Our results suggest that the solubility of TDP-43 is largely influenced by its cognate single-strand DNA (ssDNA) or RNA (ssRNA) rather than hnRNP, which is known to associate with TDP-43 C-terminus. The direct interaction between the refolded TDP-43, purified from E.coli, and ssDNA were further characterized by Circular Dichroism (CD) as well as turbidity and filter binding assay. In addition, ssDNA or ssRNA failed to prevent the aggregation of the F147L/F149L double mutant or truncated TDP-43 (TDP208–414). Consistently, these two mutants form aggregates, in contrast with the wild-type TDP-43, when expressed in Neuro2a cells. Our results demonstrate an intimate relationship between the solubility of TDP-43 and its DNA or RNA binding affinity, which may shed light on the role of TDP-43 in ALS and FTLD.The aggregation of TAR DNA-binding protein (TDP-43) has been shown as a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) since 2006. While evidence has suggested that mutation or truncation in TDP-43 influences its aggregation process, nevertheless, the correlation between the TDP-43 aggregation propensity and its binding substrates has not been fully established in TDP-43 proteinopathy. To address this question, we have established a platform based on the  The 43 kDa TAR DNA-binding protein (TDP-43) and its C-terminal proteolytic fragments have been identified as the major component of inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with unbiquitin-positive inclusions (FTLD-U) in vivo and in vitro studies TDP-43 contains two highly conserved RNA-recognition domains, RRM1 and RRM2, which are involved in DNA or RNA binding, preferentially with sequence enriched in UG or TG dinucleotide repeats 182–414), which contains only RRM2 and glycine-rich domains, remains soluble in a cellular environment Recently, it has been shown that the disruption of DNA or RNA binding through mutation or RRM1 truncation may alter the solubility and localization of TDP-43. Nuclear bodies or aggregates appear in the nucleus when DNA binding ability is abolished or after the treatment of the nucleus with RNAse A E.coli TDP-43 after refolding in the presence and absence of ssDNA. To further elucidate the implication of the DNA or RNA binding effects within cell, we have also transiently expressed truncated (TDP208–414) and double mutants of TDP-43 (F147L/F149L) in Neuro2a cells, a mouse neuroblastoma cell line. Our results have provided new evidence that the aggregation process of TDP-43 is not only influenced by C-terminal fragment of the protein but also by its binding partners. These underscore an important consideration that should be included in the design of in pathologic and therapeutic studies against ALS and FTLD.It is well known that bacterially expressed recombinant TDP-43 is intrinsically aggregation-prone and may form fibrillar aggregates 208–414 including the additional encoded FLAG-tag at the 5′-end, were ligated between the XhoI-NotI restriction enzyme sites in the pCMVTNT vector  and the NcoI-XhoI enzyme sites in pIVEX2.3d vector , respectively. For TDP-43 expression in E. coli, pET17b plasmid was used. All the constructs were sequenced by Tri-I Biotech, Inc. (Taiwan) and the inserted sequence of the recombinant TDP-43 was verified.The DNA sequence encoding human TDP-43 was introduced into several plasmids for different applications. For cell transfections and cell-free reactions, the genes of TDP-43 and TDPE.coli extract  were applied to generate the TDP-43 proteins and provide a cellular mimicking environment to study the TDP-43 protein aggregation. After the synthesis was completed at 30°C for 2 h, the cell-free reactions were further incubated at either 30 or 37°C (1–4 h) for the formation of the TDP-43 aggregates. To evaluate the influence of ssDNA/RNA, various oligonucleotides  repeated sequences with 6 to 24 nt lengths and 4–10 µM concentrations were added into the cell-free reaction immediately after the synthesis. At different time points after incubation, the samples were divided into supernatant and pellet fractions by centrifugation at 16,100×g for 30 minutes at 4°C. The pellet was then dissolved by SDS-PAGE loading buffer  and the proteins in the supernatant were precipitated by acetone followed by re-suspension with the SDS-PAGE loading buffer. The TDP-43 in both fractions was detected by western blotting with a primary anti-FLAG M2 monoclonal antibody  and a secondary anti-mouse antibody conjugated with HRP . The antibodies-TDP-43 complexes were detected on Biospectrum® AC image system  using a chemiluminescent substrate for HRP . The blotting signals were analyzed by Image J  and the fractions of TDP-43 protein in supernatant were obtained by the equation S/(S+P), where S and P denote the detected signals of the TDP-43 in the supernatant and pellet fractions, respectively. All the experiments for each sample were repeated three times.The eukaryotic cell-free system derived from the rabbit reticulocyte lysate  and the prokaryotic cell-free system derived from the The native and refolded TDP-43 as well as mutants (2–3 µM) were dissolved in phosphate buffer and incubated at room temperature. The CD spectra of the resulting protein solutions were recorded in 1 mm quartz cuvette on the J-815 CD spectrometer . All data were collected from 200 to 260 nm with the scanning speed of 100 nm/min. Three scans were averaged for each sample.6 or (TG)6] were incubated in refolding buffer at 25°C with a continuous agitation (1400 rpm) in the thermomixer . At different time points after incubation, the turbidity of TDP-43 proteins was detected by measuring the absorbance at 600 nm in a UV-spectrophotometer (Beckman Coulter) at room temperature. The filter binding assay was followed with the published protocols 12 was added into the protein solution with different concentration  at 37°C. After 1 h, the mixture was loaded onto the nitrocellulose membrane  presoaked with buffer containing 0.4 M KOH, rinsed with water, and washed with diluted buffer . The fluorescence signal on the membrane was obtained from the Typhoon TRIO imager .The refolded TDP-43 proteins (3 µM) in the absence or presence of different oligonucleotides . Comparing with the TDP-43 in the absence of ssDNA, the lower turbidity of TDP-43 with (CA)6 suggests that the size of aggregates may be efficiently reduced and this effect is even more stronger in the case of (TG)6 (6 (>60 min) than with (CA)6 (<30 min) or control. On the contrary, co-incubation of ssDNAs with the purified mutant TDP-43 (F147L/149L) had no significant effect on the protein aggregation process (6/(CA)6 are similar to those without exogenous DNAs (The binding of the ssDNA on wild type/mutant TDP-43 was confirmed by the filter binding assay with 6-carboxyfluorescein (FAM)-labelled (TG)L/F149L) . We had of (TG)6 . The obs process . The curous DNAs .In an attempt to characterize the morphology of the refolded TDP-43 in the presence and absence of single-strand oligonucleotides, we had observed the TDP-43 structure by electron microscopy (EM) with secondary antibody conjugated on gold nanoparticles. Instead of fiber-like aggregates, only amorphous and/or irregular aggregates could be found in our refolded TDP-43 . However208–414) TDP-43 in the Neuro2a cells (208–414), but not wild type TDP-43. Interestingly, most of the aggregates of the mutant F147L/F149L are found to be round in shape  in the nucleus rather than in the cytosol, suggesting that the presence of nuclear localization sequence (NLS) results in the location of this mutant in the nucleus, as expected, and the loss of DNA/RNA binding of this mutant effectuated aggregates. On the other hand, the truncated TDP208–414, which did not contain the NLS sequence, has failed to enter through the nucleus membrane and formed linear aggregates  in the cytosol. The expression level for wild type, mutant (F147L/F149L), and truncated (TDP208–414) TDP-43 are similar in the Neuro2a cells as shown on the SDS-PAGE with western blotting , and N-terminally truncated  which lacks NLS sequence and RNA recognition motifs  As pointed out from the previous literature, the intrinsically disordered proteins/domain may transform into ordered structure or start folding upon binding to its cognate RNA C) may interact with nucleic acids in vitro and is transformed into a β-sheet rich structure similar to its disease-causing isoform (PrPSC). Moreover, the interaction between PrP and nucleic acid may result in the partial unfolding of the prion protein and the formation of amyloid-like structure TDP-43 may cooperate with many partners  to participate in mRNA splicing, degradation, stabilization, translation and transportation within cells. We had shown that the cognate ssDNA and ssRNA rather than the hnRNP may directly regulate the solubility of TDP-43 and proposed that the binding partners/collaborators might help to diminish the intrinsically aggregation-prone tendency in TDP-43. Recently, it was also shown that the oxidative stress promoted TDP-43 cross-linking via cysteine oxidation near the RRM2 domain and induced cytoplasmic stress granules (SGs). Similarly, they have shown that stress-induced cysteine cross-linking impaired TDP-43 splicing function that could be due to the abrogation of the RNA binding ability in TDP-43. This would functionally mimic the RNA-binding deficient mutants Figure S1The solubility of human TDP-43 expressed in E. coli cell-free system. (A) The western blotting signals of TDP-43 generated at various temperatures in the supernatant (S) and pellet (P) fractions. (B) The solubility test of generated TDP-43 in the presence and absence of DNA under the incubation at either 37 or 24°C.(DOC)Click here for additional data file.Figure S2Expression of human TDP-43 protein in the rabbit reticulocyte cell-free system (TNT Quick Coupled Transcription/Translation Systems). (A) The reactions were analyzed by western blotting with anti-FLAG antibody in the absence (lane 1) and presence (lane 2) of the plasmid encoding for the FLAG-tagged TDP-43. Proteins in the cell-free reaction were stained by either ponceau S or coomassie blue to confirm a similar amount of the samples were analyzed. (B) Time course (1–4 h) of the FLAG-tagged TDP-43 expression in the cell-free system at 30°C. After 2 h, the protein synthesis was completed. The production of TDP-43 was quantified by western blotting and analyzed by Image J.(DOC)Click here for additional data file.Figure S3Quantification of FLAG-TDP-43 protein in the rabbit reticulocyte cell-free system (TNT Quick Coupled Transcription/Translation Systems). (A) Different concentrations of purified FLAG-trigger factor protein were loaded on the SDS-PAGE as a standard to quantify the expressed FLAG-TDP-43 in the cell-free system. (B) The TDP-43 protein concentration of the E.coli cell-free system is diluted at five times and has been used to compare the TDP-43 protein expression in the Rabbit cell-free system. All proteins were analyzed by western blotting with anti-Flag antibody.(DOC)Click here for additional data file.Figure S4DNA binding assay for refolded TDP-43 proteins. 0.1 µM FAM-labeled (TG)12 (left) and (CA)12 (right) were mixed with TDP-43 FL (wild type and mutant) at indicated concentrations. The mixtures were filtered through a nitrocellulose membrane, and ssDNA/protein complex trapped on the membranes were probed with florescence dye. The image was obtained by Typhoon TRIO variable mode imager .(DOC)Click here for additional data file.Figure S5EM images of refolded TDP-43 in the absence and presence of single-strand DNA. Both (A) small and (C) large aggregates are found in the refolded TDP-43 samples. Similar aggregates are found in the refolded TDP-43 in the presence of single-strand DNA, (TG)12, as shown in (B) and (D). The red arrows indicate the gold nanoparticles for immunogold staining. The small bars indicate 100 nm in (A), (B) and (C), and 500 nm in (D).(DOC)Click here for additional data file.Methods S1Supporting methods.(DOC)Click here for additional data file.


