice , arisingout mice . Reducin defects . Mice lang aorta –37. Pitx and gut , 37–40 a and gut , 39, 41.pression , and Pitelopment . Ts65Dn;Tbx5 haploinsufficiency are combined. We suggested previously that a universal response deficit to Sonic hedgehog signaling due to trisomy could explain many of the clinical features of DS [Pitx2 expression [Pitx2 expression seen here.The left-right signaling pathway is affected when trisomy and es of DS ; hedgehopression –47. Exprpression . A respoTbx5 dosage show increased expression of the Adamts1 gene. Adamts1 is important in the development of the extracellular matrix in endocardial cushions [Tbx5 dosage may contribute to the risk of heart defects by a role in transcriptional regulation of the Adamts1 gene, a regulator of the VEGF pathway.Finally, we discovered that mice with reduced cushions , and is cushions . It is icushions . ReducedTbx5+/− mice relative to either genetic defects in isolation. The mechanism likely involves reduced expression of Pitx2, a gene with known roles in laterality. In addition, we demonstrated that a change in Tbx5 dosage affects transcription of Adamts1, suggesting it is a target of TBX5 in normal heart development in vivo.Defects in left-right patterning likely contribute to the significant increase in overriding aorta in Ts65Dn;Tbx5 dosage and trisomy interact to affect the outcome of heart development, potentially altering left-right patterning of the heart. We provided evidence of weak Pitx2 expression, important for left-right patterning, in the Ts65Dn;Tbx5+/− mice. We have also provided evidence that TBX5 plays a role in regulation of the trisomic Adamts1 gene. A valid TBX5 binding site was shown upstream of the Adamts1 locus and mutation of the site significantly affected transcription.We have shown that ad libitum. Ts65Dn mice (B6EiC3H-a/A-Ts(1716)65Dn) were obtained from The Jackson Laboratory and maintained as an advanced intercross on the B6;C3H background. Tbx5 heterozygous null mice (Tbx5+/−) [Tbx5+/− mice onto a C57BL/6J background for at least five generations. All data was taken after mice had been backcrossed for at least five generations. Progeny in Tables Tbx5+/− mice on a B6 background with wild type B6 mice (obtained from Jackson Laboratory) or B6;C3H mice bred in our lab. Progeny in Tables Tbx5+/− males on a B6 background. All procedures were approved by the Institutional Animal Care and Use Committee.Animals were maintained in a virus and antibody-free facility with food and water Tbx5+/−)  were kin3H-a/A-Ts71665Dn) Tbx5 genotyping, three primers designed to amplify either the wild type or null alleles were added together in each reaction [Tbx5 genotyping are as follows: 3 F2: 5′-CCCAGCGGCAGGCGTAGAC -3′; Loxp-F: 5′-GCAGCGCAGTCCTCACCAG -3′; Loxp-R: 5′-AAATTCCAACCCCTTCCACAGAT -3′. The PCR was done under the following cycling conditions: 94 °C 3 min,  for 35 cycles, 72 °C 10 min.We extracted genomic DNA from tail tips of mice by heating at 90 °C for 2 h in 10 mM NaOH, 0.2 mM EDTA and used for genotyping by PCR. Sequences of primers for Ts65Dn genotyping are as follows: C17F: 5′-GTGGCAAGAGACTCAAATTCAAC-3′; C16R: 5′-TGGCTTATTATTATCAGGGCATTT-3′; IMR5: 5′-AAAGTCGCTCTGAGTTGTTAT-3′; IMR6: 5′-GGAGCGGGAGAAATGGATATG-3′. For Ts65Dn genotyping, PCR was done under the following cycling conditions: 95 °C 3 min,  for 31 cycles, 72 °C 5 min. For Tbx5+/− crosses at postnatal day 0 (P0) within hours of birth. We euthanized pups and removed and fixed thoraxes in 10 % formalin for at least 48 h. Tissues were embedded in paraffin, sectioned at 7 μm and stained with hematoxylin/eosin using standard methods. The heart morphology in each animal was scored under a dissecting stereomicroscope  by at least two individuals blinded to genotype. Pictures were taken using the NIS-Elements Br software .We collected the progeny of Ts65Dn x Tbx5+/− mice . Virtual Histology™ is a micro-CT based method that allows for high resolution imaging. Specimens were stained in a proprietary solution before being scanned at 20 μm resolution with a volumetric micro-CT instrument. Using this method tissues are left intact and imaging analysis can be used to create “virtual” histological sections.Virtual Histology™ was performed on six Ts65Dn;We examined 109 genes that are trisomic in Ts65Dn mice for localization of expression in the heart using the following databases: EMAGE gene expression database , VisiGenTbx5 was quantified using a Taqman FAM-labeled pre-designed assay (Mm0195728_m1) from Applied Biosystems and normalized to a Gapdh VIC-labeled assay (Mm99999915_g1) using Taqman Gene Expression master mix. POWER SybrGreen master mix was used for all other expression assays . Primers used are as follows: Adamts1-F: 5′-CACGTGTGACACTCTCGGAA-3′; Adamts1-R: 5′-CGTGCGGCATGTTAAACACA-3′; Gapdh-F: 5′-TGCACCACCAACTGCTTAG-3′; Gapdh-R: 5′-GATGCAGGGATGATGTTC-3′; Pitx2-F: 5′-GCAGCCGTTGAATGTCTCTTC-3′; Pitx2-R: 5′-GTCCGTGAACTCGACCTTTTT-3′. PCR was done under the following cycling conditions: 95 °C 15 min,  for 40 cycles, followed by a melt curve analysis between 95 and 60 °C. Expression of candidate genes was normalized to Gapdh. Fold change expression values were determined using the delta delta Ct method [We extracted total RNA from the hearts of embryos using the RNeasy mini kit . cDNA synthesis was carried out with the First-Strand cDNA synthesis kit  using 1 μg of total RNA as template. PCR was carried out on a 7500 Real-Time PCR System . t method .Tbx5 cDNA clone from the I.M.A.G.E. consortium and subcloned it into the pcDNA3.1+ vector , putting it under the control of the CMV promoter. NIH-3T3 cells were transfected with the pcDNA-Tbx5 construct using Lipofectamine 2000 . Stably transfected cell lines were selected with 1 mg/ml Geneticin . We maintained the cells in Dulbecco’s Modified Eagle Medium with high glucose (4.5 g/L) , 10 % fetal bovine serum, 1 mg/ml Geneticin, 1X glutamine and penicillin/streptomycin at 37 °C and 5 % CO2.We obtained a Adamts1 that was bound by TBX5 in a ChIP study [Adamts1 promoter. The following primers were used to mutate the putative TBE site and insert an EcoRI restriction site, Primer 1: 5′-CACAGCTCGTCACTCTGGGAATTCAAGACGCCGAAACAGCGCTG-3′; Primer 2: 5′-CAGCGCTGTTTCGGCGTCTTGAATTCCCAGAGTGACGAGCTGTG-3′. We transfected the constructs into NIH-3T3 cells constitutively over expressing Tbx5 . Luciferase levels were measured in a 1450 Wallac Jet MicroBeta liquid scintillation and luminescence counter  using the Dual Luciferase Assay Kit .We amplified the portion of the promoter region of IP study , by PCR in situ hybridization using traditional dioxigenin (DIG)-labeled RNA probes detected with alkaline phoshatase (AP)-conjugated anti-dioxigenin antibody using nitro blue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl-phosphate toluidine-salt (NBT/BCIP) substrate. DIG RNA labeling mix, anti-DIG-AP antibody, blocking reagent, NBT/BCIP were all purchased from Roche Applied Science  and used according to the manufacturer’s instructions with minimal modifications. The Pitx2 antisense and sense probes were described earlier [E13.5 embryos were fixed in 4 % paraformaldehyde for 1 h, rinsed in 1X PBS three times for 5 min and then dehydrated in 25, 50 and 70 % ethanol for 1 h each. After dehydration the embryos were stored at −20 °C in 70 % ethanol until embedded in paraffin and sectioned according to standard methods. We performed  earlier . Images t-test using delta Ct values. All tests were 2-tailed and P < 0.05 were considered significant.The incidence of various heart defects for different genotypes was compared by Fisher’s test using GraphPad Prism version 5. A 2×2 contingency table was made comparing the two groups for each separate phenotype. The relative quantification of gene expression from different genotypes was compared by the student’s


