Small nucleolar (sno)RNAs are required for posttranscriptional processing and modification of ribosomal, spliceosomal and messenger RNAs. Their presence in both eukaryotes and archaea indicates that snoRNAs are evolutionarily ancient. The location of some snoRNAs within the introns of ribosomal protein genes has been suggested to belie an RNA world origin, with the exons of the earliest protein-coding genes having evolved around snoRNAs after the advent of templated protein synthesis. Alternatively, this intronic location may reflect more recent selection for coexpression of snoRNAs and ribosomal components, ensuring rRNA modification by snoRNAs during ribosome synthesis. To gain insight into the evolutionary origins of this genetic organization, we examined the antiquity of snoRNA families and the stability of their genomic location across 44 eukaryote genomes.We report that dozens of snoRNA families are traceable to the Last Eukaryotic Common Ancestor (LECA), but find only weak similarities between the oldest eukaryotic snoRNAs and archaeal snoRNA-like genes. Moreover, many of these LECA snoRNAs are located within the introns of host genes independently traceable to the LECA. Comparative genomic analyses reveal the intronic location of LECA snoRNAs is not ancestral however, suggesting the pattern we observe is the result of ongoing intragenomic mobility. Analysis of human transcriptome data indicates that the primary requirement for hosting intronic snoRNAs is a broad expression profile. Consistent with ongoing mobility across broadly-expressed genes, we report a case of recent migration of a non-LECA snoRNA from the intron of a ubiquitously expressed non-LECA host gene into the introns of two LECA genes during the evolution of primates.Our analyses show that snoRNAs were a well-established family of RNAs at the time when eukaryotes began to diversify. While many are intronic, this association is not evolutionarily stable across the eukaryote tree; ongoing intragenomic mobility has erased signal of their ancestral gene organization, and neither introns-first nor evolved co-expression adequately explain our results. We therefore present a third model — constrained drift — whereby individual snoRNAs are intragenomically mobile and may occupy any genomic location from which expression satisfies phenotype. O-methylation of ribose on a broad assortment of RNAs, including ribosomal RNA (rRNA) [O-methylation modification sites may predate the diversification of the three domains of life [Small nucleolar RNAs (snoRNAs) constitute a major class of small RNA in eukaryotes. There are two broad types of snoRNA, which differ in structure and function. H/ACA snoRNAs are characterized by a double stem-loop structure, and many members of this class are involved in guiding pseudouridylation of other functional RNAs, most notably rRNA ,2. Most  (rRNA) . While t (rRNA) , includi (rRNA) , the ant (rRNA) -8. Moreoof life ,10. Howeof life ,12, therof life ,14. Alteof life . As partof life ,17.While it is difficult to definitively establish the timing of emergence of snoRNAs, the introns-first model does generate testable predictions. One prediction is that these early RNA-world snoRNAs may still be housed within the introns of the protein-coding genes postulated to have evolved around them. If such an association has been preserved, host genes should be among the oldest genes, traceable to the origin of templated protein synthesis ,17. A stSome snoRNAs have been shown to be intragenomically mobile ,20, withIn order to better understand the evolutionary dynamics of snoRNAs, we sought to address the following questions. 1. Do any snoRNA families trace back to the LECA? 2. Do eukaryotic snoRNAs show evidence of homology with archaeal snoRNA-like sRNAs? 3. Is the intronic location of snoRNAs evolutionarily stable across the eukaryote tree? We report that dozens of snoRNA families can be traced to the LECA. However, none of these LECA snoRNA families show significant levels of similarity with archaeal sno-like RNAs, precluding firm placement of individual snoRNA families in the common ancestor of these two groups. In agreement with previous studies , we find). Of these, 40 of 42 families/clans were C/D box snoRNAs . Using Dollo parsimony, we reconstructed the pattern of snoRNA conservation across the eukaryote tree using a five supergroup phylogeny, with the root of the eukaryote tree between unikonts and bikonts -26. ThisCharacterized snoRNAs are heavily biased towards plants and animals  Figure b so thesRfam clans may potentially provide an additional source of information about snoRNA relationships, and could reflect family expansion through processes such as duplication  or retroArabidopsis. We find support for recent expansions , but no family can be traced to duplication events predating the diversification of eukaryotes. Instead, it appears that the division of clans into families may be artefactual for these three cases, and, in the case of the SNORD61 clan, the clan likely represents the orthologous group . We then examined overlap between predicted LECA modification sites and Rfam families or clans previously determined Figure  to be trArabidopsis. Under the unikont/bikont rooting, Arabidopsis is on the other side of the root from human and yeast, so this gives us an independent means of predicting putative LECA modifications. Table Arabidopsis, yeast and human. Of 60 previously reported sites conserved across these three species [As these target sites are predicted using a computational strategy, we sought to establish whether they gave reasonable correspondence to independently verified target sites and their cognate snoRNAs -36. We tspecies -37, our O-methylation, and two further putative LECA-snoRNAs belong to the H/ACA class (which we did not map against rRNA) - so are automatically excluded from this intersect for this reason. In summary, a stringent, total evidence-based approach , we compared the results of all three methods Figure . The int Figure  identifiO-methylation at an equivalent site to yeast snR70 [Archaea and eukaryotes both carry an equivalent H/ACA and C/D RNA-based guide machinery, with a conserved set of associated proteins ,12. Howet snR70 . Our anat snR70 , and sout snR70 . The aliO-methylation and pseudouridylation sites on rRNA are conserved across all three domains of life [O-methylation and pseudouridylation was protein-enzyme based as in extant bacterial lineages, with the snoRNA-based system evolving in response to a need for more extensive modifications in archaea and eukaryotes [Hemiselmis andersenii nucleomorph [Available data are consistent with snoRNA-based rRNA processing/modification being present in the common ancestor of archaea and eukaryotes ,12. Howeof life , and we of life . Howeveraryotes ,14. It iaryotes ,17 . This yielded 9117/98871 (7.6%) orthologous intron loci that can be traced to LECA  of snoRNA-containing host genes in our study can themselves be placed in the LECA , we combed our dataset for intronic snoRNAs that could be unambiguously attributed to gain. Figure Under the RNA world hypothesis, that genetically-encoded proteins and DNA genomes were preceded by functional RNAs and RNA genomes, it has previously been suggested that many RNA-based processes may directly trace back to this early stage in the evolution of life. Such arguments have largely been made on the basis of comparisons of functional RNAs ,52, thouWe aimed to specifically examine the antiquity of snoRNAs using comparative genomics. Our results confirm that individual snoRNA families can be placed in the Last Eukaryotic Common Ancestor. Using a conservative total-evidence approach, incorporating Rfam family conservation, a blast-based comparative genomics approach to identifying target modification sites, and comparison with experimentally-mapped