Oral manifestations of the sarcoidosis are rare. We report a case of a systematic sarcoidosis revealed by a nodule of the palate.To emphasize on mucosa localization of sarcoidosis when addressing nodular lesion of the palate in order to allow an appropriate treatment.Mrs M. L a 29-year-old woman, presented an isolated nodule of the soft palate. The nasofibroscopy was normal, the histology showed a non specific inflammatory infiltrate. Two years later, the patient presented papulonodular lesions of the face associated with anosmia and a second stage dyspnea of NYHA.Oral sarcoidosis is rare. Labial and lingual effects are at the forefront; effects of the palate remain exceptional. The lesions may take the form of a nodule, ulceration or perforation. Treatment based on systemic corticosteroids, sometimes with the addition of immunosuppressives or synthetic antimalarials.Sarcoidosis of the soft palate is rare and probably underdiagnosed. The risk of perforation or occurrence of systemic localization requires a careful examination of the oral mucosa in all patients with sarcoidosis.None Declared.


We carried out an analysis of intron length conservation across a diverse group of nineteen mammalian species. Motivated by recent research suggesting a role for time delays associated with intron transcription in gene expression oscillations required for early embryonic patterning, we searched for examples of genes that showed the most extreme conservation of total intron content in mammals.Gene sets annotated as being involved in pattern specification in the early embryo or containing the homeobox DNA-binding domain, were significantly enriched among genes with highly conserved intron content. We used ancestral sequences reconstructed with probabilistic models that account for insertion and deletion mutations to distinguish insertion and deletion events on lineages leading to human and mouse from their last common ancestor. Using a randomization procedure, we show that genes containing the homeobox domain show less change in intron content than expected, given the number of insertion and deletion events within their introns.Our results suggest selection for gene expression precision or the existence of additional development-associated genes for which transcriptional delay is functionally significant. One of the salient features of eukaryotic genomes is the pervasive presence of introns, comprising up to 95% of transcribed primary protein-coding sequences in mammals . The funHes7) gene is involved in the control of somite formation through oscillatory patterns of gene expression [et al.[in vivo the impact of removing the introns from the mouse Hes7 gene. They found that expression of the mutant Hes7 gene occurred approximately 19 minutes earlier than for the wild-type and that this reduction in gene expression delay resulted in abolition of oscillations and segmentation defects. Given that the delay associated with transcribing introns appears to play a crucial role in the functioning of this gene, the length of the introns may be evolving under purifying selective pressure. Moreover, further examples of genes with highly conserved intron content across species may reveal more genes for which transcriptional delay is an important aspect of gene regulation. We investigated the conservation of the intron content of Hes7 across a diverse set of nineteen mammals and carried out a search for other genes for which total intron length shows evidence of evolutionary conservation. In such cases introns are likely to play an important functional role and, in a subset, time delays associated with transcribing introns may be a significant aspect of gene regulation.Negative feedback loops with a time delay can result in oscillating patterns of gene expression, which can be exploited by living organisms as biological time-keeping devices. This appears to be particularly important in development . The haipression . RecentlHes7 resulting, at least in part, from intron transcription has been proposed to play a key role in somite formation in animal embryos by establishing an oscillating pattern of gene expression [Hes7 introns across a diverse set of mammalian species  between human gene models from Ensembl and sets of orthologous genes, obtained from OrthoDB [Hes7 . Restricting also to orthologous groups represented in at least half of the species, only 11 other orthologous groups  had intron content within 10% of the human gene in as high a proportion of the orthologues as the Hes7 group. Remarkably, of the corresponding 12 human genes (including Hes7), five are annotated with the gene ontology (GO) term GO:0007389 (pattern specification process) from the biological process component of GO, defined as “any developmental process that results in the creation of defined areas or spaces within an organism to which cells respond and eventually are instructed to differentiate”. Using the DAVID functional annotation tool [–5) and remains significant following correction for multiplicity of testing using the Benjamini-Hochberg method (FDR = 0.03). Six of the genes were annotated with the Swissprot and Protein Information Resource (SP-PIR) keywords term ‘developmental protein’ (FDR = 0.003).To determine whether intron content, and thus potentially transcription time, of  OrthoDB . Becauseion tool ,17, thisWe also used a more relaxed conservation threshold and identified a larger set of 144 genes with relatively conserved intron content among the genes with between one and five kilobases of intron. Genes for which more than 50% of the orthologues have intron content within 10% of the intron content of the human member of the group were included in this group, again restricting to orthologue groups represented in at least half of the species. We again found evidence for a set of genes with conserved intron content which was very highly enriched for genes involved in development, identified using DAVID Table . Only thTo investigate evolution of intron length through insertion and deletion in greater detail we considered changes in intron length along the human and mouse lineages following divergence from their last common ancestor. Genomic sequences at ancestral nodes of the mammalian phylogeny were obtained from Ensembl. These sequences were inferred using a probabilistic method that has been shown to have high accuracy for the inference of insertion and deletion events. We mapped insertion and deletion events to introns.Hes7 across the mammalian panel, we found qualitative support for both effects. The total amount of insertion and deletion was lower in these genes but there were also some genes with substantial insertion and deletions in balance . We applied this procedure to the set of homeobox domain proteins and found that the change in intron content of these genes along both the human and mouse lineages was far less than expected, given the number of indels inferred to have occurred in each lineage and under the null assumption that the ratio of insertions and deletions and size distribution of these events did not differ for these genes compared to the rest of genes in the size class. In the case of human, the mean absolute change in intron content was 450 bp per gene, whereas the median of this value in the randomized data was 846 bp per gene. The randomized values were less than or equal to the observed value in 87 of 10,000 randomizations (one-tailed p = 0.009). In mouse the mean absolute change in intron length was just 9 bp per gene. The median of this quantity across randomizations was 398 bp per gene. The values in the random data were lower than or equal to the observed data in 33 of 10,000 randomizations (one-tailed p = 0.003).We also identified the complete set of genes in size class one that were annotated with a homeobox domain by Interpro . To invecis or trans (e.g. non-coding RNAs) regulatory elements within introns of these genes, which would be disrupted by insertions and even more so by deletions. However, the presence of regulatory elements would tend to increase the intron length [Hes7 are shown as figure From our results it appears that there is selection to prevent large changes in intron content in specific sets of genes, notably in genes involved in development and especially in early embryonic patterning. This selection could result from the presence of n length , yet we Hes7 are relatively short (< 2 kb) and RNA polymerase II processes nucleotides at a rate in the order of 2 kb per minute [To explain our observations, conservation acting on functional elements would, perhaps, have to be balanced with selection for rapid induction, as rapidly induced genes have been shown to have short introns . If the r minute ,23. ThisSome theories of intron evolution have focussed on the energy cost associated with introns. Reduced intron lengths in highly expressed genes were proposed to result from selection for efficiency . In suppHomo sapiens, Bos taurus, Canis familiaris, Ornithorhynchus anatinus, Equus caballus, Erinaceus europaeus, Gorilla gorilla, Loxodonta africana, Monodelphis domestica, Myotis lucifugus, Microcebus murinus, Mus musculus, Oryctolagus cuniculus, Sus scrofa, Spermophilus tridecemlineatus, Tarsius syrichta, Tursiops truncatus, Vicugna pacos and Felis catus. Species were selected to sample a broad range of mammalian evolutionary history. A phylogenetic tree of these taxa, obtained from the interactive tree of life [Complete sets of gene models were downloaded from Ensembl release 62  via BioM of life ,26 is pr of life . We extr of life ,17. For Genomic multiple sequence ancestor alignments, inferred using the Ensembl Enredo-Pecan-Ortheus pipeline , were doFor each intron size class we used the complete set of insertion and deletion events within the introns to define the null expectation of events in that intron size class. To test for evidence of purifying selection acting on the intron content of a gene or a set of genes we considered all of the insertion and deletion events in the gene(s) under consideration and for each event sampled an insertion or deletion from the total set of events in the introns of genes in that class. Given a set of insertions and deletions randomly sampled in this way we calculated the change in intron content implied by this set of insertions and deletions (sum of insertion lengths minus the sum of the lengths of the deletions), separately along the human and mouse lineages. This was repeated 10,000 times and in each case the implied change in intron content in the randomized data was compared to the change in intron content, given the actual insertions and deletions inferred to have occurred. The number of times the absolute value of the change in intron content in the randomized data was less than or equal to the absolute value of the change in intron content in the observed data was used to calculate a p-value, with a separate p-value calculated for the human and mouse lineages. These p-values indicate the probability of observing as little or less variation in the intron content of a gene or set of genes, given the number of insertion and deletion events that have occurred and under the assumption that these indel events have the same distribution as events in other genes in the same intron size class.CS conceived the project. CS and PK performed the analysis and co-wrote the manuscript.The authors declare that they have no competing interests.


