The intercalated and the derived exfoliated OMMT have been deeply characterized, mainly by X-ray diffraction analyses. Monolithic composite aerogels, with large amounts of both intercalated and exfoliated OMMT and including the nanoporous-crystalline δ form of syndiotactic polystyrene (s-PS), have been prepared, by scCO2 extractions of s-PS-based gels. Also for high OMMT content, the gel and aerogel preparation procedures occur without re-aggregation of the exfoliated clay, which is instead observed for other kinds of polymer processing. Aerogels with the exfoliated OMMT have more even dispersion of the clay layers, higher elastic modulus and larger surface area than aerogels with the intercalated OMMT. Extremely light materials with relevant transport properties could be prepared. Moreover, s-PS-based aerogels with exfoliated OMMT could be helpful for the handling of exfoliated clay minerals.Supercritical carbon dioxide (scCO Over the last decades, clay polymer nanocomposites (CPN)  constitute effective ways to increase dispersion and delamination in polymer/clay nanocomposites  can be achieved by scCO2 treatments on pure organoclays, where alkali counterions have been exchanged with long-chain alkylammoniums  -dimethylammonium (2HT) was purchased from Laviosa Chimica Mineraria S.p.A.13C nuclear magnetic resonance characterization showed that the content of syndiotactic triads , used for clay dispersions, was purchased from Aldrich and used without further purification.2 at 40°C and 200 bar, for 16 and 32 h under quiescent conditions, in order to promote the diffusion of the supercritical fluid into the clay interlayer space. The system was then rapidly depressurized (1 min) to atmospheric pressure and the expansion of the scCO2 between the layers caused the clay exfoliation.Exfoliated clay samples were obtained by using a SFX 200 supercritical carbon dioxide extractor (ISCO Inc.). Organically modified clays  were processed in scCODispersions of the clays in DCB were obtained with both as received and exfoliated samples. A clay dispersion was initially prepared by adding the appropriate clay amount in 5 ml of DCB. The mixtures were homogenized for 1 h under magnetic stirring and sonicated in a 5000 mL batch bath ultrasound (Badelin Sonorex RK 1028 H) for 1 h.sPS/clay gels were prepared, in hermetically sealed test tubes, by heating the clay dispersions above the boiling point of the solvent until complete dissolution of the polymer and the appearance of a transparent and homogeneous solution had occurred. The hot solution was then cooled to room temperature, where gelation occurred. For instance, 655 mg of sPS and 5 mL of 2 wt % clay dispersion were mixed to obtain clay/polymer gels. The overall amount of polymer and clay in the gels was generally fixed to 10 wt %.T = 40°C, P = 200 bar, extraction time t = 6 h. The prepared s-PS/clay aerogels present a weight composition ranging between 96/4 and 50/50. The aerogels, as prepared from gels with an overall polymer-clay content of 10 wt %, present a porosity close to 90%.Aerogels were obtained by treating sPS/clay gels with a SFX 200 supercritical carbon dioxide extractor (ISCO Inc.) using the following conditions: P of the aerogel samples can be expressed as:For monolithic aerogels with a regular cylindrical shape, the total porosity can be estimated from the mass/volume ratio of the aerogel. Then, the percentage of porosity pol is the density of the polymer matrix and ρapp is the aerogel apparent density calculated from the mass/volume ratio of the monolithic aerogels.where ρDhkl correlation length of crystals was determined applying the Scherrer equation:Wide-angle X-ray diffraction (WAXD) patterns with nickel filtered Cu-Kα radiation were obtained, with an automatic Bruker D8 Advance diffractometer, in reflection. The intensities of the WAXD patterns were not corrected for polarization and Lorentz factors, to allow an easier comparison with most literature data. The K is the Scherrer constant, λ is the wavelength of the irradiating beam , βhkl is the width at half height, and θhkl is the diffraction angle. The instrumental broadening, b, was determined by obtaining a WAXD pattern of a standard silicon powder 325 mesh (purity >99%), under the same experimental conditions. For each observed reflection with βhkl < 1°, the width at half height was evaluated by subtracting the unavoidable instrumental broadening of the closest silicon reflection from the experimental width at half height, βhkl, using the following relationship:where: The internal morphology of the aerogels was characterized by means of a scanning electron microscope . Samples were prepared by fracturing small pieces of the monoliths in order to gain access to the internal part of the specimen. In fact, the external lateral surfaces of most samples were found to be flat and free of porosity. Low energy was used (5 keV) to obtain the highest possible surface resolution. Before imaging, all specimens were coated with gold using a VCR high resolution indirect ion-beam sputtering system. The samples were coated depositing approximately 20 nm of gold. The coating procedure was necessary to prevent surface charging during measurement and to increase the image resolution.Nitrogen adsorption at liquid nitrogen temperature (77 K) was used to measure surface areas of OMMT and polymer/OMMT aerogels with a Nova Quantachrome 4200e instrument. Before the adsorption measurement, OMMT powders were degassed at 100°C under vacuum for 24 h, while polymer/clay aerogels were degassed at 40°C, in the same conditions. The surface area values were determined by using 11-point BET analysis.The differential scanning calorimetry (DSC) of OMMT powders and polymer/OMMT aerogels was carried out under nitrogen from 0 to 300°C at a heating rate of 10°C/min on a TA instruments DSC 2920.E′ was obtained, as a function of temperature, at a frequency of 1 Hz and an amplitude of 0.03 mm. The heating rate was 2°C/min in the range of 0, +100°C.Dynamic-mechanical properties were studied using a Triton dynamic-mechanical thermal analyzer. The spectra were recorded in the three-point bending mode, on samples with the following dimensions: length 15 mm, width 10 mm, and thickness 2 mm. The modulus l reflections (up to l = 12) that indicate a high degree of order perpendicular to the clay layers and an interlayer spacing of 3.5 nm, the pattern shows well defined weak peaks, corresponding to the typical 020, 210, and 060 in-plane MMT periodicities   pattern in the 2\upvartheta range 2–80° of the OMMT, with 40 wt % of 2HT, is reported in Figure 2 are shown in Figures l peaks as well as of the rotator order peak (100r) are strongly reduced , not yet reported in the literature, assures the maintenance of a long-range order in the clay layers. In this respect, it is worth adding that the half-height width of the 060 reflection, after exfoliation, remains equal to 0.45° indicating a correlation length D060 = 28 nm.In summary, the described long-term scCO2-treated OMMT, can be obtained by DSC scans . An additional reason for this choice is the easy obtainment of aerogels exhibiting s-PS nanoporous-crystalline δ  patterns of s-PS based aerogels, containing intercalated and exfoliated OMMT, are shown in Figures l reflections of the OMMT are not detected for the aerogels with low clay content (4 and 8 wt %) while for higher clay contents (20 and 50 wt %) a narrow and intense 001 reflection is present, while the 002 and 003 reflections of the starting clay have disappeared. Moreover, the 001 reflection is markedly shifted with respect to its original position (from 2θ = 2.6° up to 2θ = 3.8°), indicating a decrease of the interlayer spacing from d = 3.5 nm down to d = 2.3 nm.All patterns of Figure 2 extraction, for low OMMT content, could lead to clay exfoliation, as already observed for scCO2 treatment of the neat OMMT in Figure The results of Figures r ≈10 J/g ≈20 J/gOMMT).Additional information on the structural organization in the s-PS/OMMT aerogels comes from DSC analyses. In particular, DSC heating scan of a 50/50 by wt. s-PS/as-received-clay aerogel is shown in Figure d001) from 3.5 nm down to 2.3 nm, with only partial loss of the hydrocarbon rotator order in the interlayer space.The combined information of the X-ray diffraction patterns of Figures l clay reflections, while show the isolated weak 060 in-plane clay reflection . However, aerogels with the exfoliated clay Figure  are muchAlso the SEM images of the two aerogels are completely different. In fact, the SEM of the aerogel including the intercalated OMMT is dominated by the micrometric OMMT particles Figure  while thThe results of the SEM analyses suggest that also the large difference in the visual appearance between the two aerogels of Figure DMA analyses indicate that aerogels based on the exfoliated clay present an elastic modulus definitely higher than for those based on intercalated OMMT (36 MPa vs. 15 MPa).SBET, as obtained by N2 adsorption data at 77 K, for the intercalated and exfoliated OMMT, as well as those of the corresponding aerogels, are compared in Table SBET of the neat s-PS aerogel presenting the same porosity is shown in the last row of Table SBET of the OMMT is rather low and is substantially increased for the exfoliated OMMT (SBET = 18 m2/g). The s-PS/exfoliated-clay aerogels present values of SBET (281 m2g−1) much higher than those of the s-PS/intercalated-clay aerogels (166 m2g−1) and not far from those observed for pure s-PS aerogels (312 m2g−1). This indicates that, also for this high OMMT content (20 wt %), the exfoliated clay not only does not disturb the formation of the nanoporous crystalline phase but also does not alter the amorphous aerogel porosity.Surface areas A schematic representation of the sPS/exfoliated-OMMT aerogels is shown in Figure 2-induced exfoliation of OMMTs has been conducted mainly by X-ray diffraction and DSC characterization techniques. The starting material is a MMT intercalated with ammonium cations bearing two long hydrocarbon tails. Suitable scCO2 treatments led to exfoliation of the OMMT and also led to a complete loss of long-range order in the packing of the hydrocarbon tails of the cationic surfactant. The maintenance of hk0 reflections (mainly of the isolated 060 reflection), not yet reported in the literature, assures the maintenance of a long-range order in the clay layers. DSC scans of the intercalated OMMT present a reversible transition that corresponds to the loss of rotator order of the hydrocarbon chains in the interlayer spacing while those of the exfoliated OMMT do not present any thermal transition up to 100°C. This confirms the absence of any 3-D order for the exfoliated clay.A thorough investigation of scCOd001) from 3.5 nm down to 2.3 nm, with only partial loss of the hydrocarbon rotator order in the interlayer space. For aerogels with high content of the exfoliated OMMT, the gel and aerogel preparation procedures allow to maintain clay exfoliation without re-aggregation, as instead observed for other common polymer processing.Monolithic composite aerogels, filled with large amounts of both intercalated and exfoliated OMMT, have been prepared, starting from s-PS-based gels. In particular, for aerogels with high content of the intercalated OMMT, the preparation procedure brings to a reduction of the basal spacing (2g−1) much higher than those of the s-PS/intercalated-clay aerogels (166 m2g−1) and not far from those observed for pure s-PS aerogels (312 m2g−1). This indicates that, also for high content, the exfoliated clay does not alter the aerogel porosity.A strict comparison between s-PS monolithic aerogels with a porosity of 90% and a OMMT content of 20 wt %, as obtained by using intercalated or exfoliated OMMT, has been also reported. Although the two aerogels present similar degree of polymer crystallinity (≈40%) as well as the same polymer crystalline form , aerogels with the exfoliated OMMT are much more homogeneous than aerogels with the intercalated OMMT. This difference, clearly apparent both on visual inspection as well as on SEM analysis, is due to micrometric and nanometric size of intercalated and exfoliated clays, respectively. Aerogels based on the exfoliated clay also present elastic modulus definitely higher than those based on intercalated OMMT. Moreover, s-PS/exfoliated-clay aerogels present values of surface area , could be helpful to an easier handling of exfoliated OMMT, removing the risks connected with inhalable nanoparticles.