sites, we report that a minimum of 25 C/D type snoRNAs can be placed in the LECA.Based on a conservative estimate of snoRNAs in LECA, we found three LECA snoRNAs for which a positionally-conserved modification was present in archaea. While the archaeal counterpart was known for all these cases, we deemed the sequence and secondary structure similarities across domains to be insufficient for an inference of homology to be made. While the equivalence of the modification-sites is striking, our analysis was unable to provide stronger evidence in favour of a common origin for individual RNA families. It may simply be the case that, given that snoRNAs are short, evolutionary signal, if it did exist, has been erased over such timescales.Finally, we aimed to establish whether the intronic location of snoRNAs is consistent with snoRNA gene mobility, with selection favouring colonisation of introns in genes with broad gene expression, or whether position is evolutionarily conserved. We find that, despite intron position being conserved across the eukaryote tree (suggestive of an intron-rich LECA), none of the snoRNAs residing in these putative LECA introns are positionally stable at this evolutionary depth. A specific prediction of the introns-first model is that snoRNAs are found in the introns of the most deeply-conserved protein-coding genes. Evolutionary signal consistent with this model requires the intron, the snoRNA associated with that intron, and the position of the snoRNA within the intron to all be traceable to the LECA. Our analysis therefore rejects this strict version of the introns-first hypothesis. As per the introns-early vs introns-late debate ,54,55, aWe suggest that our results may instead better fit an alternative evolutionary model which we call 'constrained drift' Figure . In thisOur analysis of expression profiles of human genes bearing intronic snoRNAs Figure  indicateUsing an evolutionary analysis, we have shown that the snoRNA apparatus was well-established in the LECA. While numerous snoRNAs can be traced to the LECA, the intronic location of individual snoRNAs is not stable over large evolutionary timescales, with no evidence for positional conservation traceable to the LECA. The data presented here fit a model of ongoing mobility over shorter evolutionary timescales ,32, withWe built a custom database, based on a local installation of the EnsEMBL PanCompara database . AdditioOur snoRNA dataset derives from release 10 of the Rfam database . ReleaseThe genomic location of snoRNAs was determined in several complementary ways. First, existing annotations were imported from the EnsEMBL database ,64. ThesWe then checked the literature for additional snoRNAs not yet included in Rfam, and added these to our data set  to identify additional snoRNA candidates across all genomes (unless such annotations were readily available from EnsEMBL) and added these to the final dataset. A full list of annotations with genomic coordinates is available from the authors upon request.We also used the (unmodified) Rfam annotation script  snoRNAs  were therefore predicted through a comparative approach that utilised blastn from the Blast package . To ensutabases -36 and ptabases  were addtabases .All putative rRNA-C/DsnoRNA interactions were binned into loci based on their center position (+/− 2 nucleotides). Loci restricted to a single species were discarded. We then reconstructed the ancestral state for all modification sites across species for families that we previously identified as likely LECA-candidates. Only those candidate sites whose phylogenetic distribution was compatible with presence in LECA were considered, resulting in a total of 40 deeply conserved sites. To gauge the accuracy of this approach, we compared predicted, conserved sites with previously reported methylation sites from specialist databases -36 . Second, we manually inspected the primary sequence and checked it for conservation of sequence and structural features using the R-coffee algorithm  and RNAahttp://www.ebi.ac.uk/arrayexpress, accession E-TABM-145; [i x ln(Pi)), where Pi is the proportion of expression in tissue i, and Pi = Ei/T; Ei is the expression of the gene in tissue i and T  is the sum of all expression values for tissues .Expression data from the human transcriptome atlas  were obtBM-145; . To estisnoRNA, small nucleolar RNA; LECA, Last Eukaryotic Common Ancestor.The authors declare that they have no competing interests.Conceived and designed analyses: MPH, AMP. Performed analyses: MPH. Analyzed the data: MPH, AMP. Wrote the paper: MPH, AMP. Both authors read and approved the final manuscript.Supporting Information.Click here for fileTable listing mapped snoRNA interactions across SSU/LSU rRNA alignments.Click here for fileTable listing snoRNA-containing genes traceable to LECA on distribution.Click here for fileTable of RNA-seq expression data for MRPL3, NDC1 and UBA2PL across Amniotes.Click here for file


We report the formation and characterization of graphene dispersions in two organosilanes, 3-glycidoxypropyl trimethoxysilane (GPTMS) and phenyl triethoxysilane (PhTES) as new reactive solvents. The preparation method was mild and easy and does not produce any chemical modification. The dispersions, which exhibit the Tyndall effect, were characterized by TEM and Raman spectroscopy to confirm the presence of few-layer graphene. Concentrations as high as 0.66 and 8.00 mg/ml were found for PhTES and GPTMS, respectively. The latter is one of the highest values reported for a dispersion of graphene obtained by any method. This finding paves the way for the direct synthesis of polymer nanofiller-containing composites consisting of graphene and reactive silanes to be used in sol–gel synthesis, without any need for solvent removal, thus preventing graphene reaggregation to form graphite flakes. Graphene, with its unique physical properties-4, is onN-methyl pyrrolidone[N-vinyl caprolactam[One of the most common methods to obtain graphene by this technique involves the dispersion and exfoliation of graphene oxide. This marolidone, isocyanrolidone, N-vinylrolactam, a commerolactam, and an rolactam, thus, anSi(OR)n4 − , where R is an alkyl, aryl, or organofunctional group, and OR is generally a methoxy or ethoxy group, which can react with the hydroxyl groups and liberate methanol or ethanol. Organosilanes are used in coating applications for surface treatment, as additives in paints, inks, and adhesives, and as reactive intermediates for silicone resin syntheses and organic resin modifications: The alkoxy group can be exploited for the linkage with inorganic substrates, pigments, fillers and hydroxy functionalized polymers. Organosilanes are used as precursors for the synthesis of inorganic–organic hybrid materials[Analogously in the present work, we used reactive organosilane compounds to obtain stable graphene dispersions. Organosilanes are characterized by the basic structure Raterials. In partaterials. Moreoveaterials.In the present work, for the first time PhTES and GPTMS were used for dispersing graphene by direct graphite sonication, without any chemical manipulation. The resulting material was thoroughly characterized by using Raman and UV spectroscopies. Moreover, transmission electron microscopy was used for the statistical analysis concerning the distribution among mono and few-layer graphenes.Phenyl triethoxysilane, 3-glycidoxypropyl trimethoxysilane and graphite flakes (+100 mesh) were purchased from Sigma Aldrich  and used as received without further purification.i.d. 15 mm) and placed in an ultrasonic bath  for 24 h. Then, after they were centrifuged for 30 min at 4,000 rpm, the gray to black liquid phase containing graphene was recovered.Mixtures containing various amounts of graphite flakes and organosilane (5.00 g) were put in a tubular plastic reactor  in order to directly weigh the amount of dispersed graphene and determine the actual graphene concentration.