P = 0.010), as assessed by low E-cadherin and high vimentin expression. The overall survival of PD-L1+ patients with EMT features was significantly worse than those without EMT features (P = 0.007). In an independent validation cohort (N = 91), as well as in HNSCC cases of The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia, high PD-L1 expression was also associated with the high probability of an EMT signature, referred from the GEO dataset, GSE4824. Survival analysis confirmed PD-L1+/EMT+ patients had a poorer prognosis than PD-L1+/EMT- patients in the TCGA cohort. PD-L1 positivity can thus be divided into two categories according to the absence or presence of EMT. PD-L1 expression is also independently associated with EMT features in HNSCC.Virus-associated malignancies and sarcomatoid cancers correlate with high PD-L1 expression, however, underlying mechanisms remain controversial. We evaluated the correlation between PD-L1 expression and epithelial-mesenchymal transition (EMT) in head and neck squamous cell carcinomas (HNSCC). Tumor tissues from 50 patients with HNSCC were evaluated for PD-L1 by immunohistochemistry, which showed 32 (64.0%) were PD-L1 positive (PD-L1+). Interestingly, PD-L1 expression was significantly associated with EMT ( Discovery of programmed death-ligand 1 (PD-L1) expression in tumors has encouraged research toward more efficient immunologic methods of conquering cancer , 3. The CD274 gene amplification at 9p24.1, which encodes PD-L1 [CDH1) down-regulation and vimentin (encoded by VIM) up-regulation, also correlated with PD-L1 induction [Viruses and epithelial-mesenchymal transition (EMT) are associated with high PD-L1 expression . PD-L1 ues PD-L1 . Anotheres PD-L1 –16. PD-Les PD-L1 . Moreovees PD-L1 . EMT chanduction . HoweverHead and neck squamous cell carcinoma (HNSCC) is a suitable model to investigate the clinicopathologic features associated with PD-L1 up-regulation. Recent advances in genomics have shown that mutational HPV-positive and -negative tumor profiles, which have retained and lost p16 expression, respectively, clearly differ –22. AlthIn the current study, we analyzed 1) PD-L1 expression according to the EMT and p16 statuses of patients with HNSCC, and 2) their clinical implications. A gene expression signature associated with EMT was obtained from the publicly available database, GEO4824 . This siP = 0.013, Figure P = 0.559). The vimentin H-score was significantly higher in PD-L1+/p16- compared with PD-L1-/p16- patients (P = 0.014). However, this trend was not observed in p16+ patients . The number of patients allocated to PD-L1-, PD-L1+/EMT-, and PD-L1+ /EMT+ groups were 32, 32, and 27, respectively , where PD-L1 positivity was significantly higher in patients with EMT-positive tumors  or progression-free survival . However, p16-/EMT+ displayed significantly higher PD-L1 expression compared to p16-/EMT- or p16+/EMT- . However, OS rates were not significantly different, although the 3-year OS rate differed between PD-L1+/EMT+ (40.2%) and PD-L1+/EMT- .OS and PFS rates for PD-L1+/EMT+ patients were significantly worse compared to those for PD-L1- patients from TCGA  and paranasal sinus cancer (N = 2) were included; of these, two patients showed a non-keratinizing type pathology. Therefore, caution is needed in the direct application of the current results to further clinical studies. However, significant statistical results were obtained for the independent validation cohort, as well as for TCGA and the CCLE open databases. The clinical outcomes of anti-PD-1/PD-L1 inhibitor therapy is lacking in this study, although overall prognosis according to PD-L1 expression and its up-stream mechanism should be understood before researching clinical outcomes of the target agent. Although the prognostic impact of PD-L1 expression was not reported, we clearly showed PD-L1 up-regulation according to two distinct up-stream pathways, EMT and HPV/p16, influenced the overall prognosis. The association of EMT with PD-L1 in human cancer is a novel finding in this study.In conclusion, PD-L1 expression is associated with EMT, an independent up-stream pathway distinct from HPV/p16 association. EMT-associated PD-L1 expression confers a significantly poorer prognosis compared to PD-L1 expression not associated with EMT. Clinical investigations using anti-PD-1/PD-L1 inhibitors in patients with EMT-associated PD-L1 up-regulation are warranted.Medical records were retrospectively reviewed for patients diagnosed with locally advanced HNSCC and treated at Seoul National University Hospital between December 2004 and November 2012. Fifty patients with paraffin-embedded tumor samples were included. Firstly, we analysed the association of PD-L1 and EMT in 50 HNSCC patients (training cohort). To confirm preliminary findings, we enrolled a further 91 HNSCC patients , and performed similar analyses.Treatment decisions were determined by a multidisciplinary team , 45. Pat® mAb . IHC was performed using the Ventana Benchmark XT system . When tissue sections showed diffuse and strong nuclear and cytoplasmic staining in ≥ 70% of tumor cells, this was considered positive for p16 [Representative, formalin-fixed, paraffin-embedded tissue blocks from each case were submitted for immunohistochemistry (IHC) using the following antibodies: mouse anti-p16 (E6H4) monoclonal antibody , mouse anti-E-cadherin (36B5) mAb , mouse anti-vimentin (V9) mAb  and rabbit anti-PD-L1 (E1L3N) XP for p16 . For E-chttp://linus.nci.nih.gov/BRB-ArrayTools.html) was used in the analysis of gene expression data [P values were two-sided, and considered significant if P < 0.05. A false discovery rate were applied to control type I errors. All statistical analyses and data generation were carried out using R version 3.1.3 (http://www.r-project.org) and STATA version 12 .The BRB-ArrayTools software program (ion data . A heatmion data . To asseion data  and cellion data , 75 geneion data , and a pion data , 50. Briion data . The robion data . A compa


QLP and LLP) as a function of carrier density are proposed, and the control parameters are defined. Finally, the proposed analytical model is compared with experimental data which indicates good overall agreement.Graphene is an attention-grabbing material in electronics, physics, chemistry, and even biology because of its unique properties such as high surface-area-to-volume ratio. Also, the ability of graphene-based materials to continuously tune charge carriers from holes to electrons makes them promising for biological applications, especially in lipid bilayer-based sensors. Furthermore, changes in charged lipid membrane properties can be electrically detected by a graphene-based electrolyte-gated graphene field effect transistor (GFET). In this paper, a monolayer graphene-based GFET with a focus on the conductance variation caused by membrane electric charges and thickness is studied. Monolayer graphene conductance as an electrical detection platform is suggested for neutral, negative, and positive electric-charged membrane. The electric charge and thickness of the lipid bilayer ( The cAlthough the phospholipid bilayer frequently works well, it can be damaged, and some superfluous materials can penetrate it. Phospholipids have two ends; the first is hydrophilic and attracts water; and the second is hydrophobic and resists water . As the G-Vg characteristic of the graphene-based biosensor. A monolayer graphene-based GFET with a focus on the conductance variation caused by membrane electric charges and thickness is studied. Monolayer graphene conductance as an electrical detection platform is suggested for neutral, negative, and positive electric membrane. The electric charge and thickness of the lipid bilayer (QLP and LLP) as a function of carrier density are proposed and the control parameters are defined.Some smaller molecules, such as carbon dioxide and oxygen, can pass through without difficulty on their own, but larger molecules such as water, sodium, or magnesium cannot pass easily . The intE (k) relation of the GNR is obtained as [The monolayer graphene in an electrolyte-gated biomimetic membrane graphene transistor with a ballistic channel is assumed to monitor the changes in membrane integrity. High-carrier mobility is reported in experiments on the graphene, which is thought to be due to the totally ballistic carrier transportation in the graphene, which leads to a higher transmission probability. By applying the Taylor expansion on graphene band energy near the Fermi point, the ained as .kx is the wave vector along the length of the nanoribbon and β is quantized wave vector given by [M(E) at a given