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although some preliminary work has revealed the potential transcriptional regulatory function of the introns in eukaryotes, additional evidences are needed to support this conjecture. In this study, we perform systemic analyses of the sequence characteristics of human introns. The results show that the first introns are generally longer and C, G and their dinucleotide compositions are over-represented relative to other introns, which are consistent with the previous findings. In addition, some new phenomena concerned with transcriptional regulation are found: i) the first introns are enriched in CpG islands; and ii) the percentages of the first introns containing TATA, CAAT and GC boxes are relatively higher than other position introns. The similar features of introns are observed in tissue-specific genes. The results further support that the first introns of human genes are likely to be involved in transcriptional regulation, and give an insight into the transcriptional regulatory regions of genes. Protein coding genes in eukaryotes contain multiple introns that are cut off in RNA splicing process The characteristics of human introns have attracted considerable attention The CpG island is also known to be involved in transcriptional regulation of genes. Generally, unmethylated CpG islands are the binding sites of some important transcription factors (TFs), such as Sp1 Transcriptional regulatory elements in many genes consist of several essential motifs In this paper, we focus on investigating the intron features associated with transcriptional regulation in human genes. In order to systematically obtain the rational results, we analyze intron features not only in the entire sample genes, but also in introns of tissue-specific (TSP) genes, additionally. Firstly, we compare the intron length in various ways and find that the first introns are generally longer than other introns, consistent with the previous studies A C++ script was written to extract data from NCBI Genbank database (builder 36.2). Only genes with transcript and coding sequence (CDS) features were utilized. Intron lengths were inferred according to the mRNA location information. Since some genes have multiple transcripts , we only retained one transcript feature to avoid duplicate identification of introns for the identical genes. Thus a set of about 185,000 introns were obtained from 19770 intron-containing genes. In order to compare the intron features better, sampled introns were classified by two ways. The first classified pattern is ‘first introns’ which contain all the first introns and ‘non-first introns’ in which all other introns are classified into the same group. The second way is to classify introns based on their position specificity in genes .We retrieved the TSP genes from the HugeIndex database The characteristic length  between all sampled first introns and non-first introns were compared. For the genes containing two or more introns, we also compared the length of the first intron with the average intron length  of the same gene. Due to the large sample size and unknown distributions of the intron lengths, two-sample Kolmogorov-Smirnov (KS) tests were used to assess the significance of the length difference between the first introns and non-first introns.Nucleotide contents, especially high C+G and CG dinucleotide contents are usually thought to be related to transcriptional regulation of genes However, for large sample sizes, the exact computation may take too much time. Based on center limit theorem in statistics, the binomial distribution has an asymptotic standard normal distribution as Namely, Choosing significant level The similar method and significant level were used to examine the difference in occurrence frequency of dinucleotides between the first introns and non-first introns.We searched potential CpG island regions in introns using the method described by Gardiner-Garden and Frommer (1987) The frequencies of introns harboring potential TATA, CAAT and GC boxes were assessed, respectively. The corresponding consensus sequences for the three motifs were TATAWAW (W represents A or T), CCAAT and GGGCGG.The average intron lengths of the entire samples, HK genes and TSP genes are listed in About 2/3 of the first introns are longer than the expected length of the same gene. Except breast and colon-specific genes, the genes with the first introns longer than the expected length are more than the genes with the first introns shorter than the expected length . The rese longer . We can The contents of nucleotides and dinucleotides in introns are shown in  introns , the res overall . We founThe u -value analysis to investigate usages bias of nucleotides and dinucleotides in the first introns relative to position-specific non-first introns are located in the introns, we calculated the frequencies of introns that contain TATA, CAAT and GC boxes. The results show that, of all the first introns, 56.4% contain TATA boxes; 66.2% and 56.2% contain CAAT and GC boxes, respectively. However the percentages of the non-first introns that contain TATA, CAAT and GC boxes are 49.9%, 52.6% and 20.6%, respectively. This indicates that the three TFBSs occur more frequently in the first introns than in the non-first introns. In particular, the occurrence frequency of the GC box in the first introns is nearly 36% higher than that in the non-first introns, in accordance with previous finding that the first introns contain a larger degree of GC dinucleotide and CpG island than the non-first introns. The frequencies of TATA and CAAT boxes in the first introns are only 6.5% and 13.6% higher than those in the non-first introns. Nevertheless, since nucleotides A and T and their dinucleotide compositions and the CA dinucleotide are under-represented in the first introns, it is reasonable to think that the contents of TATA and CAAT boxes in the first introns are richer than those in the non-first introns.Similar results are also found in introns of the TSP genes . In all Our study also reveals that there are differences in frequencies of TATA, CAAT and GC boxes with respect to the intron position . The notIt is noted that, we determined the first intron of a gene only based on one transcript of the gene. However, most genes have alternative transcription start sites which maybe affect what is annotated as the ‘first’ intron and the characteristics of the first introns. To counter these problems, we random chose two groups of alternative transcripts from sampled genes and performed the similar analysis for the features of introns. Excitingly, the scores of intron features have no significant difference between two collections of transcripts (data not shown). For instance, the Using the introns derived from nearly 20000 intron-containing genes, we analyze the sequence features associated with transcriptional regulation. The various characteristics of the first introns within these genes suggest that the first introns are most likely to be involved in transcriptional regulation. Consistent with previous observations Another feature is that C, G and their dinucleotide compositions are over-represented in the first introns, whereas A, T and their dinucleotides are under-represented. In addition, nearly 70% of the first introns contain the CpG island, not only higher than the other introns but also higher than the estimated percentage in entire human genes (40∼50%) Obvious differences in the distributions of TATA, CAAT and GC boxes between the first introns and the non-first introns are also observed. Generally, the frequencies of these three TFBSs are higher in the first introns than in the non-first introns, suggesting the potential for the first introns to bind TFs and participate in gene expression regulation.One additional finding of this study is the differences in sequence features between introns within HK genes and TSP genes. The HK genes have shorter introns than the TSP genes. In addition, the frequencies of CpG islands and GC boxes are higher in the HK genes than those in the TSP genes, whereas the HK genes contain fewer TATA and CAAT boxes than the TSP genes. The results hint that there are the different mechanisms of transcriptional regulation between the HK and TSP genes. For example, one possibility is that there are more CG-rich regulatory elements in the HK genes but more AT-rich elements in the TSP genes since the differences of contents of CpG island and TATA box between introns of HK and TSP genes. Besides, there are also differences among the introns within different kinds of TSP genes, indicating the laws of transcriptional regulation between these groups of TSP genes are not the same. Since the differences between HK genes and TSP genes, our next plan is to detect the transcriptional regulatory elements located in the first introns of HK genes and each group of TSP genes and explore the transcriptional regulation mechanism concerned with introns in these genes.Our results support the hypothesis that the first introns may contain transcriptional regulatory elements and provide intriguing hints into the regulatory potential of the first introns. Therefore, the promoter regions might be extended to at least the first introns while studying the transcriptional regulations of genes.Table S1List of houskeeping and tissue-specific genes. (XLS)(XLS)Click here for additional data file.Figure S1Characteristic lengths of introns in housekeeping and tissue-specific genes. (TIF)(TIF)Click here for additional data file.Figure S2Frequencies of introns overlapped with at least one CpG island in housekeeping and tissue-specific genes. (TIF)(TIF)Click here for additional data file.Figure S3TATA, CAAT and GC boxes frequencies of introns in housekeeping and tissue-specific genes. (A) Frequency of introns with TATA boxes. (B) Frequency of introns with CAAT boxes. (C) Frequency of introns with GC boxes. (TIF)(TIF)Click here for additional data file.