α: from a known volume of initial dispersion, several dilutions were done and the absorbance at a wavelength of 660 nm was measured[α (see Results and discussion) value.The second aliquot of the above dispersion was analyzed by UV analysis with a Hitachi U-2010 spectrometer . The above gravimetric data allowed us to determine the absorption coefficient measured,17,24. ATEM measurements were performed on a JEOL JEM-2100 TEM instrument , with a LaB6 filament, with an operating voltage of 200 kV.For the TEM analysis, the solutions were sonicated for 5 min and then cast directly on the 200 mesh cooper grid; eventually, the solvent was evaporated at ambient conditions for 24 h.Analyses were performed on graphene flakes obtained after vacuum filtration of dispersions on PVDF filters (pore size 0.22 μm), with a Bruker Senterra Raman microscope , using an excitation wavelength of 532 nm at 5 mW. The spectra were acquired by averaging five acquisitions of 5 s with a ×50 objective.The feasibility of using organosilanes as effective liquid dispersing media for graphene was investigated. In particular, PhTES and GPTMS were tested with the aim of preparing graphene dispersions that could be directly utilized for further uses without recovering graphene in the solid state, thus avoiding any possible restacking of it to pristine graphite and compromising any previous successful exfoliation process.As described in ‘Methods’ section, the procedure used was as simple as possible and envisaged the direct sonication of graphite without any chemical manipulation. A first indication of the nanometric dimensions of the dispersed graphene particles was provided by the occurrence of the Tyndall effect. Both GPα and set-up a reliable method for the determination of graphene concentration in the above media, UV and gravimetric analysis were carried out (see ‘Methods’ section). Figure−1·m−1 was respectively found.With the aim of determine the best initial graphite/liquid medium ratio that allows obtaining the highest graphene concentration, a series of dispersions with different amounts of graphite was prepared for each of the two organosilanes. In order to find the absorption coefficient The concentrations of graphene in GPTMS and PhTES as functions of the initial graphite concentration are reported in FiguresTEM was used to investigate the state of the graphene particles dispersed in organosilanes; indeed, this technique is usually employed for the investigation of graphene dispersions,12,14,23A statistical analysis on TEM data was performed in order to verify the exfoliation, thus analyzing carefully the edge of the graphene flakes and measuring the number of layers presented in each flake. At thisThe flakes present good exfoliation degree with an average number of layers of 2.7 for GPTMS and 2.4 for PhTES; standard deviation was about 0.4 and 0.5, respectively. In both cases, only 14% of the flakes were present more than three layers. Moreover, no more than five layers have been counted in very few flakes thus indicating a narrow dispersion. These results confirm that the exfoliation process was very effective.−1, the 2D band at 2,696 cm−1, and the disorder-related D peak at approximately 1,346 cm−1 are evident. Similarly, graphene obtained from GPTMS shows the G band at 1,574 cm−1, the 2D band at 2,701 cm−1, and the disorder-related D peak at approximately 1,345 cm−1. The shape and position of the 2D peaks  but, if especially compared with that of most the reported data,22,26,27The authors declare that they have no competing interests.DN prepared the first dispersions of graphene in organosilanes and also drafted the manuscript. RS and VA performed dispersion analysis. SS developed the dispersion method initially found by DN together with this latter. MP carried out the Raman analysis. LP and JMK performed TEM and statistical analysis. AM coordinated the work. All authors read and approved the final version of the manuscript.


Botryosphaeria dothidea is a widespread and economically important pathogen on various fruit trees, and it often causes die-back and canker on limbs and fruit rot. In characterizing intraspecies genetic variation within this fungus, group I introns, rich in rDNA of fungi, may provide a productive region for exploration. In this research, we analysed complete small subunit (SSU) ribosomal DNA (rDNA) sequences of 37 B. dothidea strains, and found four insertions, designated Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506, at three positions. Sequence analysis and structure prediction revealed that both Bdo.S943 and Bdo.S1506 belonged to subgroup IC1 of group I introns, whereas Bdo.S1199-A and Bdo.S1199-B corresponded to group IE introns. Moreover, Bdo.S1199-A was found to host an open reading frame (ORF) for encoding the homing endonuclease (HE), whereas Bdo.S1199-B, an evolutionary descendant of Bdo.S1199-A, included a degenerate HE. The above four introns were novel, and were the first group I introns observed and characterized in this species. Differential distribution of these introns revealed that all strains could be separated into four genotypes. Genotype III (no intron) and genotype IV (Bdo.S1199-B) were each found in only one strain, whereas genotype I (Bdo.S1199-A) and genotype II (Bdo.S943 and Bdo.S1506) occurred in 95% of the strains. There is a correlation between B. dothidea genotypes and hosts or geographic locations. Thus, these newly discovered group I introns can help to advance understanding of genetic differentiation within B. dothidea. Tetrahymena, are also known as ribozymes Group I introns, a class of RNAs with autocatalytic activity that was initially discovered in Botryosphaeria dothidea (Moug. : Fr.) Ces. & De Not. was designated as the type species by Barr et al. B. dothidea displays high levels of diversity. Moreover, Ma et al. B. dothidea collected from pistachio and other plant hosts in California based on their differences in growth rate, virulence on hosts and fungicide sensitivity. Peng et al. The filamentous ascomycete B. dothideaB. dothidea. To characterize the nature of these SSU insertions, we performed sequence alignments and BLAST analysis, and eventually inferred their secondary structures. Finally, we investigated variable distribution of the insertions within our B. dothidea isolates to assess their roles as indicators for intraspecific genetic diversity.Previous studies have integrated several molecular methods  to determine genetic variation and the population structure of Botryosphaeria dothidea which were studied, 32 originated from various plant hosts throughout China. The remaining five strains, regarded as the out-group, were provided by International Collection of Microorganisms from Plants (ICMP) in New Zealand, Centraalbureau voor Schimmelcultures (CBS) in the Netherlands, and Fruit Tree Research Experiment Station of the Ministry of Agriculture, Forestry and Fisheries (MAFF) in Japan. Detailed information about strains is provided in Of 37 strains of For DNA extraction, mycelium of the tested strains was first transferred from culture plates to 1.5-ml EP tubes and then ground with a tissue homogenizer. Subsequently, an aliquot of macerated mycelium was suspended in 600 µl of 2% CTAB buffer and incubated for at least 30 min at 65°C. Finally DNA was extracted using a phenol-chloroform extraction process and resuspended in 50 µl of 1×TE buffer .5′-GCTTGTCTCAAAGATTAAGCC-3′) and SSU-R (5′-CAAAGCAACAGAGGTAGGTAC-3′) was designed according to the conserved 18S regions of species available in GenBank that were closely related to B. dothidea. PCR was performed in a reaction mixture of 25 µl containing approximately 10–30 ng fungal genomic DNA, 10×Taq buffer with (NH4)2SO4, 1.5 mM MgCl2, 0.2 µM of