energy which is dependent on the sub bands location can be calculated. By taking the derivatives of wave vector k over the energy E (dk/dE), the number of the mode M(E) is written aswhere given by . Based oL is the length of the nanoribbon. A higher transmission probability causes a higher carrier conductance from source to drain, as provided by the Boltzmann transport equation [where equation ,3:q is the electron charge, Planck's constant is shown by h, E is the energy band structure, M(E) is the number of modes, f is the Fermi-Dirac distribution function and T(E) is the transmission probability. On the other hand, because of the ballistic transport T, the possibility of one inserted electron at one end that can be conveyed to other end is considered equivalent to one (T(E) = 1). The number of modes in accordance with the Landauer formula with respect to the conductance of monolayer graphene can be written aswhere l, k is the wave vector, and where the length of the graphene channel is shown with parameter x = (E - Eg)/kBT and the normalized Fermi energy is η = (EF - Eg)/kBT. Thus, the general conductance model of single-layer graphene obtained is similar to that of silicon reported by Gunlycke [VDS). As shown in Figure where Gunlycke . AccordiVg,min to measure the smallest conductance of the graphene layer, which is identified from the transfer characteristic curve. In total, the Vg,min shift (at the Dirac point) can be considered as a good indicator for lipid bilayer modulation and measurement. Nevertheless, the magnitude of the voltage shift from both positive and negative lipids is comparable when this shift is measured from the position of the minimum conductivity of bare graphene. As shown in Figure Vg,min on the device, and the hole density value can be estimated as decreasing. A negatively exciting membrane demonstrates a very small enhancement in conductivity and a positive change in the Dirac point compared with that of exposed graphene.This is because of an enhancement in the remaining pollution charges caused by the negatively charged membrane. A detection-charged lipid bilayer can be obtained based on a detectable Dirac point shift. In light of this fact, the main objective of the current paper is to present a new model for biomimetic membrane-coated graphene biosensors. In this model, the thickness and the type of coated charge as a function of gate voltage is simulated and control parameters are suggested. Subsequently, to obtain a greater insight into the role of both the thickness and the type of lipid bilayer, GFET modeling is employed to identify the relationship between the conductance and the voltage of the liquid gate, where two electrodes of the sensor, as shown in Figure As depicted in Figure α) and thickness factor (β) are suggested. Subsequently, for better understanding of the role of the lipid bilayer, FET modeling is employed to obtain an equation describing the conductance, electric charge, and thickness, where the suggested structure of the GFET is shown in Figure GLP is considered to be a function of electric charge (QLP) as follows. GLP = GNeutral + αQLP where electric charge factor (α) is assumed, GLP is the channel conductance of graphene with biomimetic membranes of different surface charges, and QLP is the electrical charge of the membrane. Consequently, the supposed conductance model of the graphene-based GFET channel can be written as.Different ions can be adsorbed by changes in the membrane's electric charge and thickness, and subsequently, the sensor will be capable of attracting the ions in the solution which have caused a transformation in the conductance of the graphene-based biosensor. Dependent upon the channel conductance in the biomimetic membrane-coated graphene biosensor, it is concluded that GLP is a function of electric charge and thickness, where GLP is the channel conductance after adding the lipid bilayer. The focus of the present paper is to demonstrate a new model for GFET to measure changes in the membrane's electric charge and thickness. In other words, the conductance of the GFET device as a function of different electric charges and thicknesses is simulated and an electric charge factor  and membrane thickness as follows:Furthermore, the proposed model is strongly supported by the experimental data. According to Figure β) and LLP are the thickness parameter and thickness of the adsorbed lipid bilayer, respectively. In the non-saturation region, the GFET conductance model is involved as a result of gate electrical energy and the perfect conductance-voltage related to the graphene channel of the GFET device, which leads to the modified conductance as:where  indicates the same trends as those reported by [Vg,min with increasing membrane thickness. Comparison of the experimental data depicted with the theoretical data in Figure Vg,min.In Figure GLP and LLP parameters, respectively, in agreement with the reported data which is shown in Table Vg,min did not shift further at greater membrane thicknesses due to the saturation current density of the injected carrier concentration by the charged lipid bilayer.In the suggested model, differently charged lipid bilayers and membrane thicknesses are demonstrated in the form of m,min belongs to the graphene-based biosensor, the control parameter with respect to the iteration method is suggested as:According to the saturation region of the presented conductance model and given that gl1 = 0.4157 and l2 = -0.543. In addition, α for the neutrally, negatively, and positively charged membrane is assumed to be 0, 1, and -1, respectively. Consequently, the justified model for the interaction of charged impurity and the consequence of charged lipid membranes in a biomimetic membrane-coated graphene biosensor is proposed aswhere Vg,min of the ambipolar FET occurs due to the electronic devices on both the n-doping and p-doping materials.The proposed model, coupled with the experimental data, is shown in this work to confirm that the conductivity of the graphene-based biosensor is changed by the electric charge and membrane thickness of the lipid bilayer. In a nutshell, electrolyte-gated graphene field-effect transistor structure was used after chemical vapor deposition (CVD) as the electrical transduction stage because of its high electrical conductivity, optical transparency, and large area, given the likelihood of manufacturing a dual-mode optical and electrical detection system for detecting the changes of membrane properties. Based on what has been discussed, one could firmly claim that, in response to changes of the charged lipid membranes and charges of biomimetic membranes of different thicknesses, a significant shift in QLP) and thickness of the adsorbed lipid bilayer (LLP) is presented. A dramatic decrease in the minimum conductance related to the gate voltage  by both changing the electrical charge from negative to positive and decreasing the lipid thickness has been reported. In the presented model, the Vg, min variation based on the adopted experimental data as an electrical detection platform is considered and the sensor control parameters are defined. The presented model confirms the reported experimental data and in addition facilitates the employment of alpha and beta as biosensor control parameters to predict the behavior of graphene in graphene-based biosensors.The emerging potential of nanostructured graphene-based biosensors in the highly sensitive and effective detection of single-base polymorphism or mutation, which is thought to be the key to diagnosis of genetic diseases and the realization of personalized medicine, has been demonstrated. In a lipid bilayer-based biosensor, the graphene carrier concentration as a function of the lipid bilayer can be modeled. In this research, the total conductance of graphene as a function of the electric charge (The authors declare that they have no competing interests.MJK wrote the manuscript and contributed to the analytical modelling of the presented FET via MATLAB software. Dr. FKCh and Dr. MTA revised the manuscript and coordinated between all the contributors. HKFA, MR, and AH organized the final version of the manuscript. All authors read and approved the final manuscript.