The 2D peak variation originates in that the three effective sub-modes correlate with the light polarization through different relations. We develop a method to quantitatively analyze the positions, intensities, and polarization dependences of the three sub-peaks. The analysis reflects the local strain, which changes with detected area of the graphene film. The measurement can be extended to detect the strain distribution of the film and, thus, is a promising technology on graphene characterization.We report the strain effect of suspended graphene prepared by micromechanical method. Under a fixed measurement orientation of scattered light, the position of the 2D peaks changes with incident polarization directions. This phenomenon is explained by a proposed mode in which the peak is effectively contributed by an unstrained and two uniaxial-strained sub-areas. The two axes are tensile strain. Compared to the unstrained sub-mode frequency of 2,672 cm Recenologists-19. The ologists-22. In aologists-25. Receologists-30.2 are defined by photolithography on an oxidized silicon wafer with oxide thickness of 300 nm. Reactive ion etching is then used to etch the squares to a depth of 150 nm. Micromechanical cleavage of HOPG with scotch tape is then used to deposit the suspended graphene flakes over the indents, as shown in the schematic of FigureSuspended graphene are fabricated by mechanical exfoliation of graphene flakes onto the oxidized silicon wafer. First, ordered squares with areas of 6 μmΦ) are shown in FigureΦ is defined as the included angle between the incident polarization direction and the analyzer. The clear anti-symmetric spectra with different polarization angle can be observed in the spectra. Fitting of the 2D peaks is done using a double-Lorentzian function for Φ of (a) 0° and (b) 90°.Polarized Raman spectra of 2D modes under incident lights with different polarization angle (−) and 2,660 (2D+) cm−1, while that for Figure−) and 2,662 (2D+) cm−1. To systematically analyze the sub-peak of the 2D modes, the spectra of 2D modes with different polarization angle can be fitted by double-Lorentz function, and the 2,647 and 2,660 cm−1 by average of all the peak positions of 2D+ and 2D− modes with different polarization angles. The 2D0 showed the peak position at 2,672 cm−1 as an original 2D peak in the unstrained graphene. The peak positions of 2D+ and 2D− modes compared with an original 2D peak both red shifts. The results showed that the 2D+ and 2D− modes are both tensile strains on suspended graphene. To understand the strain effect of the suspended graphene, fitting the 2D peak of graphene by a triple-Lorentzian function whose three peaks correspond to the sub-modes of 2D+, 2D−, and 2D0 with Φ = 40°, which is just an example of all the spectra, is shown in FigureThe 2D peak originates from four-step Stokes-Stokes double-resonance Raman scattering. AccordiI(2D+), I(2D−), and I(2D0) as functions of Φ is shown in Figure+ and 2D− sub-bands which have the peak positions of 2,647 and 2,660 cm−1, respectively, showed a prominent sinusoidal intensity modulation with a period of 180°. Both the modulation of the 2D+ and 2D− bands can be fitted by a function of cos2(θA − θP), where θA and θP are the polarization angles of the analyzer and polarizer, respectively. Both the intensities of 2D+ and 2D− modes are the maximum when Φ = 0° and minimum when Φ = 90°. This result of suspended graphene is very different from those of the supported graphene by previous research[+ and 2D− modes can be obtained.To systematically analyze the intensities of the fitting sub-peaks, the plots of research. Compare+ and 2D− can be calculated by employing the Grüneisen parameter[0 is the 2D peak position at zero strain and Δω is the shift caused by the strain of ε. For the uniaxial strain,+ and 2D− are estimated as 0.44% and 0.93%, respectively. Based on these results, the distribution of strain on the suspended graphene can be obtained through our analysis.The strains of the 2Darameter (γ) for graphene. In addigraphene. Hence t+ and 2D− sub-bands having the peak positions of 2,651 and 2,661 cm−1, respectively, showed a prominent sinusoidal intensity modulation with a period of 180°. Both the modulation of the 2D+ and 2D− bands can be fitted by a function of cos2(θA − θP), where θA and θP are the polarization angles of the analyzer and polarizer, respectively. Both the intensities of 2D+ and 2D− modes are the maximum when Φ = 1° and minimum when Φ = 91°. Using the same calculation, the stains of+ and 2D− are estimated as 0.44% and 0.93%, respectively. The result in FigureAnother interesting phenomenon measured from our sample can be observed in FigureWe have explored the suspended graphene by polarized Raman spectroscopy. In the exploration, the polarization direction of the incident and scattered light are variable and fixed, respectively. The position and intensity of the graphene's 2D peak is modified by the incident polarization, and the modification is explained by a proposed biaxial-strained model. In this model, the 2D peak is contributed by three effective areas related to unstrained and two tensile-strained graphene, respectively. The two strains are uniaxial and in the same directions. The strength of the strains is quantified through our analysis. This analytical method can be used to probe strain and help us understand the situation of suspended graphene. Hence, this method provides great application potential on graphene-based electrical and optical devices, whose performance usually relies on strain.The authors declare that they have no competing interests.CWH and BJL carried the experimental parts: the acquisition, analysis, and interpretation of data. CWH also had been involved in drafting the manuscript. HYL and CHH performed the analysis and interpretation of data. They also had been involved in revising the manuscript. FYS and WHW  prepared the samples, suspended the graphene using micromechanical method, and captured the OM and AFM images. CYL has made substantial contributions to the conception and design of the study, and the critical revision of the manuscript for important intellectual content. HCC, the corresponding author, had made substantial contributions to the conception and design of the study, and had been involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript.CWH received his BS degree in Electronic Engineering from the National University of Kaohsiung, Kaohsiung, Taiwan, in 2008. He studied his MS degree in 2008 and PhD degree immediately in 2009. Currently, he is a PhD candidate in the Department of Photonics, National Cheng Kung University, Tainan, Taiwan. He focuses on the property of graphene and surface plasmon resonance of nanoparticles.BJL received his BS degree in Physics from the National Chung-Hsing University, Taichung, Taiwan, in 2010. He received his MS degree in Photonics from the National Cheng Kung University, Tainan, Taiwan, in 2012. His research interests mainly include Raman measurement of graphene. He is in compulsory military service now.


This study aims to quantify the spontaneous proliferation of PBMCs using flow cytometry. PBMCs were cultured for 24 to 120 hours in the presence of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). For comparison, PBMCs were also cultured with [3H]thymidine. The cutoff values for spontaneous proliferation were >0.06 for the division index and >5.8% for the percentage of divided cells. Sixty-two percent of HTLV-1-infected individuals presented spontaneous proliferation of PBMCs, which was detected in the first 24 hours. Moreover, proliferation was detected in CD4+ and CD8+ T-lymphocyte subsets. A positive correlation was found between the division index and [3H]thymidine incorporation. This method may prove useful to better understand the phenomenon of spontaneous proliferation of PBMC of patients infected with HTLV-1.The spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) is a hallmark of the human T-lymphotropic virus (HTLV) type-1. Cell proliferation is usually measured using a [ A proliferation assay based on cell staining with 5,6-carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry analysis has been described as an alternative approach thymidine . After this period, the content of the plate was harvested to determine the [3H]thymidine incorporation using a Beta Radiation Counter . Results were expressed as mean counts per minute.Additionally, cells from 11 HTLV-1-infected individuals were cultured for 5 days and pulsed overnight with 1 P value of less than 0.05 denoted a statistically significant difference. Graphad Prism 5.0 software was used for all statistical analyses.Data were expressed as percentages, means, standard deviations, medians, and 25% and 75% percentiles. Groups were compared using the Mann-Whitney test and Kruskal-Wallis test. Correlations were evaluated by Spearman correlation. A The cutoff values to define spontaneous proliferation were >0.06 for the cell division index and >5.8% for the percentage of divided cells, which represents three times the division index mean and three times the percentage of divided cells in PBMCs from uninfected controls. Under these criteria, 62% of HTLV-1-infected individuals presented spontaneous proliferation in PBMCs as measured by the cell division index, while 47% presented proliferation using the percentage of divided cells .P = 0.9) (Cell proliferation was detected in the first 24 h and remained constant over 120 h (P = 0.9) .+ and CD8+ T-lymphocyte subsets were similar to those defined in the PBMC quantification: >0.06 for the cell division index of cells and >5.2% for the percentage of divided cells. Under these criteria, 50% of HTLV-1-infected individuals presented spontaneous proliferation of CD4+ T lymphocytes, while 30% presented spontaneous proliferation of CD8+ T lymphocytes , as well as between the percentage of divided cells and [3H]thymidine incorporation  (A positive correlation was found between the division index of cells and [= 0.004) .3H]thymidine incorporation assay. The cell division index (average number of cell divisions) showed a better correlation than percentage of divided cells, since it may determine more accurately the number of cell divisions occurring during culture [This study determined the parameters to quantify the cell proliferation of PBMC from patients infected with HTLV-1 by flow cytometry. The results obtained indicated that there was a strong correlation between the intensity of spontaneous proliferation measured by the cell surface stain CFSE and the [ culture .3H]thymidine incorporation assay, which requires three to four days of culture. The intensity of proliferation remained stable over a culture period of 120 hours. Sixty-two percent of the individuals infected with HTLV-1 had spontaneous proliferation of PBMCs. This proportion was similar to that found by other authors using the [3H]thymidine incorporation assay, which described that 50% to 67% of individuals infected with HTLV-1 has spontaneous proliferation of PBMC thymidine incorporation assay, classically used to evaluate proliferation in the context of the HTLV-1 infection, the assay using cell staining with CFSE provided a greater number of parameters for statistical analysis is less laborious and expensive process and safer, since it does not require the use of radioactive substances [Compared with [bstances –24. ThisThe spontaneous proliferation of PBMC is assessable using flow cytometry. This method may prove useful to better understand the phenomenon of spontaneous proliferation of PBMC of patients infected with HTLV-1.The authors declare that there is no conflict of interests.