Moreover, the output properties of the graphene transistor show significant current saturation, and the graphene transistor can be modulated using the local graphene gate. These results clearly show that the device is well suited for analog applications.We report a high-performance graphene/Si field-effect transistor fabricated via rapid chemical vapor deposition. Oligolayered graphene with a large uniform surface acts as the local gate of the graphene transistors. The scaled transconductance,  Finally, the carrier transport mechanism in the GFET is discussed.Graphene is a single-atom-thick carbon film,2 that h scaling,4. To da scaling, nanoele scaling, molecul scaling, graphen scaling, and sol scaling,11, have scaling, cut-and scaling, the epi scaling,14, chem scaling, and oth scaling,17. The 2, and placed at the center of the furnace. Prior to deposition, the furnace was evacuated to 10−2 Pa, heated to 300°C, and maintained at this temperature for 30 min to remove any moisture from the mixture. The furnace was then heated to 700°C for the deposition. A 300-μm SiO2 layer was thermally oxidized for 30 min using high-purity oxygen gas followed by annealing at 900°C for 40 min. The source and drain sections were formed by doping the Si with B atoms via exposure to an Ar gas flow carrying an analytically pure C3H9BO3 doping agent into the reactive chamber at 900°C for 20 min, followed by annealing at 950°C for 30 min. After doping, a mixture of CH4 gas (99.999%) and Ar gas at a volume ratio of 1:10 was introduced into the reaction chamber at the same temperature (950°C). CH4 decomposed to give a mixture of C, H+, and H2 when the vapor-phase CH4 and Ar gases flowed through the furnace; the C atoms condensed on the Si substrates to form the graphene film under a working pressure of 50 Pa. The growth process was carried out for 2 min, and the samples were then annealed at 1,000°C for 30 min. To observe the surface morphology and structure of the graphene film, a few graphene reference samples were concurrently deposited. Subsequently, the graphene film was capped with a 300-μm SiO2 layer, and the graphene and SiO2 layers above the source and drain sections were removed. Finally, the GFET was completed by depositing Al electrodes on the gate, source, and drain sections via evaporation. The morphologies and structures of the samples were characterized using optical microscopy, atomic force microscopy (AFM), and Raman spectroscopy. The electronic properties were assessed via Hall effect measurement (HMS-3000) and micro-current  measurements.Figure 4 at 950°C (the decomposition temperature of CH4 is about 900°C) absorbed, condensed, and formed islands on the Si substrate. The graphene film was formed by the combination of many islands over time.Figure −1 and a G-band peak at 1,590 cm−1. The intensity ratio of the peaks (I2D/IG) is 14, which confirms that the graphene film comprises a few layers and is of high quality. The surface carrier concentration, carrier mobility, and current–voltage (I-V) properties of the graphene film were determined via Hall effect measurements. The carrier concentration of the samples is about 1010 cm−2, while the electron mobility is 5.1 × 104 cm2 V−1 s−1, which is very close to the known ideal value of 2 × 105 cm2 V−1 s−1[I-V behavior of the graphene film is shown in Figure I-V behaviors presented in Figure Figure 2 V−1 s−1,4. The sIg, versus gate voltage, Vg, behavior of the graphene/Si transistor is shown in Figure Ids, versus the source-drain voltage, Vds, under different gate voltages; the GFET exhibits a nearly linear plot when Vds is less than 2.5 V for different gate voltages. As Vds increases above 2.5 V, Ids tends towards saturation. The results indicate that the IV plot for a Vg of 4 V is optimal as a ratio of current switch, Ion/Ioff, of 100 is obtained. Moreover, the GFET has a smaller threshold voltage, and the Ids-Vds plots of the graphene transistor show significant current saturation characteristics, which is typical of Si FETs but rare for GFETs. It has been suggested that the velocity saturation of the carrier at higher biases may lead to the current saturation phenomenon in graphene transistors[2 layer that supports the graphene channels[The gate current, nsistors. The satchannels,21. The Ids versus Vg at a Vds value of 1 V , of the GFET as a function of the gate voltage. A peak transconductance of 3 mS/μm was obtained at a Vds value of 1 V in this device. Significantly, this scaled transconductance is nearly one order of magnitude better than that of the reported graphene transistors[gm is strongly dependent on the gate voltage. The branch where gm is less than zero represents p-type transport that is dominated by holes; when gm becomes positive with increasing gate voltage, the channel becomes n-type.Moreover, we determined the transfer characteristics of  Figure . The plonsistors. The sig4 cm2 V−1 s−1, the scaled transconductance of the graphene transistors exceeds 3 mS/μm, and the ratio of the current switch, Ion/Ioff, is as high as 100. The fabricated GFET shows current saturation characteristics which demonstrate that two-dimensional graphene devices can be used for analog and radio-frequency circuit applications without the need for bandgap engineering.High-performance graphene/Si transistors were fabricated by rapid chemical vapor deposition. The electron mobility of the graphene film is 5.1 × 10The authors declare that they have no competing interests.XM designed the structure of the graphene transistor, analyzed the results, and wrote the manuscript. WG participated in the fabrication of the graphene film on the substrate. JS fabricated the drain, source, and gate of the transistor and participated in the analysis the results of the transistor. YT measured the electrical properties of the transistor. All authors read and approved the final manuscript.XM is a professor and PhD degree holder specializing in semiconductor materials and devices, specially expert in nanoscaled optical-electronic materials and optoelectronic devices. WG is a graduate student major in fabrication of new semiconductor nanometer materials. JS is a lecturer and PhD degree holder specializing in semiconductor devices. YT is an engineer specializing in optoelectronic measurements.


The costs and benefits of spliceosomal introns in eukaryotes have not been established. One recognized effect of intron splicing is its known enhancement of gene expression. However, the mechanism regulating such splicing-mediated expression enhancement has not been defined. Previous studies have shown that intron splicing is a time-consuming process, indicating that splicing may not reduce the time required for transcription and processing of spliced pre-mRNA molecules; rather, it might facilitate the later rounds of transcription. Because the densities of active RNA polymerase II on most genes are less than one molecule per gene, direct interactions between the splicing apparatus and transcriptional complexes (from the later rounds of transcription) are infrequent, and thus unlikely to account for splicing-mediated gene expression enhancement.The serine/arginine-rich protein SF2/ASF can inhibit the DNA topoisomerase I activity that removes negative supercoiling of DNA generated by transcription. Consequently, splicing could make genes more receptive to RNA polymerase II during the later rounds of transcription, and thus affect the frequency of gene transcription. Compared with the transcriptional enhancement mediated by strong promoters, intron-containing genes experience a lower frequency of cut-and-paste processes. The cleavage and religation activity of DNA strands by DNA topoisomerase I was recently shown to account for transcription-associated mutagenesis. Therefore, intron-mediated enhancement of gene expression could reduce transcription-associated genome instability.Experimentally test whether transcription-associated mutagenesis is lower in intron-containing genes than in intronless genes. Use bioinformatic analysis to check whether exons flanking lost introns have higher frequencies of short deletions.The mechanism of intron-mediated enhancement proposed here may also explain the positive correlation observed between intron size and gene expression levels in unicellular organisms, and the greater number of intron containing genes in higher organisms.This article was reviewed by Dr Arcady Mushegian, Dr Igor B Rogozin (nominated by Dr I King Jordan) and Dr Alexey S Kondrashov. For the full reviews, please go to the Reviewer's Reports section. Saccharomyces cerevisiae, intron-containing genes produce more copies of RNA than intronless genes [S. cerevisiae also showed that introns could enhance transcription  having >2 RNA polymerase molecules/gene, and 0.86% of genes  having >1 RNA polymerase molecules/gene [S. cerevisiae have >1 RNA polymerase molecules/gene , the 2.37 × 10-3 molecules/kb can be converted into 0.044 molecules/gene, a value which is lower than the 0.096 molecules/gene in yeast. From this we can estimate that genes that have recruited multiple active RNA polymerase II molecules are probably also infrequent in mice.Although genome-wide data on the densities of RNA polymerase II are not yet available for other species, we can roughly estimate them by steady-state mRNA abundance and mRNA stability. In yeast, the median abundance of mRNAs is 1.38 copies/cell ; the medIn both yeast and mice, therefore, direct interactions between splicing factors and the later rounds of transcription are probably infrequent events. The splicing-mediated enhancement of gene expression might be attributed to the indirect interactions occurring between splicing factors and later rounds of transcription. If future studies show that multiple active RNA polymerase II molecules on a single gene are common and direct interactions between splicing factors and later rounds of transcription are not infrequent, indirect enhancement of the later rounds of transcription by splicing would contribute less than we envision here. Nevertheless, indirect enhancement of the later rounds of transcription mediated by splicing should be explored if there is evidence for it.In the following sections, we first propose a possible mechanism for splicing-mediated indirect enhancement of the later rounds of transcription, and then explore potential answers to the following questions: Is there any difference between the enhancement of gene expression by introns and strong promoters? What are the costs and benefits underlying the enhancement of gene expression by intron splicing?It has been documented that transcription generates positive supercoiling ahead of the transcriptional assembly and negative supercoiling behind the assembly if the DNA is topologically closed -52. In eIn eukaryotic cells, topoisomerase I has another function; acting as a kinase to phosphorylate the serine/arginine-rich (SR) proteins like SF2/ASF ,57. WhenS. cerevisiae have more active RNA polymerase II molecules attached to them and higher nascent transcription rates than intronless genes  revealed an approximate bell-shaped relationship between intron size and gene expression levels [nrb activates topoisomerase I to