each dNTP, 5 pmol of each primer, 1 U Taq polymerase and sterile ultrapure water. The PCR conditions consisted of an initial denaturation at 94°C for 2 min, followed by 35 amplification cycles of denaturation at 94°C for 35 s, annealing at 49°C for 30 s, elongating at 72°C for 3 min and then one final step at 72°C for 10 min.In order to obtain the entire SSU rDNA sequence, the primer pair SSU-F (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) were used to amplify the entire internal transcribed spacer (ITS) region ITS1 (PCR products (5 µl) were electrophoresed in 1% agarose gel in the presence of EZVISION ONE, DNA DYE & BUFFER, and visualized under UV light. Acquired unpurified PCR products with clear target bands were sent directly to Sangon Biotech (Shanghai) Co, Ltd. for sequencing.http://blast.ncbi.nlm.nih.gov/Blast.cgi) to preliminarily determine their attributes.To locate intron insertion positions, the obtained sequence data of amplicons were aligned with a reference SSU sequence without insertions by using Clustal X 2.0 software http://mfold.rna.albany.edu/?q=mfold) Mfold Web Server (http://exon.biology.gatech.edu/eukhmm.cgi) http://blast.ncbi.nlm.nih.gov/Blast.cgi). Alignment of amino acid sequences was carried out by Clustal X 2.0 and edited by BioEdit version 5.0.9 Open reading frame (ORF) prediction and translation were performed by the online programme GeneMark.hmm . The 2600-bp fragments revealed 366- and 401-bp insertions at positions 943 and 1506, respectively, except for one with a 742-bp insertion at position 1199 , KC702792 (Bdo.S1199-A), KC702793 (Bdo.S1199-B), KC702794 (Bdo.S1506).Pairwise comparison was made among the four insertions, which divided them into two sets. A homology (70% identity) was revealed between Bdo.S943 and Bdo.S1506, indicating that they belonged to the same class. In contrast, Bdo.S1199-A and Bdo.S1199-B were highly similar in sequence (99% identity), although unlike Bdo.S1199-A, Bdo.S1199-B lacked a 610-nt-long region ranging from 650∼1259 nt and had a triplet of nucleotides GGA inserted at position 1301; in addition, an A/G polymorphism at position 407 and a G/A polymorphism at position 1296 were found  .Scytalidium dimidiatum  N. dimidiatum (>90% identity) of Bdo.S1506 made it difficult to directly define this insertion, the similarity of Bdo.S1506 to Bdo.S943 led us to regard it as a group IC1 intron as well. In addition, we used the entire 1349-bp Bdo.S1199-A for searching in GenBank and obtained four blast hits of high similarity (19–22% query coverage and 82–83% identity), all of which were described as ∼390-bp group IE introns of S. dimidiatum  BLASTN analyses of these insertion sequences were performed in order to identify their nature subsequently. The best match (79% query coverage and 91% identity) obtained for Bdo.S943 corresponded to an entry (GenBank accession number: AY307355) described as the group IC1 intron in Compared and aligned with group IC1 introns described previously Analogously, we constructed the secondary structure of Bdo.S1506 by consulting Bdo.S943 . The fouWe predicted the secondary structure of Bdo.S1199-A by its first domain  in referS. dimidiatum (GenBank ID: AAP73783), with an E-value of 0.014. Amino acid sequence alignment (100 (90X) Cys191 (4X) Cys196 (4X) His201. Furthermore, the motif coordinating the second zinc ion corresponded to the sequence Cys216 (26X) Cys243 X His245 (3X) Cys249. The two conserved zinc-binding motifs characterized by a series of Cys and His residues were named His-Cys box. Moreover, additional conserved residues were regarded as the catalytic sites of the enzyme. Bdo.S1199-A, therefore, proved to be a group IE intron containing a putative His-Cys box HEG, regardless of whether the gene can still be expressed.On the second domain of Bdo.S1199-A, we identified a protein sequence of 259 amino acids which began with an AUG initiator codon at position 478 and ended with a UAA terminal codon at position 1323. BLASTP analysis indicated similarity to the His-Cys box HE of lignment  of the plignment  revealedAccording to the previous result of pairwise alignment, we determined that Bdo.S1199-B presented the same secondary structure as Bdo.S1199-A in spite of the large sequence difference in their so-called protein-coding regions. Deletion of a large fragment in the Bdo.S1199-B made it unlikely that an ORF would occur on its terminal ∼350-nt area. Therefore, see B. dothidea each possessed one or more of the introns Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506  possessed only Bdo.S1199-A, Bdo.S943 and Bdo.S1506 coexisted in the 18S region of 11 B. dothidea strains , and of the remaining two strains (5%), one (genotype III) had no introns and the other (genotype IV) contained Bdo.S1199-B . It was confirmed that C90 and G119 were present in the strains of both genotype I and III, T90 and gap119 were present in the strains of genotype II, and C90 and gap119 were present in the genotype IV strain. This result indicated that the four genotypes of B. dothidea determined by introns can be also partially differentiated by ITS polymorphisms.Two single nucleotide polymorphisms (SNPs) were found by scanning ITS sequences of all the Botryosphaeria species. In our study, however, four types of insertions were found at three different sites of the nuclear SSU rDNA in Botryosphaeria dothidea. In spite of their significant structural discrepancy, all the SSU insertions were identified as group I introns. Distribution of these introns among the 37 B. dothidea strains provided clear evidence of genetic diversity within this species.There have been no previous reports or descriptions of group I introns in Among the four introns, both Bdo.S943 and Bdo.S1506 were found to belong to subgroup IC1 with the typical characteristics of group I intron, namely, size ranging from 250–500 nt and approximately ten paired elements B. dothidea so that they could co-evolve with all the other inherent genes of this species. This is necessary because a unique mechanism of group I introns, called “reverse splicing”, could enable group I introns to spread laterally among unrelated species, which may lead to erroneous interpretation of genetic structure of populations. Our sequence analysis results indicated that all three group I introns had homologs at the corresponding insertion sites in N. dimidiatum, a close relative of B. dothidea within the same family (Botryosphaeriaceae). Consequently, these introns share a common ancestor and they are relatively conserved in evolution, even though we do not yet know their rate of replication or rate of invasion of species or populations. On the other hand, group I introns are considered to be neutral or slightly deleterious to the host’s fitness thanks to the mechanism of self-splicing, which can help them release from pre-mRNAs and make the subsequent translation process proceed normally Before using the above group I introns as markers to evaluate intraspecific genetic diversity, it is necessary to verify that each of the three introns  already existed in the ancestral B. dothidea surveyed, the most reasonable explanation is a cyclical loss and regain of introns among populations over the course of evolution Exophiala dermatitidis and Phialophora verrucosaE. dermatitidis, the invasive genotype A and B strains from the natural environment were differentiated by the distribution of ribosomal introns. In P. verrucosa, 34 strains were classified into five genotypes according to intron distribution, and some correlation between genotypes and geographic location was inferred. For B. dothidea, four genotypes were established according to the patterns of intron distribution. Distinct from the other three types, genotype III was found only in Switzerland, which may have resulted from its evolution in a geographically isolated environment. Compared