Transcriptional interference has been recently recognized as an unexpectedly complex and mostly negative regulation of genes. Despite a relatively few studies that emerged in recent years, it has been demonstrated that a readthrough transcription derived from one gene can influence the transcription of another overlapping or nested gene. However, the molecular effects resulting from this interaction are largely unknown.in silico chromosome walking, we searched for prematurely terminated transcripts bearing signatures of intron retention or exonization of intronic sequence at their 3′ ends upstream to human L1 retrotransposons, protein-coding and noncoding nested genes. We demonstrate that transcriptional interference induced by intronic L1s (or other repeated DNAs) and nested genes could be characterized by intron retention, forced exonization and cryptic polyadenylation. These molecular effects were revealed from the analysis of endogenous transcripts derived from different cell lines and tissues and confirmed by the expression of three minigenes in cell culture. While intron retention and exonization were comparably observed in introns upstream to L1s, forced exonization was preferentially detected in nested genes. Transcriptional interference induced by L1 or nested genes was dependent on the presence or absence of cryptic splice sites, affected the inclusion or exclusion of the upstream exon and the use of cryptic polyadenylation signals.Using Our results suggest that transcriptional interference induced by intronic L1s and nested genes could influence the transcription of the large number of genes in normal as well as in tumor tissues. Therefore, this type of interference could have a major impact on the regulation of the host gene expression. Genes in mammalian chromosomes are distributed between gene-poor and gene-rich territories. While individual genes in gene-poor regions can be regulated independently without interference from others, genes in gene-rich regions can be regulated via complex network of positive and negative interactions. Gene-gene interactions, occurring at transcriptional level, have been recently recognized as a widespread and unexpectedly complex process, termed transcriptional interference (TI) One interesting example, a gene within a gene, or so called nested gene, has been in the focus in several recent studies  insertion into an intron of the host gene. Human genome contains 80–100 retrotranspositionally competent L1s Transcriptional activity of human L1 is determined by convergently arranged sense (SP) and antisense (ASP) promoters, located in its 5′ untranslated region (5′ UTR) in positions +1 to +101 and +400 to +600, respectively Here we studied the effect of L1 SP- and ASP-induced TI on human genes. We also extended our studies to protein-coding and noncoding (nc) nested genes, i.e. a category of genes, for which only a limited information about transcriptional regulation is available. Our results show that intron retention, forced exonization of intronic sequence and cryptic polyadenylation at the 3′ ends of prematurely terminated transcripts are the major effects induced by intronic L1s or nested genes. These effects were deduced from aberrant endogenous transcripts expressed in different human cell lines and tissues and confirmed in minigene transfection experiments.in silico chromosome walking with UCSC Genome Browser  A and B. The term intron retention, used here, refers to the unspliced intron, which is retained in the 3′ end of spliced exons most likely due to the premature termination of Pol II possibly induced by L1. Similarly, a different molecular effect, forced exonization of the intronic region upstream to L1, describes TI effect, which may be caused by either transcriptional activity of L1 or TFs bound to L1. Exonization depends on the presence of upstream cryptic acceptor splice site.Using Table S1). Of the L1s analyzed, intron retention was observed in 22 genes (45 %) and forced exonization in 19 genes (39 %). Both, intron retention and forced exonization were observed in 8 genes (16 %). Cryptic polyadenylation was detected in 13 genes (27 %). Of these genes, 5 showed intron retention, 3 exonization and 5 had both effects.Our genome-wide search revealed 50 intronic L1s in 49 genes which had a tandem arrangement with respect to the host gene transcription and possibly caused intron retention, forced exonization and cryptic polyadenylation in the upstream region (Table S2). None of this type of ESTs were found in 17 genes (35 %). However, for the remaining 31 genes (63 %) we detected additional potential TI effects in a single intron of 15 genes and in two or more introns in 16 genes. For these 31 genes intron retention was observed in 10 genes (32.5 %), exonization in 11 genes (35 %) and both effects in 10 genes (32.5 %). The occurrence of aberrant ESTs in these cases may be explained by the presence of either repeated DNA  or a putative nested gene, which could influence the transcription across the intron . Indeed, their direct influence, i. e. intron retention, exonization, termination or polyadenylation within the repeated DNA or nested gene was detected in 18 genes (58 %). Of 49 genes, only in one case (KIAA0586) we were unable to explain the intron retention in mRNA AK302718 (marked with ? in Table S2). From these data we conclude that in addition to L1, other repeated DNAs as well as nested genes, located in introns of host genes, could possibly cause TI effects in these genes.In order to determine if the L1s analyzed are the only potential inducers of TI, we searched for additional aberrant expressed sequence tags (ESTs) in our dataset of 49 genes  and selected putative single exon nested genes, for which at least one TI-predicting EST was found. Representative examples of one protein-coding and nc nested genes are shown in  C and D. Our search revealed 104 nested genes, of which 94 (90 %) were potential nc genes and 10 (10 %) were protein-coding (Table S3). Of these protein-coding genes, 4 were detected earlier (Table S1) also revealed that in the case of nested genes exonization was more prevalent (about 2-fold) than in the case of intronic L1s (see above).Despite the fact that biological function of the vast majority of nc RNA transcripts, an abundant class of RNAs (over 55 00)  earlier Table 1.Table S4). Of the 74 host genes analyzed, 26 (35 %) showed no additional aberrant ESTs demonstrating that in these cases the nested gene was the only potential TI inducer. The next large group of 43 host genes (58 %) showed variable number of aberrant ESTs in different introns; intron retention in 15 genes, exonization in 14 and both effects in 16 host genes. Their presence in these introns may be explained by the TI effect induced by repeated DNA  similar to that described above for L1-containing genes. Consistent with this explanation, their direct influence, i. e. intron retention, exonization, premature termination or polyadenylation within the repeated DNA was detected in 17 cases (40 %). Finally, we also detected 5 host genes , which expression gave rise to aberrant ESTs (exonization in 1 case and intron retention in 4 cases) in other introns (marked with ? in Table S4). Their presence in these few cases remained unknown. Taken together, these data show that in addition to intronic nested genes, pseudogenes or repeated DNAs can potentially induce the major TI effects compared to minor effects (43 vs 5), caused by other unknown factors .To test whether the potential TI effect in host gene expression is specific only to the intron, wherein the nested gene is located, we searched for aberrant ESTs over the entire length of each host gene (Table S3). For these genes, we tested the presence of normal transcripts for host and nested and aberrant transcripts for host genes in various human tissues. Widespread transcription of host and nested genes was detected in all tissues analyzed, which in most cases matched the potential TI effect induced by nested gene  were randomly chosen  is located in chromosome 17q24.2, consists of 39 exons and encodes a transporter protein which transports different molecules through extra- and intracellular membranes ABCA9, a full-length L1 PA3 is located in intron 25 and it was previously shown that this L1 has a functional ASP ABCA9 exon 23  is located in chromosome 11q23.1, consists of 19 exons and encodes a protein involved in neural cell adhesion NCAM1 (Table S1) and may interfere with its transcription by causing intron retention and cryptic polyadenylation about 2 kb upstream to L1 (TXNDC12 (thioredoxin domain containing 12) is located in chromosome 1p32.3, consists of 7 exons and encodes a thioredoxin domain-containing protein involved in redox regulation and defense against oxidative stress TXNDC12 contains a tandemly oriented single exon gene (KTI12), which encodes a chromatin associated protein of unknown function  , cf. lanes 1 and 5–7). Consistent with this result, a small (ΔASP292) or a large (ΔASP613) deletion encompassing L1 ASP region , lanes 1 and 3). This transcript was absent in negative control  and in ΔSV40 experiments (lanes 5–6), suggesting that SP activity was dependent from the SV40 promoter activity. Finally, SV40 spliced transcripts of the expected size (319 bp) with retained intron 23 were detected for all constructs, except for ΔSV40 . Since this method is based on solution hybridization and allows quantitative and simultaneous detection of different (un)spliced transcripts, contributions of each promoter or transcription unit can be determined.  