Drosophila melanogaster to examine the roles that capping, splicing, cleavage/polyadenylation, and telescripting  might play in shaping exon-intron architecture in protein-coding genes. Our findings suggest that the distance between subsequent internal 5′ splice sites (5′ss) in Drosophila genes is constrained such that telescripting effects are maximized, in theory, and thus nascent transcripts are less vulnerable to premature termination. Exceptionally weak 5′ss and constraints on intron-exon size at the gene 5′ end also indicate that capping might enhance the recruitment of U1 and, in turn, promote telescripting at this location. Finally, a positive correlation between last exon length and last 5′ss strength suggests that optimal donor splice sites in the proximity of the pre-mRNA tail may inhibit the processing of downstream polyadenylation signals more than weak donor splice sites do. These findings corroborate and build upon previous experimental and computational studies on Drosophila genes. They support the possibility, hitherto scantly explored, that mRNA-associated processes impose significant constraints on the evolution of eukaryotic gene structure.mRNA-associated processes and gene structure in eukaryotes are typically treated as separate research subjects. Here, we bridge this separation and leverage the extensive multidisciplinary work on  Protein-coding genes in eukaryotes have a paradoxical structure. They contain sequences that need to be removed from precursor messenger (pre-m)RNAs to enable the synthesis of functional proteins. These intragenic noncoding sequences, known as spliceosomal introns , a nonprWhile several genetic and epigenetic factors influence intron recognition and splicing . The 5′sRNA splicing is not an isolated process in the cell. Rather, it interacts tightly with several co- and post-transcriptional processes, including mRNA capping, and 3′ end cleavage/polyadenylation, among others. It is well established that these molecular interactions are instrumental for the accurate processing of nascent transcripts . What isFirst, empirical evidence shows that at the pre-mRNA 5′ end the cap-binding complex (CBC) enhances the association between U1 and the cap-proximal 5′ss in human and yeast .Drosophila) that 5′ss-bound U1 protects pre-mRNAs from premature cleavage/polyadenylation at nearby polyadenylation signals (PAS) . Our working dataset contains 7256 protein-coding genes with at least two introns , for a total of 35,896 introns.We extracted information about gene structure using in-house python (python.org), bash (shell language), and R scripts (r-project.org). We randomly selected one isoform (rather than focusing only on the longest isoforms), and considered only protein-coding genes. Our dataset includes constitutively and alternatively spliced introns that contain no nested genes and whose size is equal to, or greater than, 32 nt . Finally, to focus on RNA splicing mediated by the major spliceosome, we discarded introns with noncanonical splice sites , we estimated subsequent medians of 2000 observations for the remaining variables, using a step size of 1. So, each point on the graph is supported by exactly 2000 observations. Sliding-window analyses were conducted using default functions  in R. All the reported correlation coefficients are based on raw data.We used a sliding-window approach to illustrate the trend of the examined correlations, similar to that described in e.g., the sense DNA strand, Ns) is equal to the number of their reverse complements on the opposite strand . The degree of DSA supplies a measure of the selective pressure exerted on a motif of a particular DNA strand  using published protocols  equivalent numbers of introns and exons. Any intron in a group had the same probability to be chosen, and to be associated with any following (second) exon. A nonparametric two-sample Kolmogorov-Smirnov test was used to measure the level of discrepancy between the actual and the random size distributions. Kolmogorov-Smirnov P-values were estimated for each of the 100,000 simulations. An overall unique P-value was estimated as N/100,000, i.e., the number of times that the test was nonsignificant  divided by the number of simulations.We tested the hypothesis that the size of the introns at specific positions , and that of their next exon, are nonrandomly associated. To this end, we first partitioned our dataset into three groups, https://github.com/GildasLepennetier/GildasSources.The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article. All of the scripts are publicly available through GitHub at At the pre-mRNA 5’ end, the CBC enhances the association of U1 with the cap-proximal 5′ss . Separate analyses of first, internal, and last introns, however, reveal that the positive relationship between 5′ss strength and intron size is relatively weak, though statistically significant, at the gene 5′ end . Moreover, the association between first intron size and the 5′ most 5′ss even becomes negative when introns are near the CBC   . AlthougP < 0.001) (P < 0.001). In contrast, the strength of 5′ss and the size of the upstream exon are not correlated in the gene body , and they are only weakly correlated at the gene 3′ end . These results suggest that the putative splicing-enhancing effects of the CBC in Drosophila might vanish with distance. Our observations hint at a maximum interval over which the putative splicing-enhancing effects of the CBC might extend . Beyond this distance, both the strength of first 5′ss and first exon length are tightly and positively coupled with first intron size.It has also been reported that the splicing-enhancing effects of the CBC may be distance dependent (< 0.001) . The rob< 0.001) —whereas Drosophila, as well as human and mouse, 5′ss-bound U1 counteracts premature termination by cleavage/polyadenylation at the nearby PAS, a process known as telescripting  (P < 0.001) (P < 0.001) (P < 0.001), and 2) weakly increases when second exons are larger than 200 nt . These observations demonstrate that intron-exon structure may differ dramatically at the gene 5′ end. They reveal constraints that fall within the action range of telescripting in Drosophila, i.e., the sizes of introns and their next exon covary such that the median length of the IpE unit is ≤ ∼500 nt  the size of first IpE units, and 2) the relationship between the size of introns to their next exon within these units. We examined first IpE units whose 5′ end donor splice site resides either within or outside the 5′-most 250 nt separately . This partition might help uncover putative interactions between the processes of capping and telescripting. We found that the size of cap-proximal IpE units is uniform (median: 553 nt) irrespective of the strength of the upstream 5′ss  . In cont< 0.001) . Further< 0.001) , the rel< 0.001) . More spUnder a model where selection pressure keeps two subsequent 5′ss at a distance that minimizes the risk of premature cleavage/polyadenylation, the CBC-enhanced recruitment of U1 should produce two effects: 1) it should compensate for the weak strength of cap-proximal splice sites, and 2) it should maximize the protective effects of telescripting at the gene 5′ end. Under this same model, large (>> 500 nt) IpE units are at major risk of premature cleavage/polyadenylation, unless additional protective features come into play (see below).e.g., when exon size is ≤ 400 nt: 14,428 observations; Kendall’s tau = –0.07, P < 0.001), whereas they are positively, albeit very weakly, correlated beyond this range   and 735 nt (3′-end), Kruskal-Wallis test, P < 0.001]. Finally, the size distribution of internal IpE units differs significantly from a set of distributions generated by randomly sampling introns and exons from our dataset (P < 0.001) . These observations suggest that there are constraints on the internal intron-exon structure of protein-coding genes in D. melanogaster that approximate the action range of telescripting in this species  . AdditioThe median size of internal IpE units increases alongside the strength of the 5′-end 5′ss, ranging from 294 nt (weak 5′ss), through 328 nt (weak to moderate) and 374 nt (moderate to strong), to 513 nt (strong 5′ss) . This trDrosophila introns is indeed negative (–0.06) , and ∼4-fold more negative compared to the DSA value of two AATAAA anagrams, TAAAAA, and