remove negative supercoiling [Another insight from the results of Fox-Walsh et al.  is that xpressed , a trendxpressed ,87,89,90n levels . With inn levels . Besidesn levels . For exarcoiling . The splrcoiling . It is tS. cerevisiae, the energetic cost of a long intron in a highly expressed gene is a burden visible to natural selection [If introns only confer the benefits proposed by ourselves and others -10, the election .The authors declare that they have no competing interests.DKN conceived the hypothesis and wrote the original draft; YFY collected the genome and expression data and modified the manuscript; both authors read and approved the final text.Dr Arcady Mushegian, Stowers Institute of Medical Research, USANe. What promotes intron persistence in all eukaryotes, aside from small Ne, is an open question. The authors argue that a factor here is the ability of one of the SR proteins to inhibit topo I activity, thus reducing mutation rate. This is an interesting hypothesis compatible with some of the observed data on correlation between intron length, expression strength, polymerase occupancy, etc. I request, however, that the others state more explicitly their opinion on when in the course of evolution this inhibition arose - do I understand it correctly that it had to be an ancient property, and if so, has this been borne out by pinpointing the origin of the SR factor in question, or by showing that this is a general property of many SR factors, not the serendipitous advantage of this particular one?The origin of eukaryotic introns is most likely explained from the mechanistic point of view by group II intron invasion from the mitochondrial ancestor, and from population point of view by weak purifying selection in populations with small Authors' response: This is a very important question raised about our hypothesis. We would also like to be able to see the potential benefit(s) that might have driven the origin and evolution of spliceosomal introns. Unfortunately, we are unable to speculate further about this at this time, because very little is known about the origin and early evolution of spliceosomal introns and SR proteins. Further evidence is required to offer a more explicit opinion.Dr Igor B Rogozin, NCBI/NLM/NIH, USA (nominated by Dr I King Jordan)The paper discusses various issues related to the positive correlation between intron size and gene expression which is observed in unicellular organisms and some multicellular organisms. This correlation is not particularly strong and have various explanations, for example, longer introns may be more efficiently spliced out or may be splicing is important for an efficient transport of mRNA. The authors suggested their own hypothesis:"If splicing could inhibit topoisomerase I DNA cleavage and religation activity, eukaryotic cells could avoid the dark side of topoisomerase I, while maintaining a high level of transcriptional activity. However, cells lacking introns and splicing activity do not necessarily exhibit obvious growth rate defects. Indeed, the deletion of most introns has been found to have no significant effects on cell growth, and, in the laboratory setting at least, introns appear to be nonessential . If our I think that by the "the genetic risk" the authors mean the increased rate of spontaneous mutations. In general, I do not think that the transcription-associated mutagenesis is different from other sources of spontaneous mutations. I do not see any connection between the transcription-coupled mutagenesis/repair and introns. Some unicellular eukaryotes have a few introns, prokaryotes without introns (self-splicing introns) are doing just fine. I do not think that they are under any "genetic risk". Thus the transcription-coupled mutagenesis/repair is unlikely to be an important factor in evolution of the exon/intron structure.Authors' response: We consider that our hypothesis may provide some insight about the correlation between intron size and gene expression levels. The main question we want to address is the correlation observed between the presence of an intron and the effect it might have on the gene expression level; such a correlation has been found in many genome-wide bioinformatic analyses and transgenic analyses [analyses ,107-111.We also do not know if any connection between transcription-coupled mutagenesis/repair and introns exists. But, in the light of such a hypothesis, we would seek to investigate this further.There are two possible explanations here. The first is the widely held opinion that introns are slightly deleterious, and so the presence/absence of introns depends mainly on the efficiency of natural selection. The second is that introns are abundant in some organisms (like humans) and some genes from intron-rare organisms  because of the distinctiveness of these organisms and these genes. These organisms may be less able to tolerate genetic risk than others. And these genes  may be less tolerant of genetic risks than other genes. In S. cerevisiae, only 3.1% of the nuclear genes contain introns, but the majority (75.8%) of cytoplasmic ribosomal protein genes have introns . In addition, Dr. Rogozin and colleagues have reported that evolutionarily conserved genes tend to have more introns [Certainly, these observations are consistent with our hypothesis, but not proof per se. However, there is, to the best of our knowledge, no convincing evidence to reject the hypothesis that introns are retained in some organisms and some genes because of their intolerance of genetic risk."Some unicellular eukaryotes have a few introns, prokaryotes without introns (self-splicing introns) are doing just fine." ata from , accesse introns . CertainThe authors suggested two ways to test the hypothesis:"If our hypothesis is correct, splicing inhibits topoisomerase I DNA cleavage activity, thus reducing the frequency of transcription-associated short DNA deletions. That is, transcription-associated mutagenesis would be much lower in intron-containing than intronless genes. Experimental approaches that block intron recognition and splicing would strengthen the validity of transcription-associated mutagenesis.Because topoisomerase-I-associated damage causes mainly short DNA deletions ,73, thisHowever, the authors did not try to find any support for the hypothesis. I think that if the authors did not do the suggested analyses by themselves, nobody is going to do it.Authors' response: We thank Dr. Rogozin for reminding us of this. We did not want to write a research article with a very long introduction, but preferred to formulate a hypothesis and then (after some studies) write a concise research article on the subject.I suggest to readers of this paper to consider it as a review paper rather than a hypothesis paper.Authors' response: We do not completely disagree with this suggestion. In fact, we ourselves have often derived more benefit from reading the background and introduction sections than the hypothesis section of some hypothesis papers.Dr Alexey S Kondrashov, Department of Ecology and Evolutionary Biology, The University of Michigan, USAThis reviewer provided no comments for publication.


AbstractLabeninae, Apechoneura seminigrasp. n., is described. Specimens were collected from the Amazon Rainforest of Colombia.A new species of the ichneumonid subfamily  Labeninae is a subfamily of Ichneumonidae containing approximately 150 described species classified in four tribes and 12 genera. Compared with other subfamilies, this group is quite well-known worldwide , Apechoneura was considered part of Certonotus , but after a phylogenetic study  , may also be present in Colombia because it occurs in Costa Rica  . AnotherLabeninae, 14 of the main entomological collections of Colombia were reviewed (view Appendix). The specimens described here are deposited in the insect collection of the Instituto de Ciencias Naturales (ICN), Universidad Nacional de Colombia, Bogotá, Colombia. The nomenclatural treatment, morphological terminology and taxonomic characters used here follow PageBreakDuring an undergraduate project focused on the subfamily Herrerasp. n.urn:lsid:zoobank.org:act:4CFB6077-71E1-42D0-92FB-77907767B3CChttp://species-id.net/wiki/Apechoneura_seminigra3°41'N, 70°15'W, Malaise trap, Martin Kelsey: 200 m, II-III.1989 (ICN 083474). PARATYPES: 1 female, same data as holotype (ICN 083472); 1 female, same locality, 300 m, 1.III.1988, bosque de tierra firme (ICN 083471).HOLOTYPE: Female, Colombia, Amazonas: Parque Nacional Natural Amacayacu Caño Mata Matá, Non-type material: 1 male, same locality, bosque de várzea (ICN 083473).Apechoneura by the combination of the following: head orange; mesosoma and legs mostly orange (hind leg partly black); metasoma black. Epicnemial carina absent. Metapleuron with a conspicuous sharp lateral denticle. Hind wing with first abscissa of Cu1 0.2× as long as cu-a.This species can be diagnosed from all other Neotropical Female. Fore wing length 15.0 mm.Head. Clypeus almost flat, with a weak transverse ridge near apex; malar space 0.6× as long as basal mandibular width; lower face at narrowest point 0.9× as wide as height from clypeofacial suture to level of insertion of antenna; hypostomal carina joined to occipital carina far from base of mandible; posterior ocellus separated from eye by 1.3–1.5× its own maximum diameter. Antenna with flagellomeres 1 and 2 subequal by length; subapical flagellomere slightly elongate.Mesosoma. Pronotum with upper hind margin swollen, forming a small conical projection; scutoscutellar groove broad and shallow; scutellum with three evident rugae posteriorly; epicnemial carina absent . Hind wing with apical abscissa of Cu1 arising from M apical to junction of M + Cu1 with cu-a .Apechoneura seminigra sp. n., just like Apechoneura nigricornis, lacks an epicnemial carina; this characteristic separates them from the rest of the species of the genus. As Apechoneura nigricornis, Apechoneura seminigra sp. n. possesses a conical projection on the metapleuron and lacks an indentation on tergite 7. However, the metasoma is orange in Apechoneura nigricornis and black in Apechoneura seminigra sp. n. Also, in the hind wing, the first abscissa of Cu1 is 0.4× as long as cu-a in Apechoneura nigricornis and 0.2 in Apechoneura seminigra sp. n. Although these two species are rather similar morphologically, the difference in color PageBreakpattern makes in this case their separation reliable. Apechoneura nigricornis from Costa Rica but also some material from Brazil and Peru, and Apechoneura nigricornis collected in Porce  in 1998 and no color variation compared to the holotype of this species was found.PageBreakApechoneura seminigra sp. n. is so far only known from Colombia, Amazonian Region, northwest of Leticia. According to Apechoneura and the sex association is often difficult, in part because the male specimens are less frequently collected. Despite the differences between the female specimens and the male specimen of Apechoneura seminigra sp. n., especially in the hind wing venation, they are tentatively considered here as belonging to the same species, mainly because all the specimens were collected in the same exact locality, in two consecutive years.