cf. lanes 3 and 6–8, and 13 and 16–18). We also tested potential TI effect of L1 PA3 derived from the ABCA9 intron 25 and compared it to the #11AS in ABCA Fl and retrotranspositionally active L1 RP  cf lanes 3, 9 and 10 or 13, 19 and 20). Full-length L1 PA3 and RP reduced significantly SV40 transcription, suggesting that these elements are rather difficult to transcribe ( lanes 2–4 and 9–11). Full-length L1 RP and ORFs (to a lesser extent) in sense orientation had much greater effect in TI than antisense orientation (lanes 2–3 and 5–6), consistent with previous studies Previous studies have suggested that SOX family TFs can modulate L1 SP activity  and et al. et al. The next series of results were obtained from probing transcripts, derived from the expression of transfected ABCA minigenes, with ASP and SP probes  and 5A. ThiBecause some of the SV40 transcripts contained cryptic exon  increased exon 9 skipping about 5-fold , indicating that these regions are required for efficient SV40 transcription.Using the third minigene, we investigated how a single exon nested gene KTI12 were determined. These transcripts and less abundant endogenous transcripts of the expected size (505 nt), encompassing KTI12 positions 595–1099, were detected for all four constructs . However, deletion in a putative promoter region of KTI12 showed transcription level comparable to that of TE, suggesting endogenous transcription (cf. lanes 8 and 10). In addition, KTI12 transcription was detected for ΔSV40, indicating that KTI12 activity was independent from SV40 promoter activity (data not shown). Consistent with our bioinformatic study, a 554 nt-long SV40 transcript containing exons 1, 2 and a cryptic exon (Ex) generated by using an acceptor splice site 251 nt upstream to the most 5′ transcription start site of KTI12 was detected (lane 14). Two additional transcripts, a 389 nt transcript contained alternative acceptor splice site (86 nt upstream to KTI12) and a 909 nt transcript, derived from the readthrough of the intron between exon 2 and KTI12 were also detected. Therefore, these results suggest that KTI12 decreases the efficiency of SV40 transcription by forcing exonization using alternative cryptic acceptor splice sites located upstream to KTI12.In the following series of RT-PCR experiments transcripts derived from KTI12  increased SV40 transcription about 5-fold, suggesting that KTI12 interferes strongly with the elongating Pol II (1 (cf. lanes 3 and 4) suggesting that KTI12 interferes with intron retention and forces exonization in its upstream region. Deletion of KTI12  eliminated these effects, thus further supporting this conclusion. Consistent with RT-PCR results described above  ranging from low level or endogenous transcription for ΔP to high level for Δ395. Deletions made in the initiation region (Δ115 and Δ395) did not abolish the KTI12 transcription (lanes 14 and 15) suggesting the redundancy in transcriptional initiation. Heterogeneous transcription initiation from about 300 nt interval was also supported by ESTs (data not shown).To further investigate the TI effects observed in RT-PCR experiments, we next used RPA. The following results were obtained from probing transcripts expressed from transfected minigenes using Fl and KTI riboprobes. Deletion of KTI12 promoter region (ΔP) would increase SV40 transcription. However, no such increase was observed. Instead, about 2-fold decrease in transcription was detected, while the amount of intron-retained and exonized transcripts remained unchanged . The ratio of Fl transcripts to intron-containing transcripts was comparable in Fl, Δ115 and Δ395 , but it was significantly increased in ΔKTI2, (cf. lanes 9 and 12) suggesting that KTI12 contributed to the intron retention. The high level of intron retention in ΔKTI1 (lane 10) most likely reflected transcriptional readthrough.To further analyze the essence of non-Fl transcripts shown in KTI12 promoter and initiation region to TI was difficult to interpret, our data support the conclusion that KTI12 interferes strongly with SV40 transcription by forcing exonization and causing intron retention in its upstream region. All these data suggest that, similarly to L1, a tandemly located nested gene interferes with the transcription and splicing of the host gene.In summary, despite the fact that in some deletion experiments the contribution of In this paper, we show that TI induced by L1s and nested genes may be characterized by three different effects: intron retention, forced exonization and cryptic polyadenylation Figure 8et al. In ABCA minigene, the presence of cryptic acceptor splice site upstream to L1 5′ UTR determined the efficiency of L1-induced TI, because its deletion increased intron retention about 5-fold. Therefore, the occurrence of cryptic acceptor splice sites is somehow beneficial for SV40 Pol II to guarantee processive transcription coupled with splicing across exons 23, ExSP and 3 . However, this number is clearly an underestimate and does not consider inter-individual variation and expression differences in different cell types or tissues  and nc genes was exonization (Table S3). And 16 (16 %) nested genes showed TI ESTs in both types of tissues. Similar preferential expression in normal tissues was observed for L1 (Table S1). We speculate that about 3-fold preferential expression in normal tissues indicates that TI between nested genes may be part of their normal regulation, although the possibility of misregulation in some cases cannot be excluded. Our results also suggest that TI between nested genes may be widespread and frequently restricted to different human tissues, although we cannot exclude that for some genes tissue-specific patterns (depending on the TF binding or promoter activity) may be observed.Differently from the intronic L1s, which showed comparable intron retention and exonization effects, the main effect determined for nested protein-coding . Interestingly, Zhang et al.In this study, several lines of evidence suggest that L1 or nested gene is able to cause TI in normal gene transcription. Not only ESTs, showing unequal distribution in introns of host genes, but also the orientation bias (tandem orientation being twice less frequent than convergent orientation) Although, in our experiments, we were unable to distinguish between the DNA binding of TFs and the act of transcription as a cause of TI effect in nested genes, it seems likely that DNA-bound TFs may play a role in reducing the speed of transcription elongation. In support of this kinetic model L1 retrotransposons have been shown to influence the expression of host genes by variety of effects, including insertional mutagenesis, mobilization and transcriptional regulation of cellular genes We have characterized TI between host genes and intronic L1 retrotransposons as well as between the nested genes. Based on our bioinformatic study, we first predicted the existence of TI between tandemly arranged host genes and intronic L1 retrotransposons and nested genes, and thereafter proved it experimentally using the expression of three different minigenes in cell culture. Analysis of endogenous transcripts derived from different human cell lines and tissues also supported TI between protein-coding and nc nested genes. Our results suggest that TI induced by intronic L1s and nested genes may be widespread and could influence the expression of many genes. Further analysis of TI effects between nested genes should reveal their role in the expression of a large number of host genes in normal as well as in tumor tissues. Also, it would be interesting to determine whether other retroelements  could exert similar TI effects as L1 retrotransposons and whether their effects may be due to DNA-bound TFs not just transcriptional activity or the mere presence of splicing and polyadenylation signals.In silico chromosome walking with 100 kb steps was carried out on the human genome with UCSC Genome Browser ABCA9 fragment containing exon 23 (108 bp) and its 5′ and 3′ flanking regions  was amplified from genomic DNA derived from NTera2D1 cell line using PCR with primers 22 int Dir and 23 int Rev. Similarly, NCAM1 genomic region was amplified in three overlapping fragments. First, a 1733 bp fragment A containing exon 9 (30 bp) and its flanking regions was amplified with primers 8 int Dir and 9 int Rev 2. Second, a 1908 bp fragment B containing intron 9 was amplified with primers 9 int Dir 2 and 9 int Rev 1. Third, a 848 bp fragment C containing intron 9 and L1 5′ UTR was amplified with primers L1 Dir and L1 Rev. Fragments A, B and C were used to restore a 3637 bp NCAM1 genomic region containing L1 5′ UTR (see Minigene constructs). TXNDC12-KTI12 genomic region was amplified in two fragments. A 226 bp TXNDC12 fragment containing exon 2 (61 bp) and its 5′ and 3′ flanking intronic sequences  was amplified with primers TX ex Dir and TX ex Rev. A 1896 bp KTI12 and its 5′ and 3′ flanking sequences  was amplified with primers KTI Dir 2 and KTI Rev. PCR was carried out with recombinant Taq DNA polymerase (Fermentas) according to manufacturer's protocol. For cloning, genomic fragments were treated with Klenow polymerase (Fermentas) and purified by TAE agarose gel electrophoresis using InvisorbSpin kit (Invitek). All primers used in this study were purchased from TAG Copenhagen A/S and their sequences are listed in Table S5.A 1036 bp ABCA9, NCAM1  and TXNDC12 gel-purified genomic fragments were first cloned into pBlueScript (pBS) SK+ vector (Stratagene) digested with EcoRV. KTI12 genomic fragment was cloned into pBS KS+ vector (Stratagene) digested with SmaI. Positive recombinant plasmids were isolated with alkaline lysis minipreparation method Ecl136II and HindIII, treated with Klenow polymerase and cloned downstream to the ABCA9 insert in pBS SK+ vector blunted at ClaI site. From this plasmid, ABCA9-L1 5′ UTR fragment was separated after digestion with SalI, then treated with Klenow polymerase and digested with EcoRI. The obtained fragment was further cloned into exon trapping vector pSPL3 (Invitrogen), from which a 1 kb intron fragment containing cryptic splice site at position 1134 bp was removed with NheI, after blunting the ends with Klenow polymerase and digestion with EcoRI, thus leaving appropriate sites (blunt and EcoRI) for its insertion. The final plasmid was termed ABCA Fl (7.2 kb) and contained SV40 promoter-ABCA9-L1 5′ UTR-SV40 polyA signal , named ΔASP292 and ΔASP613, were made with BspT1 and NheI, respectively. ΔYY1 (deletion +13 to +21 in L1 5′ UTR) was generated by cloning a 132 bp fragment, starting with YY1 primer sequence and ending with PauI site (obtained from PCR with primers YY1 and L1 ASP10) into ΔASP292 or ABCA Fl construct between blunted XhoI and PauI site. A 96 bp deletion (positions +1 to +96) in L1 SP was made with XhoI and KpnI and the construct was named ΔSP. Double deletions were made consecutively. A 2 bp deletion (ag) in the acceptor splice site (ΔSA) in intron 23 in ctttccagATGGTGCTAAA was made in the ABCA Fl with PCR mutagenesis. First, PCR was carried out with two primer pairs: 23 ex Dir – SA mut2, and SA mut1 – L1 SF1. The obtained two fragments were combined and amplified with primers 23 ex Dir and L1 SF1. Then, the PCR product was digested with Acc65I and inserted into ABCA Fl from which the same, but nonmutated fragment (740 bp) was removed. Sox mutations in the ABCA Fl  was removed. A 423 bp deletion in SV40 promoter (positions 68–491 nt) in ABCA Fl was made with PaeI and NcoI generating ΔSV40. Similarly, partial SV40 promoter deletion (positions 68–140 nt) was made with PaeI generating ΔSV40part.L1 5′ UTR-containing #11AS fragment (990 bp) was obtained from a recombinant pGL3 vector ABCA9 locus was amplified from genomic DNA derived from HeLa cell line using PCR with primers L1 PA3 5′ UTR Dir and L1 5′ UTR Rev. RP 5′ UTR (903 bp), RP ORFs (5121 bp) and RP 5′ UTR+ORFs fragments (6006 bp) were amplified from L1 RP clone  with L1 RP 5′ UTR Dir – L1 5′ UTR Rev, L1 RP ORF1 Dir – L1 3′ UTR Rev and L1 RP 5′ UTR Dir – L1 3′ UTR Rev primers, respectively. We were unsuccessful in amplification of the full-length L1 PA3 from the ABCA9 locus, because of high content of repeated DNAs in the 3′ flanking region. Therefore, we fused L1 5′ UTR with RP ORFs which were 98% identical to L1 PA3 ORFs. Obtained fragments were treated with Klenow polymerase and cloned into pBS SK+ vector blunted at EcoRV site with different orientation. Thereafter these fragments were digested from pBS SK+ with SalI and SmaI and subcloned