AAAAAT (data not shown). The average DSAAATAAA is also > 3-fold more negative at the gene 3′ end than at the 5′ end . Finally, levels of counter-selection of intronic AATAAA decrease  when the strength of the upstream 5′ss increases (Table S2). These patterned variations suggest that the risk of premature polyadenylation and/or the efficiency of telescripting might differ between the gene 5′ end and 3′ end, and that the strength of the 5′ss may indeed bear on the action range of telescripting.We found that the average DSA of AATAAA within  (–0.06) . In intrAATAAA = 0.21)—where it may serve to regulate gene expression . As rationalized above, the excess of cryptic PAS at the gene 5′ end may reflect efficient U1-mediated silencing of polyadenylation in this region. Elegant work by We also examined the degree of DNA strand asymmetry of AATAAA in exons . AATAAA AATAAA in first or second exons and the size of the intervening intron . This observation provides some ground to speculate on why shielded PAS at the gene 5′ end might be favored (see Discussion).Finally, we detected a positive relationship between DSASplicing can be problematic for accurate 3′ end formation in that the suppressive effects of 5′ss-bound U1s might perturb the accurate processing of downstream cleavage and polyadenylation signals, particularly when the U1-bound 5′ss is very close to the polyadenylation site . Nowhere else along the gene do we detect a significant correlation between exon size and upstream 5′ss strength . This indicates that increasing exon length significantly contributes to the positive coupling between the size of last  IpE units and the strength of the 5′-end 5′ss , then, in order for accurate mRNA 3′ end processing to take place, progressively stronger last-intron 5′ss should be coupled with increasingly large next exons.Furthermore, we found that the strength of the last 5′ss is significantly and positively correlated with the size of the last exon (after correcting for last intron size) , end 5′ss . This obP < 0.001). This observation is surprising given the exceedingly small size of last introns in Drosophila , in line with previous findings (Table S2). This negative selection notwithstanding, about 4% (n = 1417) of the surveyed introns  contain at least one GGTAAG, and the motifs GGTAAG and AATAAA co-occur more frequently 1) in > 500-nt introns compared to smaller introns , and 2) downstream from, rather than within, the first 500 nt, in ∼1000-nt introns . Finally, we observed that the AATAAA motif resides preferentially toward the intron 3′ end, downstream (rather than upstream) from GGTAAG . Taken together, these findings lend some support to the hypothesis that U1 binding to cryptic 5′ss in Drosophila’s large introns may help suppress cleavage/polyadenylation at otherwise unshielded cryptic PAS.We found that the 5′ss-related GGTAAG motif is counter-selected in introns and exons , a distant relative of D. melanogaster (∼10 million yr of sequence divergence). These analyses corroborate virtually all of the relationships and trends that we document for D. melanogaster . Two discrepancies, which we detected, do not seem to have significant consequences for our overall conclusions. First, we found no significant negative association between the size of cap-proximal first introns and the 5′ most 5′ss for D. yakuba . Second, for D. yakuba the size of cap-proximal IpE units may vary depending on the strength of the upstream 5′ss (rather than being uniform as is for D. melanogaster). The magnitude of this variation in size is marginal however .To assess the validity of our study, we replicated most of our analyses on the annotated genome of Drosophila. The principal conclusions of this study are summarized in We have presented several findings which suggest that interacting mRNA-associated processes, including capping, splicing, cleavage/polyadenylation, and telescripting might play a role in molding the structural properties of protein-coding genes in Drosophila genes that, in theory, maximize telescripting effects, thus minimizing the risk of premature cleavage/polyadenylation in nascent transcripts. At the gene 3′ end, on the other hand, our observations suggest that telescripting effects are often circumvented, so that the process of 3′ end formation is facilitated.We have detected size constraints in the internal regions and at the 5′ end of AATAAA might also indicate that intron-bound cleavage/polyadenylation factors perturb splicing, e.g., intron-bound cleavage/polyadenylation factors might disturb the recruitment of U1 at the upstream canonical donor splice site. If so, then weakly counter-selected intronic polyA sites would preferentially occur downstream of strong (rather than weak) donor splice sites, which facilitate the recruitment of U1. This scenario is consistent with previously proposed models that invoke competing splice and cleavage and polyadenylation reactions  is more pronounced in IpE units with weak 5′-end 5′ss compared to IpE units with strong 5′-end 5′ss. This observation may indicate that strong 5′ss protect pre-mRNAs from premature termination more efficiently than weak 5′ss. In line with this scenario, introns with a weak 5′ss are more likely to undergo alternative cleavage/polyadenylation compared to introns with a strong 5′ss , and rather strong at the gene 3′ end . It follows that, whereas splice site strength and intron size are overall positively correlated within a certain intragenic location, across intragenic regions this relationship may disappear . We interpret this pattern of longitudinal decoupling between intron size and splice site strength as indicating that splicing in Drosophila is assisted at the pre-mRNA 5′ end but not at its 3′ end.Our analyses reveal significant variations in the average splice site strength along the genes of Drosophila genes is that splicing efficiency differs among introns, and that first introns are more prone to be inefficiently spliced, at least in part because of their relatively weak 5′ splice sites. This view provides an additional explanation for why first introns in Drosophila are retained more frequently than others . This selection for last exon expansion would have the beneficial effects of keeping the 3′ termination signals and the upstream 5′ss-bound U1 separated by a distance that is sufficiently large to circumvent telescripting effects and, thus, to facilitate both cotranscriptional splicing and mRNA 3′-end formation. Should telescripting effects not be adequately circumvented, splicing at the mRNA tail could still be completed post-transcriptionally, which is consistent with the observed excess of post-transcriptional splicing at the mRNA 3′-end compared to the upstream regions  complicates attempts to draw conclusions concerning underlying molecular dynamics, and might yield erroneous interpretations. In consequence, we cannot definitively rule out the possibility that some of the evidence presented in support of the proposed role of mRNA-associated processes in molding gene structure may be inaccurate. With this caveat in mind, we attempted to eliminate false signals whenever possible, using partial correlations or control sequence motifs. Not only do our conclusions withstand this scrutiny, they are also sustained by the coherence of the detected patterns. These patterns are in accord with U1-dependent definition  of the variables that we have investigated in this study , and potentially extend the repertoire of these traits with novel hypotheses that can be verified experimentally. In one example, the degree of DNA strand asymmetry of cryptic PAS motifs might serve as a simple but powerful tool to detect and measure in silico the efficiency of telescripting effects within the intragenic regions of eukaryotic species.The features and the relationships that we document support the idea that intracellular processes in conjunction with population-genetic forces may shape gene structure in Drosophila may extend to other eukaryotes is an open question that warrants further investigation. Many eukaryotic species, such as humans for example, have considerably longer introns compared to Drosophila and there is compelling experimental evidence that in human and mouse U1 telescripting effects extend for ∼1000 nt (rather than 500 nt as in D. melanogaster). Human and mouse are therefore excellent candidate systems to examine. These investigations would also furnish a test of the validity and the generalizability of the patterns observed for Drosophila.The extent to which the proposed effects of mRNA-associated processes in e.g., distinct levels and types of DNA methylation (We anticipate that much will be gained from examining the features that distinguish fruit flies from other species. Between-species differences in gene structure might flag different properties of the intracellular environment (hylation , and/or