with genotype I and genotype II with a total prevalence of 95%, the fact that genotype III contains only one strain suggests a non-dominant population in China. The observation that genotype II strains were isolated only from apple and pear whereas genotype I strains had a wider host range (except pear) implies that genotype I may represent a dominant population. However, the hypothesized relationship between intron distribution and plant host or geographic origin requires testing with more data; in particular, size and geographic coverage of the sample need to be expanded.Given the fact that an irregular distribution of the four group I introns was observed among the 37 isolates of B. dothidea, a plant pathogen of substantial economic importance worldwide. We have also advanced efforts to comprehend the range of genetic diversity in this species. By using the introns as a marker, we can begin to understand the population structures of the species as well as the dominant population in a particular geographic area, which may give us some guidance to devise more efficient disease management. Second, these introns with variable distribution can be regarded as clues to analyse the evolutionary relationship among populations of B. dothidea. Because group I introns were speculated to be universal in related species of B. dothidea, they may be useful as tools to explore phylogenetics within this lineage. Moreover, some novel ideas might be proposed to control this kind of pathogens by targeting the introns. For example, drugs like pentamidine or 5-ﬂuorocystosine and antisense oligonucleotides that specifically inhibit the self-splicing of introns and kill intron-containing fungal strains have been developed Our findings are meaningful because they provide clues to patterns of evolution and distribution of group I introns in


I2D/IG ratios to confirm that our method really works. For the suspended graphene, the peak positions of G band are downshifted with respect to supported graphene, and the I2D/IG ratios of suspended graphene are larger than those of supported graphene. With data fitting into Voigt profile, one can find out the information behind the lineshapes.We provide a new approach to identify the substrate influence on graphene surface. Distinguishing the substrate influences or the doping effects of charged impurities on graphene can be realized by optically probing the graphene surfaces, included the suspended and supported graphene. In this work, the line scan of Raman spectroscopy was performed across the graphene surface on the ordered square hole. Then, the bandwidths of G-band and 2D-band were fitted into the Voigt profile, a convolution of Gaussian and Lorentzian profiles. The bandwidths of Lorentzian parts were kept as constant whether it is the suspended and supported graphene. For the Gaussian part, the suspended graphene exhibits much greater Gaussian bandwidths than those of the supported graphene. It reveals that the doping effect on supported graphene is stronger than that of suspended graphene. Compared with the previous studies, we also used the peak positions of G bands, and  Recent studies indicate that the remarkable carrier transport properties of suspended graphene with respect to supported graphene include temperature transport, magnetotransport, and conductivity [+, 2D-, and 2D0. In another work, we observed three sub-G peaks: G+, G-, and G0. The property of intensity of G+, G is similar as 2D+ and 2D peaks. The linewidth analysis with data fitting into pure Lorentzian and Voigt profiles had been applied two-photon transitions in atomic Cs [Graphene has many unique and novel electrical and optical properties -3 becausuctivity -6. The puctivity -9. Ramanuctivity -13. Receuctivity -18. Withuctivity ,20, the uctivity ,22, we utomic Cs ,24, becaI2D/IG ratio, and bandwidths of G band fitted with Voigt profile are obtained with the Raman measurements. Under our analysis, details about the effects of charged impurities on the substrate can be realized. About the strain effect or doping effect on graphene, some possible broadening mechanisms may still be responsible for deforming it, so we considered the Gaussian profile necessary.In this work, the supported and suspended graphene were both fabricated by micromechanical cleavage, and then, they were identified as monolayer graphene by Raman spectroscopy and optical microscopy. The Raman signals of suspended and supported graphene can be measured and analyzed by probing the graphene surface which contains them. The peak positions of G band, the 2 were defined by photolithography on an oxidized silicon wafer with an oxide thickness of 300 nm. Reactive ion etching was then used to etch the squares to a depth of 150 nm. Micromechanical cleavage of highly ordered pyrolytic graphite was carried out using scotch tape to enable the suspended graphene flakes to be deposited over the indents. The thickness of the monolayer grapheme is about 0.35 nm. The optical image of suspended graphene, atomic forced microscopy (AFM) image, and its cross section are shown in Figure Suspended graphene was fabricated by mechanical exfoliation of graphene flakes onto an oxidized silicon wafer, and the illustration of that is shown in Figure I2D/IG ratios and peak positions of G and 2D bands were various as graphene surface was doped by depositing silver nanoparticles on its surface. The I2D/IG ratios and peak positions can be related to the doping, and the I2D/IG ratio is more sensitive to the doping than is the peak shift. A lower I2D/IG ratio is associated with a larger amount of charged impurities in graphene. Therefore, peak positions of G band and I2D/IG ratios by integrating their respect band, G and 2D band, are obtained in Figure -1, and the I2D/IG ratios of suspended and supported graphene are around 3.9 and 2.1. The upshift of the G band reflects doping with charged impurities. The peak position of the G band of the suspended graphene is redshifted comparing to that of supported graphene, consistent with the above expectations.To understand the unique properties of graphene surface covering on the different substrates, the Raman signals of G and 2D bands of graphene were obtained in these measurements. According to previous study , the I2DI2D/IG ratios for monolayer graphene flake covering on different substrates can provide information of substrate effect. In the previous reviews, the bandwidths of G and 2D bands were usually fitted by Lorentzian function [The examination on G-band peak positions and the function -29, becafunction -32 asG and L, and γ and Γ are their bandwidths. In Figure where the Gaussian profile and Lorentzian profile are expressed as x = 0.5 μm) and suspended (x = 4.5 μm) graphene by Voigt profile are shown in Figure The bandwidth of Raman band was usually fitted and understood the situation of background of material by Gaussian function. Therefore, the G bands of supported and suspended graphene were fitted by Voigt profiles that give the Gaussian and Lorentzian profiles. The fitting results of Raman spectra of supported  and instrumental uncertainty (0.9 cm-1). The natural linewidth is just linked with the phonon lifetimes between interaction levels. On the other hand, the Gaussian bandwidths of the suspended graphene exhibit a much higher than those of the supported graphene. Some mechanisms resulted in the Gaussian bandwidth broadening and the curve is consistent with the deformation of graphene surface. Other broadening mechanisms are related to the substrate effect and the local heating effect  prepared the samples, suspended graphene using by micromechanical method, and captured the OM and AFM images. HC, the corresponding author, had made substantial contributions to conception and design, and had been involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript.