downstream to the ABCA9 insert into ABCA Fl, from which #11AS 5′ UTR was removed with NheI, after blunting the ends wiht Klenow polymerase and digestion with XhoI. These constructs were named PA3 5′ UTR, RP 5′ UTR, RP ORFs +, RP ORFs –, RP 5′ UTR+ORFs +, RP 5′ UTR+ORFs – and PA3 5′ UTR+ORFs.A 960 bp L1 PA3 5′ UTR fragment from NCAM1 genomic fragments A, B and C were ligated together as follows. Fragments A and B obtained from the appropriate recombinant pBS SK+ digested with BglII and SmaI were ligated and inserted into pBS SK+ digested with EcoRV. Fragment C, obtained after digestion with HindIII, blunted with Klenow polymerase and digested with SpeI, was inserted downstream to the fragments A–B using SpeI and NotI, blunted with Klenow polymerase. Finally, a 3637 bp NCAM1 genomic fragment cloned in pBS SK+ was digested with SacII, blunted with Klenow polymerase and digested with SalI. This fragment was further cloned into exon trapping vector pSPL3 from which a 1 kb intron fragment was removed with NheI as described above. The final plasmid was termed NCAM Fl (8.7 kb) and contained SV40 promoter-NCAM1-L1 5′ UTR-SV40 polyA signal (SpeI and Acc65I. This deletion removed L1 SP (L1 5′ UTR positions +1 to +97) generating ΔSP. ΔL1 was made by removing a 3249 bp fragment (A–B) from NCAM Fl construct with SpeI and SalI. After blunting the ends, this fragment was inserted into pSPL3 vector digested with NheI and XhoI. A 611 bp deletion of SV40 promoter (positions 74–685 nt in pSPL3) was made in NCAM Fl with SalI and Mph1103I generating ΔSV40.TXNDC12 genomic fragment, containing exon 2 obtained from recombinant pBS SK+ after digestion with XhoI and SmaI, was cloned upstream to KTI12 in pBS KS+ digested with XhoI and EcoRV site in the same transcriptional orientation. From this cloning a 2674 bp TXNDC12-KTI12 fragment obtained after digestion with XhoI and XbaI was inserted into exon trapping vector pSPL3 digested with the same restriction enzymes. As described above, from this vector a 1 kb intronic fragment was removed before cloning. The final plasmid was termed TX-KTI Fl (7.7 kb) and contained: SV40 promoter-TXNDC12-KTI12-SV40 polyA signal  generating ΔP. The entire KTI12 coding sequence (1910 bp) was removed with MluI and BamHI generating ΔKTI2. A 115 bp (Δ115) and 395 bp (Δ395) deletions were made within the initiation region of KTI12 with PCR deletion mutagenesis, similarly as described above (ABCA ΔSA) using TX-KTI pBS construct and two primer pairs: T3 – KTI Δ1 as2 and KTI Δ1 s2 – KTI Rev RT in the case of Δ115 and T3 – KTI Δ2 as and KTI Δ2 s – KTI Rev RT in the case of Δ395. PCR fragments were purified from agarose gel and amplified with fusion PCR with T3 and KTI Rev RT primers. After digestion with XhoI and KpnI these fragments were gel-purified and cloned into pSPL3 vector digested with the same restriction enzymes. Deletion of SV40 promoter (positions 72–685 in pSPL3) in TX-KTI Fl was made with PaeI and SalI generating ΔSV40.A 273 bp In cloning experiments, where deletions were made with heterologous restriction enzymes, the ends were blunted with Klenow polymerase and ligated using T4 DNA ligase (Fermentas). Restriction fragments were separated and purified from TAE gel with InvisorbSpin (Invitek) kit. Structures of the final plasmid DNAs were confirmed by restriction mapping and sequencing using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's protocol. Sequences of all Fl minigene constructs are available upon request.2) in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine calf serum, penicillin (100 u/ml) and streptomycin (100 µg/ml) (Invitrogen). Cells were plated in 6-well tissue culture dishes with appropriate density 24 hour before transfection. Transient transfection was carried out with calcium phosphate 2) was used as a negative control. All transfections were made in three parallels. After 4 hours, transfection mix was replaced by fresh medium and incubation continued 24–40 hours.Human teratocarcinoma , adenocarcinoma , and mouse neuroblastoma  were purchased from LGC Standards. Human neuroblastoma (Kelly) was a gift from A. Veske. Cells were grown at 37°C in an atmosphere containing 5% carbon dioxide (CO2) and incubated with proteinase K (Fermentas) (0.2 µg/µl) at 37°C for 2 hours. After incubation, RNA was treated with phenol and precipitated with ethanol. DNA was removed from RNA samples by DNase treatment  at 37°C for 2 hours. Finally, RNA was treated with phenol, precipitated with ethanol, dissolved and kept in TE at −20°C. The amount and quality of RNA was checked by electrophoresis of one tenth of RNA sample.Total RNA was isolated from transfected or nontransfected cells using TRIzol reagent (Invitrogen), extraction with chlorophorm and precipitation with isopropanol as described by the manufacturer's protocol. The RNA pellet was washed with 70% ethanol, dissolved in PK buffer  and kept at −20°C.Cytoplasmic and nuclear RNA fractions were isolated from Kelly and NTera2D1 cell lines using the following protocol. Cells were washed with PBS, placed on ice and lysed directly on dish with 1 ml of NP-40 lysis buffer containing 0.5% Nonidet P-40, 3 mM MgClAGC primer for SV40 exonization and polyadenylated transcripts in ABCA Fl and NCAM Fl; 9 int Rev B primer for NCAM 1–9 transcripts; KTI Rev RT primer for KTI12 transcript; EST1 Rev for SV40 exonization transcripts in TX-KTI Fl, incubated at 65°C for 5 min and chilled on ice. RT reactions were carried out with SuperScript III RTase (Invitrogen)  in a final volume of 5 µl according to manufacturer's protocol. For specific primers and oligo dTAGC incubation temperatures were 50°C and 40°C, respectively.Reverse transcription (RT) was performed essentially as follows, 0.2–1.0 µg of total RNA was hybridized with appropriate primers: pSPL3 Rev primer for SV40 and L1 SP transcripts; 22 int Dir primer for ABCA L1 ASP transcripts; 23 int Rev primer for ABCA SV40 intronic transcripts; oligo dTTable S5) and recombinant Taq DNA polymerase (Fermentas) using 30–40 amplification cycles and a 10 µl final volume. PCR products were treated with Klenow polymerase, identified by restriction mapping, cloned into pBS SK+ vector EcoRV site and sequenced. PCR amplifications of human cDNAs derived from the multiple tissue cDNA panel provided by P. Pruunsild Figure S2). DNA-free total RNAs from human tissues were purchased from BioChain and used in experiment shown in TXNDC12 and KTI12 transcripts were detected with the following primer pairs: 2 ex Dir - 7 ex Rev, KTI Dir 1 - KTI Rev RT, 2 Ex Dir - EST1 Rev and 2 Ex Dir - EST2 Rev for nested PCR (30 cycles). The primers used for the detection of host gene, nested gene, potential TI-specific transcripts induced by nested genes and PPIA (peptidylprolyl isomerase A) are shown in Table S5.PCR was conducted using different combinations of primers (listed in 32P-labeled riboprobes as described in + plasmids linearized with different restriction enzymes. Hybridization was carried out in a 8–10 µl volume. Single-stranded RNAs were removed with RNase A (40 µg/ml) and T1 (100 u/ml) (Fermentas) at 30°C (or 40°C for polyA probes) for 1 hour. RNases were removed with proteinase K (Fermentas) (200 µg/ml) in the presence of 0.5% sodium dodecyl sulfate (SDS) at 37°C for 1 hour. After phenol treatment dsRNAs were precipitated with ethanol in the presence of carrier tRNA (3 µg) and were dissolved in gel loading buffer containing 95% formamide. Protected 32P-labelled RNA fragments were analyzed on a 5% polyacrylamide gel and were detected by Personal FX phosphoimager (BioRad). Quantitation of the bands corresponding to transcripts was carried out by comparing their intensities to the reference, i.e. two-fold serial dilutions of the probe, and if necessary, size normalization was made. All RPA experiments were reproduced using total RNAs obtained from at least two separate transfections.Equal amounts of total RNAs isolated after transfection were dissolved in FA hybridization cocktail and hybridized with different Figure S1TI effects induced by L1 5′ UTR and ORFs. Quantitative detection of various minigene transcripts by RPA. Riborobes Fl1-23-3 and Int1-23 (lanes 1 and 11) are schematically shown above each panel. (A) Different types of 5′ UTR , deletion (Δ) and mutation (M) constructs and (B) RP 5′ UTR+ORFs +/−, PA3 5′ UTR+ORFs and ORFs +/− used in transfection experiments are shown on top of each lane. Their structures are mapped to the schemes above panels. Protected transcripts (marked with arrows) are schematically shown by boxes and their sizes are given in nucleotides. Dashed lines/boxes show the remaining exon(s) not protected by the riboprobe used. In the case of RP 5′ UTR + ORFs (+/−) very faint signals were detected in the original autoradiogram. TE – transfection simulated with buffer.(TIF)Click here for additional data file.Figure S2L1-induced TI determined from NCAM1 endogenous transcripts. (A) Detection of alternatively spliced NCAM1 transcripts in different human cell lines using RT-PCR. (B) Detection of endogenous NCAM1 transcripts in neuroblastoma cell line (Kelly) by RPA. Various transcripts (structures shown on the right) were detected with three different riboprobes shown above panels. Their abundance was determined from the comparison to 2-fold serial dilution of riboprobe (bottom right) after size normalization. (C) Genomic structures of the human and mouse NCAM1 containing exons 7–1. Hatched boxes show intronic regions observed in RT-PCR. (D) Detection of NCAM1 transcripts in mouse and human neuroblastoma cell lines and brain cells. Separation of the RT-PCR products with and without exon 9 are shown at the bottom of panels. Various alternatively spliced transcripts in all panels  were detected with primers specific to exons 7 and 11 and introns 8 and 9. (E) Analysis of the NCAM1 transcripts containing exons 7–11. Endogenous transcripts (Ex-exon and Int-intron) shown on the right of panels were detected by RT-PCR. Intron-containing transcripts were further amplified by nested PCR. Note that NCAM1 primary PCR product  is also visible in some lanes.(DOC)Click here for additional data file.Figure S3TI dependence on the location of L1 (A) or nested gene (B) relative to the intron retention and exonization effects in their upstream region. Data analysis from (TIF)Click here for additional data file.Figure S4TI determined from endogenous NCAM1 transcripts derived from different human cell lines and tissues.(DOC)Click here for additional data file.Table S1Prediction of TI between human genes and intronic L1 retrotransposons.(DOC)Click here for additional data file.Table S2Distribution of additional aberrant transcripts in human genes containing intronic L1 retrotransposons.(DOC)Click here for additional data file.Table S3TI between host and nested genes.(DOC)Click here for additional data file.Table S4Search for additional aberrant transcripts in selected host genes shown in (DOC)Click here for additional data file.Table S5Primers used in RT-PCR and construction of minigenes.(XLS)Click here for additional data file.