We examine the unusual dispersion and attenuation of transverse electromagnetic waves in the few-THz regime on nanoscale graphene and copper transmission lines. Conventionally, such propagation has been considered to be highly dispersive, due to the RC time constant-driven voltage diffusion below 1 THz and plasmonic effects at higher optical frequencies. Our numerical modeling across the microwave, THz, and optical frequency ranges reveals that the conductor kinetic inductance creates an ultra-broadband linear-dispersion and constant-attenuation region in the THz regime. This so-called LC region is an ideal characteristic that is known to be absent in macro-scale transmission lines. The kinetic-LC frequency range is dictated by the structural dimensionality and the free-carrier scattering rate of the conductor material. Moreover, up to 40x wavelength reduction is observed in graphene transmission lines. Transmission lines, one of the simplest forms of single-mode electromagnetic waveguides, are widely used for signaling in digital, analog, and optoelectronic systems. Transmission lines generally consist of two or more parallel conductors that guide a transverse electromagnetic (TEM) wave over many wavelengths1368910Two recent advancements have made it intriguing to examine the dispersion of TEM waves in nanoscale transmission lines: state-of-the-art fabrication processes have allowed the dimension of transmission lines to fall below the skin depth; graphene has emerged as a practical conductor with large kinetic inductance1314151617212224252427236–fold variation in dimensions (nm through cm) and 104–fold variation in frequency (10 GHz–100 THz), we examine the conditions for the kinetic inductance to produce a surprisingly broadband LC region for small-scale transmission lines. Investigating both graphene and copper conductors, we develop a map of the performance regions for 2D and 3D materials, contrast the significant differences in their scaling laws, and explain dominant physical mechanisms that limit their performance.In this article, we carry out a first-principle numerical and analytical study on the effect of material kinetic inductance on the propagation and attenuation of the transverse electromagnetic modes in nanoscale and microscale transmission lines. We explore the potential of the kinetic inductance in providing a usable broadband LC response for signaling, transducing and sensing applications. Over a vast space, spanning 10t, and the graphene ribbons are treated as an infinitesimally-thin surface conductor. At THz frequencies and below, both materials are accurately modeled with a Drude conductivityAmong various transmission line structures, we focus on the coplanar stripline (CPS) shown in 30σ0 corresponds to the bulk DC conductivity for copper and surface conductivity for graphene. For the purposes of this paper, graphene has been modeled as an infinitesimally-thin conducting surface. Scattering due to surface roughness, finite temperature effects and spatial dispersion are neglected. The effective surface conductivity  and spatial phase evolution (β), respectively. The circuit parameters C, R, R, but at low enough frequencies where R, R and The underlying physical mechanisms leading to frequency-dependent attenuation in transmission line modes are captured by the circuit parameters in the solution to the telegrapher’s equations. Since we limit our discussion to lines with lateral dimensions that are much smaller than the free-space wavelength, this analytical model is accurate and yields comparable results to ab-initio solutions to Maxwell’s equations1In contrast to the Faraday inductance, the kinetic inductance stems from a material constant, the complex-valued conductivity. The kinetic inductive impedance is proportional to the imaginary part of the reciprocal of the conductivity, while the resistive impedance relates to the real part. Physically, they contribute to the power stored or dissipated, respectively, in the conduction currents:J is the surface or volume current density produced by an electric field, E. From this equation follows an important relation between the kinetic inductance and the resistance:A (or width w in the case of graphene), but unlike the resistance, it is related to the imaginary part of the inverse conductivity, This ratio reduces to a material constant and hence, may be frequency dependent but is completely independent of the geometry of the transmission line. Therefore, similarly to the resistance, the kinetic inductance is also inversely proportional to the current carrying area, R and increases with current crowding ; lines with a dominant kinetic inductance suppress current crowding because current favors a distribution of lowest total impedance.When the conductivity is represented by a Drude model, m and Nanoscale lines are where the kinetic inductance typically exceeds the Faraday inductance. Generally, the Faraday inductance of a transmission line is scale-invariant, for 3D conductors andfor 2D conductors. For the transmission line configurations chosen in this paper, w < wK and ωoperating > Γ.In lines with conductors that are smaller than the thresholds given in w = 20 nm to 500 nm. The conductor separation (as well as the copper conductor thickness) are equal to w/2, i.e. below the threshold predicted by w = 20 nm and w = 500 nm graphene CPS lines have an LC onset frequency that is exactly equal to the intrinsic scattering rate of graphene (1 THz). In the w = 500 nm copper CPS, although R and R and First-principle modeling with Maxwell’s equations, via the finite element method (see Methods section), confirms the above conditions through the inductive and resistive impedances  that havR in w = 50 nm, exhibits a clearly dominant kinetic inductance while the w = 100 nm CPS lies on the threshold that was predicted by The above line classifications are also reflected in the spectra of ne CPS’s , regardlThe high frequency boundary of the LC region in nanoscale transmission lines is determined by a different set of mechanisms than in micro and macroscale transmission lines. In copper and other metals, for frequencies below their intrinsic scattering rate, Γ the skin depth is collisional and scales as not defined by the skin effect. At high frequencies, as In nanoscale graphene lines, the upper frequency limit for the LC region is mon band , the selFor graphene  and coppw−2 dependence in the copper CPS and a more gradual w−1 dependence in the graphene CPS. At very low frequencies in the RC region, the current distribution is highly uniform and the mode energy is concentrated predominantly in the region between the conductors (The lower bound of the LC region (the dark red curve), nductors .w−2 geometric dependence of the LC region onset. In copper at frequencies above the intrinsic scattering rate, this boundary becomes a vertical asymptote as the skin depth converges to its constant collisionless valuew−0.5 dependence, while below the graphene scattering rate, it closely follows the RC-LC boundary. This strongly suggests that graphene ribbons can exhibit edge-current crowding whenever their widths exceed 100 μm.The upper bound of the LC region (the dark green curve) is defined by the onset of the skin or edge effect region where the current shifts strongly towards the surface of the conductor . For theThe third boundary for graphene (the dark blue curve) separates the skin/edge effect region from the plasmonic region. In the plasmonic region the transmission line becomes multi-moded since the geometry is capable of supporting individual and hybridized ribbon plasmons. For graphene, this boundary occurs when the wavelength of an infinitely-wide-graphene plasmon falls below twice the ribbon width, and runs parallel to the LC-edge-effect boundary. This implies that the