Blueberry growers in Maine attend annual Cooperative Extension presentations given by university faculty members. These presentations cover topics, such as, how to prevent plant disease and monitor for insect pests. In 2012, in order to make the sessions more interactive and promote learning, clicker questions and peer discussion were incorporated into the presentations. Similar to what has been shown at the undergraduate level, after peer discussion, more blueberry growers gave correct answers to multiple-choice questions than when answering independently. Furthermore, because blueberry growers are characterized by diverse levels of education, experience in the field etc., we were able to determine whether demographic factors were associated with changes in performance after peer discussion. Taken together, our results suggest that clicker questions and peer discussion work equally well with adults from a variety of demographic backgrounds without disadvantaging a subset of the population and provide an important learning opportunity to the least formally educated members. Our results also indicate that clicker questions with peer discussion were viewed as a positive addition to university-related informal science education sessions. Having students respond to multiple-choice format questions designed to test conceptual understanding using personal response systems or “clickers” is one strategy that has been used to promote interaction and learning in K-12 and undergraduate courses  Does peer discussion improve performance of adult learners answering questions with clickers in an informal science setting? 2) Do members of a mixed demographic group benefit from peer discussion? and 3) Are clicker questions viewed as a positive addition to university-related informal science education?The University of Maine Blueberry Schools are offered at three locations in Maine  in mid-March every year. The Blueberry Schools meet once a year for three hours and attendees are awarded Maine Pesticide Applicator credits for attending the presentations at one of the locations.There were a total of five to seven speakers at each class location in 2012. Authors S.A. and F.D. each spoke for 30 minutes and were the only speakers to use a combination of clicker questions and peer discussion during their presentations. One additional speaker used the clickers to individually poll blueberry growers’ opinions on management strategies before and after his presentation. The data from the additional speaker are not included in this study.S.A. and F.D. are tenured faculty who do research on blueberry plant diseases and insect pests, respectively. S.A has given presentations at the Blueberry School for nine years and F.D. has given presentations for 22 years. Before giving the presentations reported on in this paper, S.A. was familiar with using clickers and had taught with them recently in her University of Maine non-majors biology course. F.D. had never taught with clickers before.All blueberry growers were given a clicker, but were told that participation was optional both during questions that focused on content information in the presentations and for the demographic questions. In addition, the blueberry growers were told that their responses were anonymous and that an individual could not be traced to a specific clicker serial number. We decided to keep the blueberry growers responses anonymous as this is the standard procedure used by the University of Maine Cooperative Extension for all surveys of farmers. Based on our counts of attendance versus the number of people voting on clicker questions, fewer than 5% of the blueberry growers opted out of voting with clickers during the presentation. The number of blueberry growers participating with clickers in each session were as follows: 24 in Waldoboro, 34 in Ellsworth, and 48 in Machias.Demographic information on the blueberry growers who participated is shown in Each of the presentations by S.A. and F.D. included three content questions for a total of six questions . The cliDuring the Blueberry School presentation, the peer instruction method Individual Vote) and questions answered after peer discussion are labeled Q-AD (for Question-After Discussion vote). The data set included responses from 106 blueberry growers (∼20% of the blueberry growers in the state of Maine). For data from any one question from an individual blueberry grower to be included in our study, the blueberry grower had to answer both Q-I and Q-AD. If, for example, a blueberry grower answered Q-AD but not Q-I for a question, the data point was removed for that blueberry grower for that question. Most growers answered both Q-I and Q-AD for five or six of the six questions . For this model, each grower was initially classified as: increasing his/her overall score after discussion (increase), showing no overall change before and after peer discussion (no change), decreasing his/her score after discussion (decrease) or having a perfect overall score before and after peer discussion (ceiling). For the logistic regression model analysis, the four classification categories  were collapsed into two categories: 1) those who were advantaged by talking to their peers (increased) and 2) those who were not (decreased and no change combined). Data from blueberry growers in the ceiling category were not used in this analysis. The two categories used as the response variable in the logistic model were advantage, coded as 1 and no advantage, coded as 0. The demographic variables listed in All data summaries and statistical analyses were performed with JMP  or Excel .Approval to evaluate blueberry growers’ responses to clicker questions  was granted by the Institutional Review Board at the University of Maine.105 = 7.11, p<0.0001) or the normalized change scores . Aside from the individuals who had either none or all of the questions correct both before and after discussion, the distribution of the individual blueberry growers’ scores ranged from 20% to 80% correct. This range was the same before and after discussion, but the scores shifted upwards after peer discussion, presenting a left skewed distribution after discussion as compared to a more symmetric distribution before.Overall the blueberry growers answered more questions correctly after peer-discussion (Q-AD) than before (Q-I) . This diThe Q-I and Q-AD results from the blueberry growers were compared with other published reports on undergraduate student clicker question performance in a variety of science courses including genetics, physics, and computer science courses  [4], [6]When comparing votes before and after discussion, four outcomes are possible, a blueberry grower can: 1) answer Q-I and Q-AD correctly, 2) answer Q-I correctly and Q-AD incorrectly, 3) answer Q-I incorrectly and Q-AD correctly, 4) answer Q-I and Q-AD incorrectly. The patterns of behavior for blueberry growers over all six questions shows 96% of blueberry growers who answered Q-I correctly answered Q-AD correctly after peer discussion , and number of years working with blueberries. Fisher’s exact tests revealed no significant differences in the distribution of blueberry growers in the increase, no change, decrease, and ceiling groups when they were sorted by any of the demographic variables  blueberry growers who were advantaged by talking to their peers (increased Q-I to Q-AD) and 2) blueberry growers who were not (decreased Q-I to Q-AD and no change combined). The logistic regression analysis simultaneously controls for the relationship of each demographic variable to both the benefit or lack of benefit of peer discussion. The results of the analysis showed that none of the demographic variables had a significant relationship with a blueberry grower’s status of being advantaged or not by the peer discussion activities  students learn more, 2) students are more likely to participate, and 3) instructors are better able to accurately assess student understanding In addition, we will also explore whether lower-cost polling methods such as using colored cards At the end of the presentation, we asked blueberry growers: “Were the clicker questions helpful in becoming familiar with the information presented?” 89% of the blueberry growers said yes. Feedback given after the presentations included comments such as: “Some answers to the questions really had you doubting yourself which made you put a lot of thought into which answer was correct” and “It was interactive and it kept us awake.” One blueberry grower commented after the meeting that it was one of the best meetings the person had attended in 25 years. On the other hand, one blueberry grower cautioned: “Using the clicker kept me more engaged in the presentations – but be careful not to get carried away with too many clicker questions either. They do tend to slow things down, having to wait for everyone to do their clicking.” These comments suggest that, similar to formal education settings, it is important to balance disseminating information and promoting interaction.This work shows that peer discussion improves the clicker question performance of adult learners in an informal setting, all members of a mixed demographic group benefit from peer discussion of clicker questions, and clicker questions are viewed as a positive addition to a university-related informal science education sessions. In addition, our work is aligned with recent calls to transform the way university-sponsored cooperative extension courses are designed so that lecture is de-emphasized and group learning is fostered Figure S1Text of all content questions in the presentation for blueberry growers.(PDF)Click here for additional data file.Figure S2Text of all the demographic questions in the presentation for blueberry growers.(PDF)Click here for additional data file.Figure S3Voting times for individual and after discussion votes for each question. The error bars show STD. The number of blueberry growers participating in each question is also shown. Author S.A. asked the first three questions and author F.D. asked the last three questions.(PDF)Click here for additional data file.Table S1Results of Fisher’s Exact Tests of association between demographic variables and overall trend of blueberry growers in the increase, no change, decrease, and ceiling groups.(DOCX)Click here for additional data file.Table S2Results of the Effect Likelihood Ratio Tests by demographic variable for the Logistic Regression Model.(DOCX)Click here for additional data file.


Studies that have reported cardiovascular (CVS) instability with haemodialysis (HD) are outdated and small. By analysing sessions in detail it will be possible to identify the frequency and nature of CVS instability. Hypothesis 1: haemodialysis is associated with CVS instability in the majority of sessions. Hypothesis 2: the majority of CVS changes in unstable sessions will be harmful/potentially harmful.Data were collected for 209 patients, identifying 1,605 dialysis sessions. Analysis was performed on hourly records, classifying sessions as stable/unstable by a cut-off >±20% change in baseline physiology (HR/MAP). Data from 3 hours prior to and 4 hours after dialysis were included, and average and minimum values derived. Three time comparisons were made: pre-HD:during, during HD:post, pre-HD:post-HD. If a session was identified as being unstable, then the nature of instability was examined by recording whether changes crossed defined physiological ranges. The changes seen in unstable sessions could be described as to their effects: being harmful/potentially harmful, or beneficial/potentially beneficial.Discarding incomplete data, 1,563 sessions were analysed. A session was deemed to be stable if there was no change >±20% in time-averaged or minimum MAP/HR across three time comparisons. In 1,563 sessions there was stability in 874 sessions . Hypothesis 1 is rejected. Each session had 12 potential comparisons of MAP, HR and time, therefore in the 689 unstable sessions there were 8,268 potential changes ±20% (689 × 12). There were 804/8,268 harmful/potentially harmful changes, 922/8,268 beneficial/potentially beneficial changes and 6,542/8,268 opportunities for change where none occurred. Therefore, looking at harmful/potentially harmful changes there were 804/8,268 . Looking at potentially beneficial changes this occurred in 922/8,268 , and if these were combined with the nonsignificant changes this gave a proportion of 7,464/8,268 . Therefore Hypothesis 2 is rejected.The results above are encouraging, especially given the stringent definitions of instability used. By making multiple time-period comparisons the validity of the claims of haemodynamic stability are enforced, compared to previous papers. The number of sessions and measurement points combine to add weight to our findings, supported by robust confidence interval data.