Twintrons represent a special intronic arrangement in which introns of two different types occupy the same gene position. Consequently, alternative splicing of these introns requires two different spliceosomes competing for the same RNA molecule. So far, only two twintrons have been described in insects. Surprisingly, we discovered several such arrangements in vertebrate genomes, which are quite conserved throughout the lineages.This article was reviewed by Fyodor Kondrashow and Eugene Koonin. The length of the introns also differs ranging from 209 nt for the shortest intron to 4,226 nt for the longest one. Interestingly, the U12-type intron belongs to the AT-AC type with an unusual AA terminus at the 3′ SS. Upon splicing, it produces a splice variant, which results in the Premature Termination Codon (PTC) and consequently is subjected to nonsense-mediated mRNA decay (NMD) in both the human and mouse genomes. Although the conserved motifs of minor intron are present in several vertebrate genomes, including opossum and Xenopus, we found solid evidence of the U12-type intron splicing only in humans and mice. Interestingly, in the platypus genome splicing signals for U12-type spliceosome has been muted and cannot be recognized by a minor spliceosome any longer. This may suggest that a twintron arrangement was a mediator of U12-type intron elimination from the host gene, in agreement to our original hypothesis [One of the interesting genes harboring a twintron is c for H4 . There apothesis .HNRPLL and NCBP2. Although the protein product of HNRPLL shows slight variations in the RNA recognition motif (RRM) for major and minor intron splice variants, the changes in the protein sequence and structure are insignificant , which has RRM from the 42nd to the 112th amino acid of the U12-type splice variant (PDB ID – 3FEX) [To elucidate the role of alternative SSs in protein architecture, we scanned protein splice variants with InterProScan. Most of the protein isoforms of twintrons did not show any changes in the conserved motifs and structures, except for  – 3FEX) . When thNCBP2 is expressed mainly in brain, thymus, uterus, lungs, testis, and several other tissues, whereas the U2-type splice variant is expressed mostly in tumors and cancerous tissues  definition a twintron is any set of nested introns, belonging to the same splicing mechanism or not. This is at odds with the definition used by the authors and perhaps should be resolved. Perhaps a figure that demonstrates what a twintron looks like is called for: it would have been clearer to me what the authors mean.Our definition of twintrons differs slightly from the original one and includes both nested and shifted arrangements. Although we provide a short twintron definition in the abstract, the full one is now provided in the body of the paper and accompanied by a figure.Authors’ response: Second, the authors suggest that having such nested introns that are excised by two different spliceosomes can be an evolutionary mechanism of switching between the two intron types. However, perhaps this is at odds with the apparent conservation of such a setup - if this is a “safe pathway for intron switching” then certainly it does not appear to be a neutral one. Additionally, the mechanism that could turn an internal exon into an external one in a nested situation is not immediately clear to me.We think that either of two introns can be switched off and this might be random process. We agree that presented examples don’t provide direct evidence that such a switch occurs. However, the fact that twintron arrangement is more common phenomena than anticipated provides indirect evidence that such a mechanism could be used during gene structure evolution.Authors’ response: Is there a preference for U-2 or U-12 introns to be the external ones in the nested setup?No, there’s no bias in the two types introns arrangement. In six cases U12-type intron is the internal one, while in four cases the arrangement is reversed. The rest of twintrons display shifted arrangement.Authors’ response: This is quite an interesting short paper that reports a number of previously unnoticed twintrons and most importantly demonstrates their evolutionary conservation at considerable phylogenetic depths, with the implication of functional importance of the twintron structure itself. This is the major finding of the work, and it is certainly valuable. I am less enthusiastic of the two hypotheses proposed in the article, namely that twintrons could be an important intermediate along the path of elimination of U12 introns and that multiple protein isoforms produced by expression of twintron-containing genes might play a role in carcinogenesis. The first hypothesis, which is one of the main themes in the article, is of interest but I find the evidence quite limited. The authors might wish to expand the discussion. The idea about carcinogenesis, to me, is sheer, unwarranted speculation. The presence of additional splice variants in cancer samples might be caused by a variety of factors, above all the general deterioration of regulatory processes in tumors, and have nothing to do with carcinogenesis. I am not sure this is even worth a mention.We agree that two hypotheses are highly speculative. As suggested, we have expanded the discussion of the first hypothesis and removed the second one from the manuscript.Authors’ response: The authors declare that they have no competing interests.WM conceived and designed the project. JJ and MJ performed genomic analysis and JJ drafted the manuscript. All the authors contributed to the final version of the manuscript. All authors read and approved the final manuscript.Conservation of major and minor splice sites in vertebrates. Click here for fileHNRPLL. The structure is colored based on the secondary structure: red color for alpha-helices and yellow for beta-sheets. A black arrow indicates the variable amino acids. Superimposed 3D structures of U12 and U2-type splice variants of the gene Click here for fileNCBP2. The 3D structure is colored based on the secondary structure: red color for alpha-helices and yellow for beta-sheets. RRM domain missing in the U2 splice variant is shown in blue. Superimposed 3D structures of U12 and U2-type splice variants of the gene Click here for fileExpression data of splice variants of twintrons. Click here for file


The development of transparent radio-frequency electronics has been limited, until recently, by the lack of suitable materials. Naturally thin and transparent graphene may lead to disruptive innovations in such applications. Here, we realize optically transparent broadband absorbers operating in the millimetre wave regime achieved by stacking graphene bearing quartz substrates on a ground plate. Broadband absorption is a result of mutually coupled Fabry-Perot resonators represented by each graphene-quartz substrate. An analytical model has been developed to predict the absorption performance and the angular dependence of the absorber. Using a repeated transfer-and-etch process, multilayer graphene was processed to control its surface resistivity. Millimetre wave reflectometer measurements of the stacked graphene-quartz absorbers demonstrated excellent broadband absorption of 90% with a 28% fractional bandwidth from 125–165 GHz. Our data suggests that the absorbers' operation can also be extended to microwave and low-terahertz bands with negligible loss in performance. Graphene has attracted much attention in recent years due to its extraordinary electronic and optical properties such as high electron mobility, truly atomic thickness, mechanical flexibility and saturable absorption12349101112111416192022232526272830Radar absorbing materials (RAMs) significantly reduce an object's observable radar cross-section within specific bandwidths3332In this letter, we report for the first time on the fabrication and characterization of transparent broadband absorbers consisting of several stacked multilayer graphene sheets on quartz substrates backed with a ground plate. The large-area CVD multilayer graphene films have been fabricated through a repeated etch-and-transfer process to minimise PMMA residue between the layers, thereby yielding high-quality and optically transparent multilayer graphene films. Herein, analytical expressions are developed for the reflection and absorption coefficients consisting of an arbitrary number of stacked graphene layers. These expressions serve as a practical design guide to estimate the influence of the sheet resistance and substrate parameters, as well as the impact of oblique incidence. To validate the broadband absorption, millimetre wave reflectometer experiments have been carried out with single and stacked absorbers from 110 GHz to 170 GHz. For 5-unit stacked structures, 90% absorbance can be achieved with 28% fractional bandwidth from 125 GHz to 165 GHz. The broadband absorption can be further improved by using thinner substrates with lower relative permittivity or stacking more graphene-quartz substrates at the expense of reduced optical transparency.2/Si wafers and were found, by optical and electron microscopy to be free of pin-holes. Samples were of high uniformity with >90% monolayer coverage, as confirmed by Raman spectroscopic mapping and optical microscopy2/Si substrates and etching them in an aqueous ammonium persulfate solution before finally transferring the released PMMA/graphene onto the quartz substrates. This method avoids significant PMMA residue build-up between the stacks of graphene layers yielding reduced mean sheet resistance of ~0.9 kΩ/sq for 2 L and ~0.6 kΩ/sq for 3 L. The number of graphene layers was confirmed via UV-Vis spectro-photometery. Optical transmittances of 85%–91% at 700 nm for quartz-supported 1–4 L graphene was noted  plane wave is assumed to arrive at the absorber surface with an oblique incidence angle of θ. Since the thickness of quartz is a few millimetres, high-order modes become evanescent and can be ignored in the millimetre wave regime. The graphene sheets can be modelled as thin, two-sided surfaces characterized by surface conductivity, which is governed by the intraband contributions at low-terahertz frequencies and can be expressed asω is the radian frequency, cμ is the chemical potential, Γ is the phenomenological scattering rate, T is temperature, e is the charge of an electron, Bk is the Boltzmann's constant. Although sσ is frequency dependent in sσ is dominated by the surface conductance and can be regarded as frequency independent.To predict the absorber performance, we have derived an analytical expression based on a circuit model equivalent, as shown in dβ and characteristic admittance dY, with the resistive graphene sheets represented as a shunt admittance, sY = sσ ≈ 1/sR, where sR is the sheet resistance  graphene-quartz absorber, the input admittance is given by NYin, ≈ Y0 or S11 ≈ 0; the absorption peaks correspond to reflection zeros. In this case the incident wave will go through multiple reflections and be fully absorbed by the lossy resistive graphene sheets.The highest absorption is achieved when In order to investigate the nanostructured absorber, reflection spectra are measured by a well-established free space millimetre wave reflectometery technique and then transformed to absorption spectra according to Firstly, single (N = 1) absorbers with 1–4 L graphene were measured and then two graphene-quartz samples were stacked together to construct a 2-unit (N = 2) absorber . Measurecμ = 0 eV and Γ = 7 meV, this corresponds to a sheet resistance of 5044 Ω/sq which makes the input impedance challenging to match with free space. The peak absorption is lower than 40% indicating a poor absorption. As the chemical potential increases, in steps of cμ = 0.1 eV, the sheet resistance of graphene is reduced and the peak absorption improves. When cμ = 0.3 eV and Γ = 5 meV, the corresponding sheet resistance is 430 Ω/sq, which tends toward the free space impedance. Good impedance matching leads to 100% peak absorption around 148 GHz.The measured reflection and absorption spectra of single graphene-quartz absorbers with 1–4 L graphene on quartz are compared with analytical calculations in Graphene surface conductivity is mainly dominated by the intraband contribution from microwaves to far infrared waves. The real part of the intraband conductivity, which contributes to energy absorption or dissipation3718cμ = 0.0 eV and Γ = 7 meV  and the air gap (~0.1 mm) between quartz and ground plate. The absorption peak increase marginally (+1.2%) for the 3 L case and fall slightly (−1%) for the 4 L case. Multilayer graphene can thusly be used to derive a turbostratic, stacked, artificial graphite-like material of sufficiently reduced sheet resistance capable of near matching the free space impedance; however, it is challenging to improve the sheet resistance further for samples consisting of more than 3 layers, which could be due to water residue between the layers that prevents good contact between the interfacing layers in the present samples.First, the bare quartz substrate on the ground plate was tested in the reflectometer as a background reference. A total reflection with no absorption is observed across the whole frequency range, except for the intrinsic systematic noise at high frequencies (>160 GHz). The single unit