observed edge effect is associated with the transition from quasi-TEM modes to ribbon plasmons where the field concentrates highly on the graphene and the current becomes non-uniform . In the In copper, the cutoff for guided plasmonic modes lies at much higher frequencies for a given waveguide size, or much larger waveguide sizes at a given frequency, due to copper’s larger DC conductivity. However, at microwave frequencies, the more practical boundary is the one that separates single-mode (TEM) operation form multi-mode (TEM+TE/TM) operation. If we contrast the overall performance of graphene transmission lines to their copper counterparts, we observe that graphene lines can provide a much lower onset frequency for the kinetic LC region. Furthermore, graphene’s LC region is supported in much larger lines, up to widths of about 100 μm. For larger structures, graphene’s reduced dimensionality also results in a more gradual shift towards a Faraday inductance-dominated LC response.High quality graphene with a sub-THz scattering rate would be extremely valuable for realizing nanoscale traveling-wave transducersEF = 0.4 eV, see β than those of the infinitely-wide graphene films, and therefore experience even higher cut-off frequencies. Thus, in the spectral range between 1 and 10 THz, the graphene TEM mode provides the optimal combination of wavelength-reduction, linear phase propagation, and constant attenuation.Finally, it is worth pointing out that in comparison to graphene plasmons, which are known for their exceptional wavelength reduction21In conclusion, our analysis of graphene and copper transmission lines has revealed that the well-known TEM modes enjoy a previously unnoticed broadband constant-attenuation regime, when the lateral dimensions are reduced to the nanoscale for both 2D and 3D conductors. This so-called kinetic-LC regime is induced by a predominant kinetic inductive impedance. The threshold dimension for this regime in a 3D line is defined by the collision-less skin depth of the conductor, about 100 nm for copper. Graphene transmission lines are remarkable in that their kinetic-LC region extends to relatively large micron-scale widths. The onset frequency of the kinetic-LC region is fixed at the free-carrier scattering rate, a material property, and therefore high-quality graphene with a low scattering rate would be desirable for THz/sub-THz applications. The upper frequency cutoff, on the other hand, depends on the material dimensionality: 2D graphene lines exhibit a width-dependent upper cutoff induced by plasmonic edge effects, whereas 3D lines have no upper cutoff in the spectral range in which the Drude model is valid. Although graphene lines exhibit constant attenuation at larger dimensions, copper lines of comparable widths provide lower attenuation. However, graphene’s large kinetic inductance drastically reduces the mode wavelength by 10–40 fold, approaching 1 micron at 10 THz. This deep subwavelength operation and the inherent tunability of graphene open up new opportunities for conventional signaling and sensing applications, as well as emerging applications involving metamaterials.z) being taken into account by replacing k as the eigenvalue. The circuit parameters .


Graphene films grown by vapour deposition tend to be polycrystalline due to the nucleation and growth of islands with different in-plane orientations. Here, using low-energy electron microscopy, we find that micron-sized graphene islands on Ir(111) rotate to a preferred orientation during thermal annealing. We observe three alignment mechanisms: the simultaneous growth of aligned domains and dissolution of rotated domains, that is, ‘ripening'; domain boundary motion within islands; and continuous lattice rotation of entire domains. By measuring the relative growth velocity of domains during ripening, we estimate that the driving force for alignment is on the order of 0.1 meV per C atom and increases with rotation angle. A simple model of the orientation-dependent energy associated with the moiré corrugation of the graphene sheet due to local variations in the graphene–substrate interaction reproduces the results. This work suggests new strategies for improving the van der Waals epitaxy of 2D materials. Understanding the factors that control island orientation in the growth of two-dimensional materials is likely to improve their quality. Here, using low-energy electron microscopy, the authors show that post-nucleation annealing of 2D materials can improve rotational order. Attempts to grow large, structurally pristine two-dimensional 2D) materials such as graphene and transition metal dichalcogenides are often frustrated by the fact that different rotational variants nucleate and grow, leading to a polycrystalline film1D materia34491112Here we use low-energy electron microscopy (LEEM) to observe in real time the evolution of graphene domains during thermal annealing. We show that large, micron-scale graphene domains on Ir(111) can reorient themselves to a preferred orientation by three mechanisms after nucleation. We estimate that domains aligned with the Ir(111) lattice are energetically favourable by as little as ∼0.1 meV per C atom. A simple model of the orientation-dependent energy associated with the moiré corrugation of the graphene sheet due to local variations in the graphene–substrate interaction reproduces the results. These results provide valuable guidance for the synthesis of 2D materials on current and future substrates: annealing immediately after nucleation will improve quality and this effect will be greater for substrates that induce larger corrugations in the 2D sheets.The first mechanism identified, shown in c, relative to the equilibrium adatom concentration for the graphene domain, ceq. Growth occurs when c>ceq and dissolution occurs when c<ceq. For an ideal adatom lattice gas, the chemical potential difference associated with growth or dissolution is Δμ≈kT ln c/ceq, where k is the Boltzmann constant and T is the absolute temperatureWe now turn to the cause of the simultaneous growth and dissolution. In general, growth or dissolution is dictated by the surface adatom concentration adjacent to the island, ceq compared with the R12 domain, which results in the growth of the R4 domain after the transformation, while the remaining R12 domain continues to dissolve.The relative chemical potential of different rotations can be immediately determined by combining the experimental results, as shown in The simultaneous growth and dissolution, that is, ‘ripening', is sensitive to temperature. As one sees in What is the magnitude of the driving force for a rotated domain RX to realign itself to R0? To estimate it, we use the temperature-controlled ripening experiments described above to determine the differences in equilibrium adatom concentrations between different rotations. From these adatom concentrations, we then extract the difference in chemical potentials of the islands. In previous work, the velocity of R0 domains was precisely determined as a function of the adatom concentration and temperature17ceq,R0 is the equilibrium adatom concentration for the R0 island, n is the cluster size for attachment to a kink and is equal to four, and B(T)=exp/T) nm s−1 is the kinetic coefficientA(t) plot of where −1 , where the error is from the s.d. of the dA/dt line fit. When the R8 domain was stationary from 2,800 to 3,200 s , the R0 velocity decreased to 144±36 nm h−1. Using When the R14 domain was stationary, the R0 velocity was 540±36 nm hB(T), the absolute scale of the chemical potential may be a factor of two larger or a quarter smaller.Five temperature-controlled experiments were conducted in the same manner. The estimated differences in chemical potential relative to R0 are shown in These measurements allow us to estimate the difference in chemical potential that cause the observed grain boundary motion in ab initio techniques such as density functional theory (DFT) to compute directly. To understand the origin of the orientation-dependent chemical potential differences and the extent to which they can be generalized, we make a model of the energy differences due to the corrugation of the graphene sheet, which is known to significantly change with orientation. In incommensurate, undistorted moirés, all equivalent positions of C atoms with respect to