Cyt b) genes from the GOBASE database were compiled and the evolutionary dynamics of the Cyt b gene introns was assessed. Cyt b gene introns were frequently present in the fungal kingdom and some lower plants, but generally absent or rare in Chromista, Protozoa, and Animalia. Fungal Cyt b introns were found at 35 positions in Cyt b genes and the number of introns varied at individual positions from a single representative to 32 different introns at position 131, showing a wide and patchy distribution. Many homologous introns were present at the same position in distantly related species but absent in closely related species, suggesting that introns of the Cyt b genes were frequently lost. On the other hand, highly similar intron sequences were observed in some distantly related species rather than in closely related species, suggesting that these introns were gained independently, likely through lateral transfers. The intron loss-and-gain events could be mediated by transpositions that might have occurred between nuclear and mitochondria. Southern hybridization analysis confirmed that some introns contained repetitive sequences and might be transposable elements. An intron gain in Botryotinia fuckeliana prevented the development of QoI fungicide resistance, suggesting that intron loss-and-gain events were not necessarily beneficial to their host organisms.In this study, all available cytochrome b ( Introns are widely distributed in numerous genes of viruses, prokaryotes and eukaryotes Introns Late theory argues that introns could have expanded in a fashion similar to transposable elements (TEs), have been the results of tandem duplications into exons Introns Late theory where introns might be able to spread horizontally between phylogenetically distant species of different kingdoms Intron density in eukaryote genomes varies by more than three orders of magnitude, suggesting that there must have been extensive intron gain and/or loss events during evolution Introns Early theory suggests that introns were abundant in ancestral genes and mainly evolved through intron loss Introns Early vs Late was concerned with the origin of nuclear spliceosomal introns, the details of evolution of mitochondrial group I or group II introns could provide insights.In contrast, via a double-strand breaks (DSBs)-repair process in trans to promote splicing of other group I introns in the same genome via an RNA-based mechanism known as ‘retrohoming’. The proteins encoded by group II introns are multifunctional, containing maturase, reverse transcriptase and DNA-binding functions as well as DNA endonuclease activity. The maturase activity facilitates intron splicing by stabilizing the catalytically active RNA conformation, while other functions aid in the retrohoming to allelic target sites Most group I introns harbor genes encoding so-called homing endonucleases, initiating intron mobility Monilinia spp. (anamorphs in Monilia), Cyt b genes, the targets of the Quinone outside inhibitor fungicides (QoIs), were isolated and analyzed Cyt b genes in M. fructicola, M. laxa, M. fructigena and M. yunnanensis have seven, whereas the Cyt b gene in M. mumecola has six large (>1 kb) introns. The intron/exon organization varies considerably among these species. In contrast to the large introns, the exons of Cyt b are usually small, ranging from 11 to 397 bp in length. The combined intron sequences of the Cyt b gene in M. fructicola, M. fructigena, M. laxa, M. yunnanensis and M. mumecola are 10,751, 12,380, 12,329, 11,456 and 13,027 bp, respectively. The large intron number and size make the Cyt b genes in Monilinia spp. the longest reported to date in all kingdoms of organisms. In contrast, the Cyt b gene in Blumeria graminis f.sp. tritici, the causal agent of wheat powdery mildew, is intronless, even though Monilinia and Blumeria species belong to the same fungal class Leotiomycetes. It will be interesting to know what caused the Cyt b genes in Monilinia species to harbor the largest ones in eukaryotic organisms.The number of mitochondrial introns or intron sequences may vary dramatically in different species. In recent studies on peach brown rot fungi Cyt b genes have been sequenced and annotated from thousands of species including distantly related species from different kingdoms and widely used in systematic studies or evolutionary studies Cyt b genes vary dramatically among different species. However, the dynamics of Cyt b gene introns, has not been systemically studied. In this study, we compiled all available Cyt b genes from the GOBASE database and assessed the evolutionary dynamics of Cyt b gene introns, and some introns in Monilinia and Botryotinia spp. were confirmed by Southern blot analyses.The Cyt b genes were previously isolated from peach brown rot fungi M. fructicola, M. fructigena, M. laxa, M. yunnanensis and M. mumecolaCyt b gene was isolated from the rice false smut fungus Ustilaginoidea virens. Briefly, degenerate primers were designed based on Cyt b gene sequences from phylogenetically close species, and PCR reaction was carried out to amplify the conserved region from U. virens isolate UV-8a. Once a conserved fragment was isolated, the flanking sequences were obtained using Tail-PCR or Inverse-PCR amplifications and sequenced. For cDNA amplification, total RNA was isolated using the RNeasy Plant Mini Kit  according to the manufacturer’s instructions. First-strand cDNA was synthesized using an oligo-dT primer and Superscript III reverse transcriptase  according to the manufacturer’s recommendations. Amplification of Cyt b-specific cDNA was performed using the primers designed to amplify the Cyt b sequence from translational start codon to translational stop codon. Intron numbers and precise locations were identified by comparing genomic DNA (gDNA) and cDNA sequences.The Cyt b gene and intron sequences were obtained directly from the GOBASE database  using the gene search and intron search options with Cyt b gene (cob) as gene name http://gobase.bcm.umontreal.ca/) is invalid currently. However, the accession nos. in GenBank (http://www.ncbi.nlm.nih.gov/) of all Cyt b genes showed in this study were indicated either in the table or figure legends.Cyt b gene is indicated by its “position” and “phase”. Intron position in this study refers to the position of codon at which the intron is inserted. Intron phase refers to the nucleotide position in a codon at which the intron is inserted. An intron is considered as phase “0” if it is inserted between two consecutive codons, “1” if inserted between the first and the second nucleotide of a codon, “2” if inserted between the second and the third nucleotide of a codon. The number, position and phase of introns in Monilinia spp. and U. virens were identified by comparing the gDNA and cDNA sequences. For Cyt b genes retrieved from the GOBASE database, the intron number, position and phase were determined as described below. To determine the locations of introns of the Cyt b genes, the deduced amino acid sequences of all Cyt b genes were aligned using the MegAlign program in the Software package DNASTAR . For the locations of introns to be comparable in different Cyt b genes, their relative locations corresponding to the Cyt b gene in U. virens were considered as their locations. If the locations of two introns vary by fewer than 6 nucleotides, they are called “sliding introns”. The annotation for all sliding introns were checked manually as described in rd intron of the Cyt b gene from M. mumecola.Information about intron location is important and the base for elucidating the dynamics of introns. In the present study, the relative location of an intron in the Cyt b gene cDNA sequences or intron sequences at position 164 were aligned with the program Clustal W in the software MEGA 4.0. The following settings were used to construct the phylogenetic trees: heuristic search using close neighbor interchange  with initial trees generated by random