graphene-quartz absorbers with 1–4 L graphene were then measured. A 1 L absorber has a small absorption peak around 30% at 148 GHz, which is similar to the case of  = 7 meV . In contif = (2i − 1)f0  have opposite phase difference which leads to periodic reflection zeros and absorption peaksThe single graphene-quartz absorber is based on a Salisbury screen absorber see , which epfi, = (2i − 1)f0/N and z,if = 2if0/N , respectively. As shown in Although a Salisbury screen absorber has multiple absorption bands, maximum absorption is limited to a narrow bandwidth around the resonant frequency. In order to achieve wideband absorption we instead consider a Jaumann absorber, which utilizes a series of a quarter wavelength thick dielectric layers that separate parallel resistive sheets, introducing a mutual coupling of Fabry-Perot resonators over a metallic ground. Here we use stacked graphene-quartz samples to realize a transparent millimetre wave Jaumann absorber. The zero-degree reflection phase condition can be used to predict the locations of absorption peaks and zeros . For thecμ = 0.15 eV) which corresponds to a sheet resistance of 859 Ω/sq. The calculated reflection spectra in The calculation results for stacked graphene-quartz absorbers are depicted in For a dielectric substrate, the resonance frequency will be decreased by increasing either the permittivity or thickness of the slabs. Moreover, the relative permittivity will affect the characteristic admittance of the dielectric according to 3O4 are added to graphene to result in high complex permeability values over the GHz rangeThere are two contributions to microwave absorption: dielectric and magnetic lossIn this work, in order to achieve optical transparency, we have not used multilayer graphene as a dielectric filler but as an ultra thin, lossy resistive component separated by the transparent quartz dielectric filler, backed by a ground plate, forming a Jaumann absorber. We aimed to match the impedance to free space by engineering the surface resistance characteristics of the multilayer graphene combined with the impedance transformation of the transparent dielectric filler. We are able to achieve absorption of 90% of the incident millimetre wave without necessarily taking permeability into consideration.θ < 30° do not have a significant impact on the absorption spectra. However, when θ > 60° absorption is reduced and the magnitude variations become significant for TE-polarized waves, whilst they improve for TM-polarized cases. We also observe that absorption bandwidth increases significantly when the relative permittivity is reduced from rε = 3.8 to rε = 1.1; suggesting that a dielectric with low relative permittivity is required to achieve both wide bandwidth and high absorption.Although data on oblique incidence is not presented, an analytical prediction can be obtained which we have validated in the normal incidence case . We obseBy stacking nanometre-thick multilayer graphene films and millimetre-thick quartz slabs, we have developed a facile and optically transparent broadband millimetre wave absorber. This absorber has been modelled using transmission line theory and an analytical expression has been derived to predict its performance.Each graphene-quartz structure forms a Fabry-Perot resonator. When backed with a ground plate, absorption peaks or reflection zeros occur at odd numbers of the fundamental resonant frequency. We have determined that the sheet resistance of the graphene is an important factor for determining the input impedance and free space impedance matching; here the multilayer CVD graphene films were used to achieve the required resistivity through a repeated transfer process developed to minimise PMMA residues.When multiple graphene-quartz samples are stacked together, the mutual coupling of the Fabry-Perot resonators introduces multiple absorption peaks in each band leading to broadband absorption. A 5-unit stacked graphene-quartz absorber seems adequate to counterbalance the absorption performance and the manufacturing complexity. In addition, the permittivity and thickness of the dielectric slabs provide additional degrees of freedom to adjust the bandwidth and in-band performance. Furthermore, for the case of oblique incidence, the absorber is found to be suitable for angles <30° for TE-polarized waves while good for all angles for TM-polarized waves. In order to achieve excellent absorption for wide incidence angles under TE-polarization, pyramidal or inhomogeneous structures may be used for future work.The measurements of transparent graphene-quartz absorbers have been carried out using a millimetre wave reflectometer across the 110–170 GHz band. Our measured results validate that multilayer graphene achieves reduced sheet resistance assessed by tuning the chemical potential in the calculation, and 5-unit stacked absorber achieves 90% absorbance with a 28% fractional bandwidth. Moreover, the derived analytical formulae predicted the periodic absorption spectra over a wide frequency region, and the proposed absorber can be designed to meet different requirements with the added benefit of optical transparency.2:Ar atmosphere at 4 mbar. Graphene growth was initiated by introducing 7 sccm CH4 (99.9%) under maintained H2 (99.98%) and Ar (99.998%) dilution. All samples were cooled to <300°C under ultra-high purity N2 (99.999%) before venting to atmospheric pressure. Temperatures were monitored using two type K bimetallic thermocouples and a surface infrared interferometer. Pressures and temperatures were accurate to within ±0.1 mbar and ±1°C, respectively.Graphene was grown by thermal chemical vapour deposition in a cold-walled, commercially available reactor (Aixtron Black Magic). 500 nm-thick Cu was magnetron sputtered onto 200 nm thermally oxidised Si-100 four inch wafers which were subsequently annealed at 850°C for 30 min in a 20:1500 standard cubic centimetres per minute H2/Si samples were first diced to 17 mm × 8.5 mm and then cast with 200 nm poly (methyl methacrylate)-(PMMA 950 A4) and were annealed at 180°C for 1 minute. Substrates were then immersed in an aqueous ammonium persulfate ((NH4)2S2O8, 2.2 g/100 mL DI) for 15 hours to etch the Cu catalyst. The floating PMMA-graphene films were then transferred into DI water baths using microscope glass slides and rinsed several times to remove the etchant. For monolayer graphene, the PMMA-graphene film was transferred to Saint-Gobain Spectrosil® 2200 Optical Fused Quartz substrates, and dried at atmospheric pressure for 24 hours. For monolayer samples the films were transferred to occupy only half side of the quartz substrate, leaving the other half exposed for UV-VIS spectro-photometry baseline calibration. For multilayer samples the films were transferred onto slightly larger diced wafer substrate pieces, in order to make PMMA/bilayer graphene stacks. The bilayer stacked substrates were etched again in (NH4)2S2O8 and were rinsed and transferred onto diced wafer quartz substrates, followed by acetone washes to remove the PMMA. The remaining PMMA/bilayer graphene films were transferred onto larger substrates and the process was repeated until stacked samples of up to 5 layers were processed.The graphene on Cu/SiOA HP N5244A vector network analyser and its millimetre wave extension heads were used to drive the reflectometer . The verY.H. proposed and supervised the project. W.I.M. advised on the project. B.W. carried out the absorber design and data analysis, M.T.C. synthesised the CVD graphene. H.M.T. fabricated the multilayer graphene samples and carried out sheet resistance and UV-Vis optical transmission measurements. B.Y. set up the millimetre wave measurement system. B.W., B.Y. and M.N. performed the measurements. Finally, the manuscript was prepared by B.W. and H.M.T. and revised by Y.H., M.T.C. and W.I.M.Supplementary


Yarrowia lipolytica. On the other hand, Basidiomycota and Perizomycotina have introns in their histone genes. We compared the distributions of 81, 47, 79, and 98 introns in the fungal histone H2A, H2B, H3, and H4 genes, respectively. Based on the multiple alignments of the amino acid sequences of histones, we identified 19, 13, 31, and 22 intron insertion sites in the histone H2A, H2B, H3, and H4 genes, respectively. Surprisingly only one hot spot of introns in the histone H2A gene is shared between Basidiomycota and Perizomycotina, suggesting that most of introns of Basidiomycota and Perizomycotina were acquired independently. Our findings suggest that the common ancestor of Ascomycota and Basidiomycota maybe had a few introns in the histone genes. In the course of fungal evolution, Saccharomycotina and Taphrinomycotina lost the histone introns; Basidiomycota and Perizomycotina acquired other introns independently. In addition, most of the introns have sequence similarity among introns of phylogenetically close species, strongly suggesting that horizontal intron transfer events between phylogenetically distant species have not occurred recently in the fungal histone genes.Saccharomycotina and Taphrinomycotina lack intron in their histone genes, except for an intron in one of histone H4 genes of  Eukaryotic genomic DNA is packaged with histone proteins to form chromatin Aspergillus nidulans and Neurospora crassa contain introns Aspergillus nidulans contain a poly(A) signal Interestingly, histone genes of the filamentous ascomycetes Phylogenetic tree based on histone H2B proteins shows that fungi are more closely related to animals than plants, which is consistent with the phylogeneny of organisms Schizosaccharomyces of Taphrinomycotina and Saccharomycotina) lack intron in their histone genes, except for an intron in one of the histone H4 genes of Yarrowia lipolytica  was shared with one of 2 hot spots in the histone H4 genes of Perizomycotina , and between Botryotinia fuckeliana and Sclerotinia sclerotiorum   . Those rAspergillus fumigatus lacks intron at the position 17 (hot spot)  of histone H4_2 of A. fumigatus has a sequence similarity with the 243-nt intron of N. fischeri (The histone H2A_2 gene of ot spot) . Howeverfischeri . The hision site . Howeverfischeri . In ordeLaccaria bicolor histone genes   . It sugghttp://www.ncbi.nlm.nih.gov/projects/genome/guide/fungi/) and performed the sequence-similarity search for the 102 fungal genome sequences. As the result, the distribution of most of the introns (except for some introns) is so limited among the phylogenetically closely related species (Aspergillus nidulans H4_2 intron (88-nt) has a unique insertion site ; Ashbya gossypii, Candida albicans, Debaryomyces hansenii, Kluyveromyces lactis, Pichia stipitis, Saccharomyces cerevisiae, Vanderwaltozyma polyspora, and Yarrowia lipolytica. Perizomycotina of Ascomycota (11 species); Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Botryotinia fuckeliana, Fusarium graminearum, Magnaporthe oryzae, Neosartorya fischeri, Neurospora crassa, Podospora anserine, and Sclerotinia sclerotiorum. Basidiomycota (4 species); Cryptococcus neoformans, Laccaria bicolor, Malassezia globosa, and Ustilago maydis. We extracted all core histones H2A, H2B, H3, and H4 genes from each fungal genome. The locations and the DNA lengths of introns were identified using the sequence viewer on the NCBI web site (http://www.ncbi.nlm.nih.gov/).We compared the following 24 fungal genome sequences. Taphrinomycotina of Ascomycota (1 species); Amino acid sequences of the histones were multiple-aligned using the CLUSTAL W of MEGA software http://fasta.bioch.virginia.edu/fasta_www2/fasta_list2.shtml) using 200 PRSS iterations E-value <0.02 as introns with nucleotide sequence similarity.We selected introns with nucleotide sequence similarity using the SSEARCH with Smith-Waterman similarity scores Click here for additional data file.Figure S2Alignment of fungal histone H2B proteins and the intron insertion site. The character “I” on the first row indicates the intron insertion site.(TXT)Click here for additional data file.Figure S3Alignment of fungal histone H3 proteins and the intron insertion site. The character “I” on the first row indicates the intron insertion site.(TXT)Click here for additional data file.Figure S4Alignment of fungal histone H4 proteins and the intron insertion site. The character “I” on the first row indicates the intron insertion site.(TXT)Click here for additional data file.Table S1Distribution of introns in fungal histone H2A genes.(DOCX)Click here for additional data file.Table S2Distribution of introns in fungal histone H2B genes.(DOCX)Click here for additional data file.Table S3Distribution of introns in fungal histone H3 genes.(DOCX)Click here for additional data file.Table S4Distribution of introns in fungal histone H4 genes.(DOCX)Click here for additional data file.Table S5Introns with sequence similarity. Yellow indicates the intron pair with different insertion sites in the same histone gene. Orange indicates the intron pair of different histone genes. The other indicates the intron pair with the same insertion site in the same histone gene.(DOCX)Click here for additional data file.Table S6E-value <0.02) of the Fungi Genome BLAST in the Fungal Genomes Central on the NCBI web site for each intron used in this study. The hit sequence ID with the score (bits) and E-value are shown.Results ((TXT)Click here for additional data file.