Moreover, the public health and/or academic institution databases constructed with responses to different questionnaires are usually difficult to merge, impairing necessary collaborations. We aimed to show that e-commerce concepts and software tools can be readily adapted to enable rapid collection of data after an infectious disease outbreak or environmental disaster. Here, the ‘customers’ are the epidemiologists, who fill their shopping ‘baskets’ with standardised questions.Should an emerging infectious disease outbreak or an environmental disaster occur, the collection of epidemiological data must start as soon as possible after the event's onset. Questionnaires are usually built For each epidemiological field, a catalogue of questions is constituted by identifying the relevant variables based on a review of the published literature on similar circumstances. Each question is tagged with information on its source papers. Epidemiologists can then tailor their own questionnaires by choosing appropriate questions from this catalogue. The software immediately provides them with ready-to-use forms and online questionnaires. All databases constituted by the different EpiBasket users are interoperable, because the corresponding questionnaires are derived from the same corpus of questions.A proof-of-concept prototype was developed for Knowledge, Attitudes and Practice (KAP) surveys, which is one of the fields of the epidemiological investigation frequently explored during, or after, an outbreak or environmental disaster. The catalogue of questions was initiated from a review of the KAP studies conducted during or after the 2003 severe acute respiratory syndrome epidemic.Rapid collection of standardised data after an outbreak or environmental disaster can be facilitated by transposing the e-commerce paradigm to epidemiology, taking advantage of the powerful software tools already available. When an outbreak of an emergent disease or an environmental disaster occurs, public health officials and researchers are expected to set up in real time the best possible epidemiological investigations to understand what happens, what the health consequences of the event are and how to mitigate them as quickly as possible .The three conditions for this real-time epidemiological intelligence are real-time data collection, real-time methods of data analysis and real-time dissemination of results. The main obstacle of real-time epidemiology, addressed herein, is the first step in the process: data collection. Indeed, survey design and the elaboration of the corresponding questionnaires take time , 3: unsaThe best solution to overcome the above-outlined difficulties would be that a respected international or national agency, or a scientific society, provides THE questionnaire that everybody would agree to use in the case of an epidemiological disaster. That ‘top-down’ best solution approach is obviously unrealistic. One of the reasons is that epidemiological investigation is a research activity. Researchers like to design their own tools, and good quality research needs a diversity of approaches.Herein, we describe software that achieves a ‘bottom-up’ approach aimed at accelerating and standardising data collection, while allowing epidemiologists to tailor their own questionnaires. The basic concept underlying the software was to revisit with an epidemiologist's eye the consumer's e-commerce model used daily. Indeed, not so long ago, the process of getting same-day delivery to your door of the best product, after an informed choice about all those available, was unimaginable. Today, when a customer wants to buy, for example, a new home-entertainment audiovisual system, the internet quasi-instantaneously provides all the necessary information on all possible TVs, DVD players, hard disks, speakers, and so on from a variety of manufacturers. He can choose his desired elements from each of these categories, put them in a shopping basket, and review and modify his choices before making his final decision. Then, he checks out and his system can be delivered in hours. The success of e-commerce led to the development of powerful software, some of it free, that we used in this project.We show herein that this e-commerce model and its software can be readily adapted to epidemiology. In the proposed EpiBasket concept, epidemiologists are the ‘customers’. The ‘categories of products’ are the categories of questionnaire questions . Information on which questions were used in the best high-profile papers published on similar events, information on all ‘products’ , will be available immediately. The epidemiologist devising his questionnaire will therefore be able to select from the ‘catalogue’ the best possible questions to meet his needs, put them in his ‘shopping basket’ and, once he has made up his mind, he ‘buys’ them and immediately gets his order, here the paper and/or the online version of the questionnaire. If the catalogue provides the best products on the ‘epidemiological market’, and if a community of researchers starts to collectively build and use this system, a positive byproduct should be the increased possibility of database interoperability because, in the future, epidemiologists working on the same subject will choose the questions for their questionnaires from the same catalogue.http://www.epibasket.orgIn this article, we describe the general outline of the ‘EpiBasket’ concept in three sections: the construction of the catalogue of questions by the developer, the user's point of view and the software that was used. The proposed concept is illustrated with a prototype based on the Knowledge, Attitudes, Practices (KAP) studies conducted on the 2003 severe acute respiratory syndrome (SARS) epidemic . This prThe methodology used to stock the catalogue with questions and to tag each question with information of potential usefulness for the users is described in this section. The user's point of view and the software used are addressed in Results section.The final goal is to provide users with a ‘catalogue’ of the ‘best’ questions needed to explore a given epidemiological issue . A basic assumption underlying the EpiBasket project is that most of the essential questions that are needed today were posed in the past, and that those providing the results published in the best scientific journals should certainly be considered as the best possible candidate questions. Three steps were defined to stock the catalogue with questions.The first was to identify the ‘variables of interest’ that must be acquired to investigate a given issue. This identification relied on a systematic review of the pertinent papers published in the domain. To facilitate the extraction of those variables, we supposed that they were mentioned in the tables and figures of the papers analysed in the systematic review. The manual extraction of those variables that we performed to build the prototype could subsequently be automated, with natural language-analysis tools  ‘readingIn a perfect world, the questionnaires used in each reviewed study would be made available to readers. In the real world, very few authors provide their questionnaires, hence the ‘question’ had to be inferred from the ‘variable’. Therefore, the second stage was to deduce the ‘question’ from the ‘variable’ identified in the previous step. For example, the questionnaire may have asked the date of birth, while the paper may report the corresponding results in a table showing age-class frequencies. In addition, EpiBasket can propose a recommendation for a preferred coding, with the aim of maximising the chances of interoperability among databases constituted with EpiBasket-derived questionnaires and their responses.Third, the formulation of questions was generalised, when possible, to make them reusable in broader situations than those in the source papers. It was accomplished by replacing words or patterns found in the reviewed papers by ‘root terms’ that the user would replace with the specific term that fits the event of interest. For example, the question ‘In the case of a SARS outbreak, would you avoid going to the cinema?’ provides a template for asking questions regarding avoidance behaviours during an outbreak. Now, using ‘root terms’, it is formatted as ‘In the case of #$DISEASE outbreak, would you avoid going to #$PLACE?’, where #$DISEASE and #$PLACE are the root terms to be instantiated by the user selecting this question template, for example, ‘In the case of a cholera outbreak, would you avoid going to a restaurant?’ So far, six root terms have been implemented in the current EpiBasket prototype: ‘#$DISEASE’, ‘#$DRUG’, ‘#$NUMBER’, ‘#$PERIOD’, ‘#$PLACE’ and ‘#$SOMEONE’. For example, the specific questions ‘Did you wear a mask in a public place?’, ‘Did you wear a mask at work?’ and ‘Did you wear a mask during a flight?’ were reframed as a single generic formulation, ‘Did you wear a mask #$PLACE?’.In addition to the questions extracted from the literature, a ‘User suggestions’ category was added to store user-proposed questions.Each question in the catalogue has tags aimed at helping the user construct his questionnaire.Categories to which the question belongs, time of its use , type(s) of population(s) studied , MeSH description of the term(s) used in the question.List of the published papers analysed that used the question .Each question is also tagged with a ‘value’, the equivalent of a price in e-commerce. The underlying assumption is that, when the user will have to choose among several questions , the one with the best ‘value’ will be retained. Since such a logical view is likely to be followed by most EpiBasket users, EpiBasket should facilitate better standardisation of questionnaires. At present, we arbitrarily decided to give to a question the value defined as the ‘Best IF’, that is, the highest journal IF encountered among all papers using it. This choice assumes that, if the user has to choose between two burnout scales, the one previously published in a high-profile journal will be preferred. As a consequence of this valuing system, all demographic variables  get a high value . IF can be systematically obtained from the Journal Citation Reports database, for which a fee must be paid. However, most journals provide their IF on their websites and we used those values in the prototype (see the Supplementary file). The value of 0.000 was arbitrarily assigned to questions published in journals not indexed in the Journal Citation Reports database. Researchers not wanting to take these IF into account can just ignore the ‘values’ indicated when they build their questionnaire.Each question was associated with at least one keyword. These keywords are given on the EpiBasket homepage as a tag cloud . Whenever the user clicks on a given cloud term, all questions associated with it are listed.Each paper from which the questions were extracted concerns a study conducted at a given time, either during the outbreak or after it, and a given population type. This information is available to the user to help him to identify the relevant questions for his own study.Each EpiBasket question provides the list of the other questions jointly asked in the papers that used it. EpiBasket also provides the list of questions in the catalogue that share at least one of the keywords associated with question.names of the other questions>’.To maximise the likelihood of compatibility among questionnaires, we paralleled the e-commerce approach, according to which shoppers know when they buy, for example, a book, what other books other customers also bought. Here, we implemented the function ‘users who selected this question also selected … <http://www.epibasket.org.Composing a questionnaire is viewed, from the user's point of view, as ‘shopping for’ questions, that is, filling a ‘basket’ with the questions he wants to eventually have in the questionnaire. However, while the order in which the items composing the shopping basket were added does not matter in a usual consumer's purchase, here the questions composing a questionnaire must be appropriately ordered. The EpiBasket software helps the user draft a questionnaire and to exchange his draft  with his collaborators until he decides to obtain a ready-to-use paper questionnaire, for a paper-and-pencil survey and/or an electronic file for an online survey. In addition, EpiBasket includes several tools enabling user–user and user–Editorial Committee exchanges to promote collaborative