the substrate are occupied with equal probability, independent of the layer's rotation41920These small energy differences seem beyond the capabilities of The reason for this corrugation is that the preferred distance of the graphene sheet from the substrate varies locally, depending on the lateral position of the C atoms with respect to the substrate . To maximize its binding to the substrate, the graphene sheet flexes slightly to follow a contour of minimum C-Ir potential. Due to the bending rigidity of the graphene, the C atoms are further away from their optimal positions as the wavelength of the corrugation shortens. So the short-wavelength corrugations observed experimentally for rotated graphene would lead to an energy cost.z0 of C atoms from the Ir(111) substrate is represented by a 2D sinusoid, which has opposite extrema at the atop sites and hollow sites. We take the force on each C atom to be proportional to how far the atom is displaced from the preferred distance and assume that the spring constant k is uniform. In the relaxed configuration, this force is balanced by the forces caused by bending the graphene sheet. In the continuum limit, the energy cost Eb of periodic corrugations is2324To estimate the magnitude of this effect, we construct a simple model for the observed corrugations. For simplicity, we assume that the graphene is incommensurate with respect to the Ir substrate, that is, that the possible energy reduction due to locking into a (higher order) commensurate lattice is not sufficient to overcome the energy cost of straining the graphene from its preferred lattice constant. We assume that the preferred distance λ is the mean bending rigidity, ∼1.4 eVK is the mean curvature. Our model approximates the local curvature Ki at a given atom i as being proportional to the height difference Δzi between atom i and the average of its surrounding three nearest neighbours. This gives a force on each atom equal to d is the C–C distance in graphene , we fit these two parameters to our DFT calculations for R0 shown in k to exactly reproduce the amplitude of the corrugation of the relaxed sheet and the root mean square of the forces on the flat sheet. Given these parameters, we predicted the corrugation for domains rotated by 7.5, 15 and 30 degrees. Height profiles extracted from our moiré corrugation model along the close-packed zigzag direction are shown in This model has only two parameters, the amplitude of the sinusoidal preferred C atom distance and the spring constant The agreement between this prediction and DFT calculations is striking. It reproduces the position of all C atoms in the profiles in Our results have significant implications on the nature of rotational disorder of the graphene islands on various substrates. Scanning tunnelling microscopy shows that graphene on Au(111)272829In summary, we have identified the energetically preferred graphene orientation on Ir(111). Domains can evolve to this orientation during thermal annealing by three distinct mechanisms. The driving force for the alignment can be as small as 0.1 meV per C atom, much less than the total binding strength of graphene to metal surfaces. We propose that the origin of the preferential alignment is caused by the varying degree to which C atoms can attain the preferred distance from the substrate: the graphene-bending rigidity prevents the sheet from following the short-wavelength corrugations inherent in highly rotated domains. Since graphene corrugations of similar size are ubiquitous, the driving force for the coarsening on Ir reported here is expected to be similar in other systems. Despite this small driving force for rotational alignment, our results indicate that post-nucleation annealing of 2D materials can still improve rotational order. This strategy means that high quality 2D materials can be grown on more substrates than is now recognized. Our understanding of the driving force for reorientation can guide the search for new substrates.Graphene islands were nucleated by heating the Ir substrate between 800 and 900 °C and introducing ethylene gas into the LEEM chamber. After nucleation, the ethylene flow was turned off. The temperature was measured by a W-Rh thermocouple welded directly to the substrate.The domain orientation relative to the Ir(111) lattice was identified by selected-area LEED. The moiré LEED spots are sensitive to small changes in domain orientation and were used to determine the orientation to within ±0.5°. The distance between each pair of moiré superstructure LEED spots was measured and normalized by the distance between the corresponding pair of first-order graphene LEED spots. This ratio was computed for the three pairs of spots per LEED pattern and then averaged. Over 47 LEED patterns were analyzed and orientations were assigned based on this ratio. This ratio is easily determined using a simple vector model and there was excellent agreement between the experimental and expected ratios.A, and perimeter, p, with time: ɛave=(1/pave)dA/dt, where dA/dt is the slope of a line fit to the area versus time data for the time interval in question, and pave was the average perimeter over the time interval.The average R0 growth velocity was computed by measuring island area, All DFT calculations were performed with the optB86b-vdW functional with spin-polarization and using the projector-augmented-wave GW pseudopotentials supplied by VASP. A cutoff energy of 400 eV for the plane-wave basis and the Brillouin zone was sampled using 1 × 1 × 1 and 2 × 2 × 1 k-point grids. The optB86b-vdW exchange–correlation functional was employed to account for van der Waals interactions that are required for a qualitative description of the intermolecular interactions between the graphene sheet and the Ir(111) surface. The functional is of the form: a=b=c=3.843 Å; the lattice of the surface was fixed during all simulations. The Ir(111) slab consisted of three atomic layers, which helped minimize computational cost and allowed for the full graphene moiré to be modelled. Five different graphene orientations were considered: R0 , R7.5 , R15 , R22.5  and R30 . Geometry optimizations were performed until all forces were <0.05 eV Å−1; the total energy was converged to within 1 × 10−6 eV for both k-point samplings.The Ir(111) surface was constructed from the optimized face-centred cubic structure, The binding energies per C atom of the systems were determined by the following equation:EG/Ir111 is the total energy of the system  substrate), EG and EIr(111) are the total energies of the optimized isolated graphene layer and the Ir(111) surface, and N is the total number of C atoms. The graphene deformation energies (bending energies) were determined from the difference between the single-point energy of the optimized graphene sheet on the Ir(111) surface and the fully optimized graphene sheet within the same unit cell.where E/Δk). For the deformation energies, ΔE/Δk is much smaller than the magnitude of the values. On the other hand, the relative binding energies between the different orientations  are smaller than ΔE/Δk. On the basis of this analysis, it is concluded that the predicted ordering of the deformation energies is statistically significant, but the relative binding energies between orientations is not significant. As a result, the orientation with the greatest binding energy per C atom cannot reliably be determined from these results. Increasing k-space much further is computationally intractable; moreover, each system has different lattice dimensions that affect the relative k-point spacing. Computationally determining differences of one-tenth of a meV is difficult given the various numerical approximations used within DFT .Two k-point grids were used to compute graphene deformation energies and binding energies per C atom. The larger 2 × 2 × 1 k-point grid was used to test whether the predicted energies from the 1 × 1 × 1 k-point grid were converged with respect to k-space. The results indicate that the computed graphene deformation energies are converged; however, the binding energies are not. This conclusion was reached by comparing the difference in energies with respect to k-space .Supplementary Figure 1, Supplementary Table 1, Supplementary Notes 1-3 and Supplementary ReferencesObservation of three graphene domain reorientation mechanisms using LEEM