epidemiology. First, the user must select the candidate questions pertaining to the event of interest: questions according to categories of the questions, time of study, study population and so on. For example, the user may want to consult all the questions belonging to the ‘Knowledge’ category, concerning healthcare workers and to be collected during the outbreak, which would require three clicks. Because questions are labelled with keywords and linked to papers in which they were initially mentioned, suggested associations of potential appropriate questions can be easily accessed.The user prepares a ‘wish list’ that will eventually be transformed into a final ‘buy’. A given user may constitute different wish lists, which may be shared with colleagues. These wish lists allow epidemiologists to discuss questionnaire contents upstream before impression of the final product. Finally, the user selects questions in the wish list to fill his ‘shopping basket’, which becomes the final questionnaire. The order in which selected questions are added to the basket will be the order of the questions in the final document.http://www.epibasket.org/limesurvey/admin/index.html). The user can also install on his own server LimeSurvey  constituting the generated questionnaire in any popular relational database-management system. The pdf format is available for epidemiologists wanting to conduct a paper-and-pencil survey. The csv format is proposed to those planning an online survey: users can directly import their csv file and initiate the corresponding survey online in EpiBasket  required substantial modifications/suppressions/additions. For example, in EpiBasket, the ‘orders’ attached to a given user are ‘my questionnaires’, that is, those ordered since his initial registration in the EpiBasket site. We also developed a specific source code to allow a user account to propose the implementation of a new question in the EpiBasket catalogue. All these adaptations required numerous and substantial code-source changes of the original e-commerce software.Reposition module to allow a drop-down presentation of question categories and subcategories; Filter search community edition to filter questions according to our desired criteria; Product bought by other people to enable users to examine which questions were associated in questionnaires in previous questionnaires built by EpiBasket users; and cy-related products which provides a list of those questions sharing keyword similarities with the question currently displayed.A second group of modifications concerns the relational associations between tags and questions, and between related questions to facilitate the user's navigation interface to explore questions. To do so, the following PrestaShop modules were implemented: quexml tool (quexs tool that was not considered in the current EpiBasket prototype.A third group of specific developments concerned the EpiBasket questionnaire outputs. The open-source xml tool  was implxml tool  is an opxml tool . LimeSurHerein, we described a prototype that, by adapting WEB tools and e-commerce concepts to epidemiology, should accelerate the creation of an epidemiological questionnaire at the time of a public health emergency. The strength of our approach is that it relies on and takes full advantage of past knowledge and experiences in similar events. The goal is not only avoiding that epidemiologists ‘reinvent the wheel’ when they are faced with situations similar, or partly similar, to those the past. It also strives to indirectly encourage more standardised and better quality questionnaires. Another indirect attribute of the proposed approach is to promote interactions among epidemiologists. Indeed, despite informatics advances, which make it easy, at least in theory, to devise databases easily sharable and accessible from any place, the fact is that, in practice, this is not yet reality . The maiIn this prototype, we chose to exemplify the EpiBasket concept with KAP studies after an outbreak because they are quite similar to those studies done after an environmental disaster. They represent a small part of the epidemiological investigations done during/after an outbreak and a full-size EpiBasket should provide questions for all fields concerned. After the 2003 SARS epidemic, we identified  at totalFinally, because a major objective of EpiBasket is to support collaborative epidemiology, additional tools could be added to the users’ community at very little informatics cost: for example, a registry of epidemiological investigations on outbreaks or public health disasters, where epidemiologists could register their new studies before publication with a brief description, authors’ names, affiliations and addresses. The difference with the current database on clinical trials  would beThe key message of the EpiBasket prototype is that the informatics tools that are used daily and successfully by lay people, like e-commerce and WEB 2.0 applications, have great potential to enhance the quality and timeliness of epidemiological studies, especially in the context of investigation of outbreaks or other public health events requiring a real-time, quality approach. We also think that our proposed approach can help the desired trend towards more transparency, more data sharing and more collaborations among epidemiologists.


Understanding the roles of disorder and metal/graphene interface on the electronic and transport properties of graphene-based systems is crucial for a consistent analysis of the data deriving from experimental measurements. The present work is devoted to the detailed study of graphene nanoribbon systems by means of self-consistent quantum transport calculations. The computational formalism is based on a coupled Schrödinger/Poisson approach that respects both chemistry and electrostatics, applied to pure/defected graphene nanoribbons . We theoretically characterize the formation of metal-graphene junctions as well as the effects of backscattering due to the presence of vacancies and impurities. Our results evidence that disorder can infer significant alterations on the conduction process, giving rise to mobility gaps in the conductance distribution. Moreover, we show the importance of metal-graphene coupling that gives rise to doping-related phenomena and a degradation of conductance quantization characteristics. Graphene nanoribbons (GNRs) are the most promising graphene-based nanostructures for electronic applications since they are potentially suited for band-gap engineering, maintaining the excellent electronic properties of the parent two-dimensional graphene layer. GNRs have been already synthesized by means of different pattering techniques ,2, and tElectrical characterization studies of GNRs must preliminarily consider the impact of disorder in their electrical properties, which is crucial also due the possible Anderson localization phenomena in these quasi-1D systems . MoreoveNa along the ribbon width . We use the non-equilibrium Green function (NEGF)We consider pure and defected hydrogen-terminated armchair GNRs (AGNR) of different widths and lengths (from few nanometers to approximately a micrometer) with two different contact configurations: (a) ideally contacted (same width as the conductor without defect or impurity inclusions) at the right side and the left side, and (b) end-contacted by 3D semi-infinite metallic electrodes  surfaces) at the left side whereas ideally contacted at the right side. The terminology of Ref.  is appliH0 and overlap matrices τ are written within first-principles parameterized models [and the standard Landauer-Buttiker approach for the calculation of quantum transport . Hamiltod models .gs for the evaluation of the respective self-energies Σ = τ gsτ† are calculated for the 3D semi-infinite contact with a back and forth real to k-space Fourier transform exploiting lattice periodicity [ρf deriving from the NEGF are passed to a 3D numerical Poisson solver from where the self-consistent potentials are calculated by solving ∇2USC = -ρf/ε, where H = H0 + USC in Equation 1. Electrical potential has been fixed at the metal-GNR interface (applying a Dirichelet type boundary condition) at a value Uleft SC = φm - φgr, where φm and φgr are the experimentally measured work functions for (111) metallic surfaces and graphene [T = 300 K.Metal surface Green functions iodicity . Mobile graphene , while zE) than conductance g(E) of quasi-1D GNRs. In particular n-type (p-type) impurities introduce electronic states  at enere states . The cone states . HoweverNa = 45 AGNR doped with a 0.2% density of nitrogen atoms and for systems with increasing length L, from approximately 0.1 μm to approximately 0.8 μm. An asymmetric decrease of the average conductance due to the impurity scattering can be observed for the whole spectrum. This behavior is particularly important in the energy region near the single impurity resonance states  where a mobility gap appears also for the smaller systems. We note that the pure Na = 45 AGNR is a semiconductor GNR with an energy gap of approximately 0.2 eV.In a real defected system, we expect a finite density of random distributed scattering centers and, as a consequence, the effect of multiple scattering processes should be evaluated in this kind of configuration. In Figure Na = 47 AGNR  for the same cases of Figure  calculations have shown a non-conventional scenario for the charging and the related electrostatics of the heterojunction between the 3D metal and the 1D GNRs . In part(a) The electrostatic potential, due to the work function difference, drops in a few-nanometer region near the interface and has non-zero flat value, denoting the presence of carrier accumulation throughout the GNR length;(b) Unlike the standard metal semiconductor interface, band bending is not rigid for both conduction and valence bands as a result of quantum interference  and electron confinement. For example, in the case of a metal with high (low) work function with respect to the graphene, like Au (Al), conduction  band states shift smoothly following the electrostatic potential, while discrete localized states appear with a few-nanometer spatial extension in the region where the valence (conduction) band maximally bends;(c) Quantum interference, localized states, and the metal induced gap states  characterize the LDOS in the junction region.a = N16 AGNR end-contacted with Au are plotted as a function of energy. These spectra show similar characteristics with the ones obtained considering metals with higher work functions than graphene . A Schottky barrier of the order of 0.2 to 0.3 eV can be determined by the difference between the gap in the conductance spectrum for the contacted GNRs at 0 V . Note that finite-bias conductance spectra are affected by the presence of the Schottky barrier. Indeed, while the -0.5-V case shows the alteration with respect to the small-bias case due to non-equilibrium charging (see Ref.  for a coI-V) characteristics of the junction in the case of a pure a = N16 AGNR contacted with different metals are reported in Figure I values obtained for a negative bias in the case of Pd with respect Au are due to its slighter more pronounced p-type character . Al has a lower work function with respect to graphene and the Al-GNR junction shows a quasi-ambipolar Schottky behavior . However, in the latter case, the dominant aspect is the strong scattering by the contacts and the related suppression of the contact transparency.Current-voltage (The results of this theoretical study have shown that defects, doping, and metal/graphene interfaces can non-trivially influence the conduction mechanism of graphene nanostructures, giving rise to mode transmission suppression, conductance asymmetries, mobility gap formations, and strong conductance fluctuations. The implication of these results for the electrical characterization of real structures has been discussed. A general crucial issue, which should also be taken into account when probing transport in GNRs, is that the electrostatics and the chemical bonding aspects can act complementary for the determination of transparency in graphene-based nanostructures.The authors declare that they have no competing interests.ab-inito based calculation presented in this manuscript. AL and ID developed the formalism used for conduction evaluation.AL carried out the conduction calculation and the statistical analysis for the defective system. ID carried out all the


Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded ‘introns first’ held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes. This article was reviewed by I. King Jordan, Manuel Irimia (nominated by Anthony Poole), Tobias Mourier (nominated by Anthony Poole), and Fyodor Kondrashov. For the complete reports, see the Reviewers’ Reports section. In a memorable phrase of Walter Gilbert, eukaryotes possess “genes in pieces” in which protein-coding sequences are interrupted by non-coding sequences denoted introns. The intGU(A/G)AGU sequence  at the donor splice signal is complementary to the 5’ end of the U1 snRNA, and this interaction appears to be the major requirement for splicing, h, h132], A. thaliana share ~25% intron positions. Th. Th53]. The reconstructions of the evolution of gene architecture in eukaryotes were performed on sets of orthologous genes with a single representative (or a single most conserved representative) in each of the analyzed genomes. Obviously, this type of reconstruction reflects only one facet of evolution of gene structure given that all eukaryotic genomes encompass numerous families of paralogous genes with broad distributions of the number of members. Reconstruction of parsimonious scenarios of gene structure evolution in paralogous gene families in animals, plants and malaria parasites revealed numerous apparent gains and losses of introns,142. In In contrast, comparison of the exon–intron structures of ancient eukaryotic paralogs reveals the absence of conserved intron positions in these genes Figure148]. T. T148]. The combined advances of comparative genomics and systems biology provide means to characterize genes by many features, for example expression level and connectivity in protein-protein interaction or regulatory networks. Various significant correlations have been demonstrated to exist between these variables; in particular, one of the most prominent, recurrent links is that the sequence of highly expressed genes tends, on average, to be more conserved-154. ConProbabilistic evolutionary reconstruction of gene structure yields gene-specific rates of intron gain and loss and thus provides for analysis of possible relationships between these rates and other characteristics of the respective genes. It has Drosophila have, on average, shorter introns than genes expressed at a lower level. St. St46]. It appears most likely that the Group II intron invasion was triggered by the establishment of the endosymbiosis between an α-proteobacterium and an archaeal host Figure. NotablyIndeed, it has to be emphasized that Group II introns are typical mobile elements that actively spread around the host genome when given a chance by weakness of purifying selection pressure.However, at the mechanistic level, the adaptation of the early eukaryotes to the swarms of genomic parasites , whereas the forms that never achieved high efficiency of purifying selection  followed the path to complexity.The elucidation of the general scenario of evolution of eukaryote gene architecture by no account implies that the main problems in the study of intron evolution and function have been solved. Quite the contrary, fundamental questions remains wide open.What are the mechanisms of intron loss and gain? There is very little direct evidence of any. A consensus seems to exist regarding the role of reverse transcription in intron loss although even this mechanism badly needs experimental corroboration. As for the mechanisms of intron gain, indications of the involvement of double-strand break repair notwithstanding, the study of this key problem has not even started in earnest.What are the sources of new introns? It is clear that duplication of pre-existing introns is a negligible route of intron evolution in many eukaryotic lineages although it seems to be important in some, whereas intronization of (parts of) exons appears to be a significant contribution throughout the evolution of eukaryotes. However, possible other routes of intron acquisition let alone their relative quantitative contributions remain unknown.What is the general role of introns in gene expression and function (if any) and why is it the case that new genes (such as those acquired from chloroplasts in plants) are saturated by introns at an apparent high rate? And a related question: why do a handful of introns  survive in the great majority of even the most streamlined eukaryotes? At best, only most general and largely speculative answers to these key questions are currently available. These are hard questions, and the only hope to obtain satisfactory answers is to combine comprehensive phylogenomic analysis with population genetic models and extensive experimentation.The authors declare that they have no competing interests.IBR, LC, MC and EVK wrote the manuscript which was read, edited, and approved by all authors.Reviewer #1: Dr. I. King Jordan, Georgia Institute of TechnologyIgor Rogozin and colleagues have written a comprehensive, synthetic and compelling review mainly covering the ‘evolution’ of spliceosomal introns. As in Darwin’s famous tome, the notion of ‘origin’ is actually given short shrift, but I will come to that point later. In any case, this ambitious work benefits from the broad perspective that the authors have gained over years of investigating the subject. Along with this perspective comes some inevitable bias, or perhaps it is more fair to say a favored world-view, as described below with respect to the authors position on ‘introns-kind-of-early’. But this does not represent a liability of the work in my opinion; the authors are clearly entitled to their views, and the conclusions they draw appear to be both nuanced and well-supported by the data. They cover a lot of ground herein and strike a nice balance between thoroughly reviewing the relevant literature and elucidating the most salient points from the large body of work on the subject. One of the major conclusions of this review relates to a resolution, or compromise really, of the ‘introns-early’ versus ‘introns-late’ debate that consumed the field for many years. The authors champion a merger of these two hypotheses into the ‘many introns early in eukaryotic evolution’ view, whereby the earliest eukaryotic lineages contained genomes that were already loaded with many introns and subsequent evolution was dominated by intron loss.The parts of the review that cover the origin of spliceosomal introns are the most speculative and least supported. This is not a critique per se; it may simply be the case that the study of origins must always be more speculative than the study of evolution. According to the ‘many introns early in eukaryotic evolution’ hypothesis, the earliest eukaryotic genomes were formed via massive intron invasion that resulted in genomes consisting of up to 80% intronic DNA. Crucially, the authors hold that this invasion was probably facilitated by low effective population sizes and the corollary weak purifying selection, following the influential Michael Lynch model for the non-adaptive evolution of eukaryotic genome complexity. This model accounts for population level dynamics but neglects the internal dynamics of the genome. If spliceosomal introns indeed evolved from Group II introns, as the authors maintain, then the initial intron invasion of eukaryotic genomes would have been driven, to some extent, by a kind of selfish genetic element with its own internal drive mechanism to replicate within the genome. In theory, such selfish replicators can efficiently increase in copy number even in the face of a selective cost to the host. Therefore, the early origin of introns may be attributed to an active internally driven process, rather than a solely passive drift related process, i.e. a mechanism akin to the molecular-drive concept of Gabriel Dover or the mutation bias emphasized by Arlin Stoltzfus. Such an active replicative process inside the genome could have actually outpaced selection’s ability to contain it. The authors actually touch on this notion, when they speculate as to whether introns are genomic parasites and how the host may have evolved the spliceosome as an adaptive response to intron invasion, but an explicit connection between their selfish drive to replicate and the origin of introns is not made.We agree on all accounts. Yes, it comes with the territory: discussion of origins is inevitably more speculative than the analysis of subsequent evolution. More importantly, the role of the active mobility of Group II introns certainly must not be under-appreciated, and we explicitly point this out in the revised manuscript: ‘Indeed, it has to be emphasized that Group II introns are typical mobile elements that actively spread around the host genome when given a chance by weakness of purifying selection pressure.’Authors’ response: One specific suggestion as to how the work can be improved relates to the abstract. Currently, the abstract is very short and concise, whereas the manuscript is rather long and presents a lot of material. I think it would be helpful to provide a more detailed abstract that specifically enumerates the authors’ most important points, something more like of a summary of the last two sections of the manuscript.Authors’ response: We fully agree, the original short abstract resulted from a misunderstanding regarding the limits on abstract length in review articles. In the revised article, the abstract was substantially expanded.Reviewer #1: Dr. I. King Jordan, Georgia Institute of Technology I have re-reviewed the manuscript of Rogozin et al. I am satisfied with the changes made, for the most part, and I recommend that the paper be accepted for publication in Biology Direct after the following point is addressed.I would like the authors to elaborate just a bit on their response the first comment that I made, in particular with respect to the connection between Group II intron dynamics and the evolution (emergence) of introns. I think I may have rambled a bit in my original comment and was not explicit enough. I would urge the authors to have a look at the manuscript of Donal Hickey from Genetics , which makes the point much better than I did in my comment. The population genetics models in the manuscript may be a bit simplistic by this time, but I think the ideas contained therein are highly relevant to their own work. In particular, Hickey makes an explicit connection between the genome dynamics of mobile elements, host selection pressure and the evolution of introns. The basic idea is that mobile genetic elements can spread in a population even in the face of a fitness cost to the host, and this kind of process could have resulted in the emergence and spread of introns. Below, I provide a comment in response to the authors' response to my first comment in an attempt to facilitate further discussion and consideration of this issue.Authors' response: We agree on all accounts. Yes, it comes with the territory: discussion of origins is inevitably more speculative that analysis of subsequent evolution. More importantly, the role of the active mobility of Group II introns certainly must not be under-appreciated, and we explicitly note in the revised manuscript: 'Indeed, it has to be emphasized that Group II introns are typical mobile elements that actively spread around the host genome when given a chance by weakness of purifying selection pressure.'Response: I would like the authors to further consider the possibility that mobile elements (such as Group II introns) can increase in frequency in a population, even when they impose a fitness cost on their host organisms, owing to the fact that a replicative transposition process results in a biased transmission rate relative to host genes. This idea was introduced by Donal Hickey 30 years ago, and he also connected this point to the evolution of introns (Hickey 1982 Genetics 101: 519). In other words, it is not simply a matter of weak purifying selection allowing active spread of the elements, but an effect of the element mutational dynamics introducing directional bias in the evolutionary process. This idea is very much analogous to the notion that mutation bias in the broader sense can be a cause of direction in evolution (e.g. see Yampolsky and Stoltzfus 2001 Evol Dev 3: 73).We agree that mutational dynamics of selifhs element could be an important driver of their spread. We think that once this additional exchange with the reviewer is published, the emphasis on this issue will be adequate.Authors' response: Reviewer #2. Dr. Tobias Mourier, University of Copenhagen (nominated by Dr Anthony Poole)This review provides a comprehensive overview of the current knowledge of intron evolution in eukaryotic genomes.The advent of numerous eukaryotic genomic sequences has consistently supported the 'many introns early in eukaryotic evolution' concept, as evident from the manuscript. But surely this hypothesis is not a merger from the introns early/late/first ideas . All recording of spliceosomal intron features comes from eukaryotic genomes, and regardless of how many eukaryotic genomes are sequenced, extant spliceosomal intron features will never allow one to synthesize past LECA.In the end of the manuscript, the authors present a scenario proclaiming that an intron-rich LECA is not inconsistent with the introns-late hypothesis. This is not a problem, but the structure of the manuscript may give the impression that this is a conclusion (or synthesis) directly from the current knowledge of eukaryotic gene architecture (that is nicely reviewed in the preceding text).Authors’ response: Actually, we do believe that the synthesis we present in the section of the review preceding the Conclusions follows from the comparative genomic results reviewed in the preceding sections. Certainly, not all parts of the article directly contribute to this synthesis: for instance, the discussion of the functional roles of introns is only tangentially relevant here albeit important in other respects. Nevertheless, we do maintain that in this section we present major implications of the comparative genomic study of eukaryotic gene structure.The review presents an overview of the comparative approaches taken to delineate intron-exon structures during evolution. The basis for such comparative analyses is well-aligned sequences around splice sites. If intron-exon structures to some extent evolve via mechanisms such as alternative splicing and intronization of exonic sequence, should this not result in sequences that are unlikely to meet the criteria for being included in the above analyses? I think it would be relevant to discuss the implications of this.Authors’ response: This issue is discussed in the section ’Evolutionary conservation of intron positions and routes of gene architecture evolution of eukaryotes’.Section "Functional elements and genes within introns"When discussing intronic RNA genes, I'm surprised there is no mentioning of the classical connection between vertebrate snoRNAs and introns .Authors’ response: Yes, this certainly is an important theme, and we added it to the section ‘Functional elements and genes within introns’.Very minor points:Page 11: "whereas the remaining 6 Xist" rather than "whereas remaining 6 Xist"Page 14: "and so does the strength" rather than "and so does and the strength"Page 18: should "introns are inserts or fixed" read "introns are inserted or fixed"?Authors’ response: All corrected, we appreciate the reviewer’s attention to these points.Reviewer #3. Dr. Manuel Irimia, University of Toronto (nominated by Dr Anthony Poole)Rogozin et al. have put together an impressively comprehensive review on the origin and evolution of splicesomal introns that will certainly become a major reference in the field. Overall, I found it easy and entertaining to read, as well as informative. I have only a few comments and suggestions, often regarding further literature, that I hope can help to improve the piece (listed according to their appearance in the main text):Authors’ response: We appreciate Dr. Irimia’s close attention to the details of this article. As detailed below, we found most of the suggestions fully pertinent and modified the manuscript accordingly.1) P3: The paragraph on splice site consensus sequences could provide a more detailed portrait of canonical intron signals across eukaryotes. For example, not all eukaryotes have polyT tracts between the branch point (BP) and the 3’ AG, and some fungal species even have polyT tracts upstream the BP . Also, some extremely intron-poor species intriguingly have strict GTATGT as consensus 5’sequence (including yeast), which may be worth pointing out. Finally, the 3’ consensus is closer to YAG than to CAG, at least in most species.Authors’ response: We added discussion of this issue to the revised text.2) P5: I found the (exciting) discussion on the ancestrality of U2 vs. U12 too short and a bit imbalanced. Personally, I think it is a good idea that the authors give their authoritative opinion/preference on this kind of discussions, but the opposite arguments should also be presented extensively. In this case, I think the arguments supporting an ancestral origin of U12  should be fully developed.Authors’ response: In our view, the questionable greater similarity of U12 introns to Group II introns does not immediately imply ancestral status of U12 introns. We added to the text ‘it might be tempting to speculate that the ancestral introns were of the U12 type  but have been subsequently converted to U2 introns.’3) P8: Pleiss et al. (PLoS Biol 2007) may be added supporting a global regulatory function of introns in yeast.Authors’ response: We added discussion of this important work to the section ‘Functions of introns associated with splicing’.4) P9: I missed a more comprehensive and complete review of the literature on the genome-wide dynamics of intron gain and loss in this section. For example, on the general slow paucity of intron gain, I missed references on vertebrates , flies , plants , apicomplexa , Entamoeba , Fungi ). On the opposite side: tunicates , diatoms , mitochondrial transfers . Given the overall level of comprehensiveness and detail of this review and that, as I said above, it is very likely to become a major reference in the field, I think it would be important to cite all relevant references in the main text, in particular from such an important and prolific subtopic.Authors’ response: There is indeed a lot of evidence on specific events in individual lineage. We appreciate their importance but it is hardly possible to discuss ‘everything’ in detail. That said, the revised version of the review cites all the references pointed out by the reviewer.5) P9: when commenting on ref 61, the use of the word “dispute” may give the impression that there is an ongoing controversy or a difference in opinions between the authors, which I guess is really not the case. Ref. 74 showed that most reported gains in ref. 61 were indeed losses by adding more species to the analysis that were not available by the time of the original study. This may not be clear to general readers that have not followed the specialized literature.Authors’ response: We added this explanation to the text.6) P11: the authors may wish to mention here the recent work by Cabili et al. (Genes Dev 2011), which describe >8,000 lincRNA genes, with an average of ~1.9 introns per Kbp and that are extensively alternatively spliced, with 2.3 isoforms per gene.Authors’ response: We added a brief description to the text.7) P14: I was quite surprised to read that the sequences at the 3’ of the intron behave completely different from those at the 5’. Many of the extremely intron-poor species  that show strict 5’ splice site consensus also have very strict BPs, and sometimes even very constrained branch-point-to-AG distances . I guess this apparent contradiction is due to the fact that these species are all missing from the analysis by Iwata and Gotoh , etc.).8) P14: also related to splicing signals, it would be interesting to include a comment on the effect of intron size on splicing signals  FigureAuthors’ response: We included an additional explanation in the legend: “An excess of protosplice sites in phase 0 is noticeable, however the ‘protosplice site’ hypothesis, which posits that introns are randomly inserted into protosplice sites, is unable to fully explain the observed over-representation of phase 0 introns.”10) P26: the authors may want to point out from the beginning that the “two competing hypothesis” they present are not necessarily mutually exclusive.Authors’ response: Added to the text as suggested.11) P28: ref. 158 also concludes that alternative splicing has emerged early in eukaryotic evolution, so it should be cited along with ref. 166 and not with 28.Authors’ response: Modified as suggested.12) P31: more references may be added supporting the low conservation of alternative splicing in mammals . Similarly, many other studies have dealt with the evolution of alternative splicing from the perspective of the splicing signals, not only regarding GC splicing donor sequences  and their polymorphism in human populations .Authors’ response: A brief discussion and references added as suggested.13) P32: perhaps the section “Functions of introns” would fit better before the section on alternative splicing (since the latter is one of those functions).Authors’ response: Alternative splicing is not exactly a function of introns, rather a mechanism of modulation of protein and RNA function. In the functional section we addressed specific functions of intron sequences. This might be debatable but we consider the original order of the sections acceptable.14) P33: the authors may want to add that some spectacular, functional exceptions are known to the general case that splicing occurs before mRNA is exported to the cytoplasm. For example, Buckley et al. (Neuron 2011) describe the case of some transcripts with retained introns, which drive subcellular location of the transcripts to the dendrites due to the presence of a particular transposable element within their sequence.Authors’ response: We appreciate the reviewer bringing our attention to this exiting work. Cited and briefly discussed.15) P34: the catalog of U12 introns by Alioto (Nucleic Acids Res 2007) could be referenced here.Authors’ response: Cited as suggested.16) P35: I think it could be useful to make a clearer distinction between Splice Leader (SL) trans-splicing and trans-splicing between two different genes from the beginning of the paragraph (I found it a bit confusing now). Also, the authors may wish to cite a very elegant analysis searching for trans-splicing in Drosophila using RNAseq on hybrids .Authors’ response: We agree and have included a brief discussion and references as suggested.17) P36: in this subsection I missed a paragraph on the (predictable and predictive) association between long introns and the presence of functional elements. For example, Denoeud et al. (Science 2011) found that the few genes with long introns in Oikopleura are enriched for key developmental regulators, and that those introns likely contain regulatory information. This has also been observed for many other developmental genes across metazoans  and for associated non-developmental genes (“bystander” genes) , with exciting implications for the evolution of genome architecture. Also, supporting the presence of regulatory elements, higher sequence conservation is often found in longer introns .Authors’ response: Brief discussion and references included as suggested.18) P38: the authors may add the report by Curtis and Archibald (Curr Biol 2010) to the list of different sources of spliceosomal introns.Authors’ response: Cited as suggested.Reviewer #4. Dr. Fyodor Kondrashov, Center for Genome Regulation, BarcelonaThis is a straightforward and extensive review of everything that is known about the evolution of introns and then some more. I do not have much to add in addition to what the authors have already said. The only thing that I am left wondering about after reading this review is whether or not the authors think that Group II introns in LECA were involved in the transport of mitochondrial precursor genes into what is now the cytoplasm across the novel intracellular membrane. In light of the previous reviews I would leave it up to the authors to space and moderate the level and format of speculation, even though I believe that the nice synthesis the authors have produced make the review more interesting and useful.Authors’ response: We appreciate this comment. We are not entirely clear about the exact meaning of the reviewer’s idea regarding mitochondrial genes. Is this about transfer of genes from the mitochondrial to the nuclear genome? If so, the possibility of involvement of the reverse transcriptase activity of Group II introns is intriguing but in the absence of specific evidence, one would think the main route was DNA recombination.