addition (100 reps).Maximum parsimony (MP) method was used to construct phylogenetic trees. Cyt b introns was identified by using a reciprocal BLAST to calculate the pairwise nucleotide identity of introns. Algorithm BLASTN was used for the identity search in program BioPerl with default parameters and an E-value cutoff of 1e-05. To obtain an accurate estimate of global hit statistics, a tiling of high-scoring segment pairs (HSPs) onto either the subject or the query sequence was performed. Two introns were considered as homologues if coverage (the sum of HSP length/longer one in paired introns) >30% and average pairwise identity >80%.The homology of EcoR I or Hind III and separated in 1.0% agarose gels in 0.5×TBE buffer and transferred to Hybond N+ membranes . Intronic fragments were amplified by PCR reactions with the intron-specific primer pairs listed in Southern hybridization analysis was performed mainly as described previously Cyt b gene from Ustilaginoidea virens was 9216 bp in size and contained six introns. They were located at cDNA positions (5′–3′) 201, 393, 490, 506, 562 and 823 with sizes of 1594, 1180, 1223, 1775, 1325 and 949 bp respectively. The exon and intron sequences of isolate UV-8a are available in GenBank under accession number JN204426. In other organisms, relative locations of introns corresponding to the Cyt b gene in U. virens were considered as their locations.The entire Cyt b genes were obtained from 3,276 species of four eukaryotic kingdoms. Only 92 species, including six fungal pathogens (Monilinia spp. and U. virens), had introns  and 16 had unique introns (present in only one gene). The intron numbers at individual positions varied from a single representative at 16 different positions to 32 different introns at position 131  have a certain chance of being homologous. However, the sum of HSPs of many hits only covered a limited part of either introns. Such introns should not be real homologues but just contained certain conserved regions. Thus, in the present study, only introns meeting additional criteria (coverage >30% and average pairwise identity >80%) were considered homologues.To understand the relationship between Cyt b genes had occurred. For example, at position 169, homologous introns were frequently detected from phylum Ascomycota , Eurotiomycetes  and Leotiomycetes , indicating that these introns were vertically inherited from a common ancestral intron before the divergence of these classes. These highly homologous introns were detected in all of the investigated species of Monilinia genus but not from the species B. fuckeliana of the same Sclerotiniaceae family, indicating that the intron at position 169 was recently lost in B. fuckeliana (after the divergence of Monilinia and Botryotinia genera). Intron loss events were also observed at position 67: homologous introns were detected in species from the Leotiomycetes  and Sordariomycetes , but not in the G. zeae species which is in the same order Hypocreales with U. virens under Sordariomycetes class. Besides positions 67 and 169, similar intron loss events were observed at least at positions 131, 143, 164, 260  of the intron . In the present study, most introns at common positions contained the LAGLIDADG endonuclease coding sequence  or the GIY-YIG endonuclease coding sequence  and thus should be able to move by lateral transfer. Homing endonuclease genes (HEGs) are disproportionately common in mitochondria and chloroplasts of eukaryotes. The first HEG discovered was ω (I-SceI) in the large subunit rRNA gene in the mitochondria of the yeast Volvox carteri (homologue 9), where the homologous introns Vc1 and Vc2 were located at different positions in the same gene. These observations suggested that the frequent gain-and-loss of introns occurred during evolution of Cyt b genes and some of the Cyt b introns might be TEs because they can move to new sites and undergo deletion or amplification in genomes.As shown in Cyt b intron sequences in the genome of Monilinia species. Intron My3 from M. yunnanensis is located at position 143, southern hybridization using My3 as probe showed expected bands  and M. mumecola  and M. fructicola, both species did not have homologous introns of My3 in their Cyt b genes. These results indicate that multiple copies of introns My3, Ml3 and Mm3 exist in the genomes of M. yunnanensis, M. laxa and M. mumecola, as well as in M. fructigena and M. fructicola. These introns were repetitive elements, a characteristic for TEs. Mfg2 was also confirmed to be a repetitive intron  . The presence of an intron at the position 143 has been shown to prevent the formation of the G143A mutation in some pathogens, because mutations in this codon would interfere with intron splicing and consequently prevent the formation of an active Cyt b gene S. cerevisiae, where an exon mutation at the second nucleotide upstream of the splice site of intron 4 in the Cyt b gene (COB4) did not permit correct splicing of the pre mRNA Single amino acid changes in the Cyt b alleles were discovered, one with and one without an intron at position 143 in B. fuckeliana. Southern hybridization using intron Bf3 as probe showed that this intron was absent from the entire genome of isolates lacking the intron at position 143 , the Bf3 gained event directly influenced the development of the QoI fungicide resistance. Once the isolates gained the Bf3, they lost the potential ability to develop the fungicide resistance based on the G143A mutation. Pathogens did not get any visible benefit from such an intron gain event but had negative influence for the development of fungicide resistance, suggesting that it was just one of many intron gain-and-loss events occurring neutrally and the intron gain-and-loss events were not necessarily beneficial to their host organisms.Two different tion 143 . Homolognia spp. . TherefoTable S1Primers used for intron-specific PCR amplification(DOC)Click here for additional data file.Figure S1Modification of the sliding intron position or phase. (A) The phase of intron Sc5 was modified based on one nucleotide shifting of the exon/intron sequence. (B) The phase of intron Gi3 was modified based on two nucleotide shifts that altered one codon. (C) Sliding introns can’t be modified to same position or phase. Lowercase letters indicate intron sequences, and uppercase letters represent the flanking exon sequences, the larger uppercase letters represent the codon at which individual introns located. If introns have different positions or phases, they are considered as different locations. The locations of introns were modified slightly from original information for some sliding introns. For example, the intron Sc5 at position 270 was a phase 0 intron based on the database information, but one nucleotide shifting of exon/intron sequence could make it have same phase with other introns at this position (such as Cbs2(2), Cc(2) etc. in (TIF)Click here for additional data file.Figure S2Cyt b gene introns in Monilinia spp and B. fuckeliana. Two restriction enzymes EcoR I and Hind III were used to generate the restriction profiles. Digested genomic DNA was separated in 0.8% agarose gel, and the blot was hybridized with the Mfg2 (A), Ml4 (B) and Bf3 (C) fragments. Lanes 1, 11 and 2, 12: M. yunnanensis isolates YKG10-64a and SM09-7c; 3, 13 and 4, 14: M. fructigena isolates SL10 and Mfg2-GE-A; 5, 15 and 6, 16: M. laxa isolates BEK-SZ and EBR ba11b; 7, 17 and 8, 18: M. mumecola isolates HWL10-11a and HWL10-20a; 9, 19 and 10, 20: M. fructicola isolates MPA14 and BM09-4a in A and B. For B. fuckeliana in C, 1, 5, isolate RhosimBC-4; 2,6, isolate PeachBC-1; 3,7, isolate GarlicBC-78; 4,8, isolate ViotriBC-1. The sizes (in kilobases) of marker DNA fragments are indicated on both sides  Co., Ltd).Identification of the (TIF)Click here for additional data file.


An intermediate higher refractive index overlay, deposited through Electrostatic Self-Assembly, is placed between the fiber cladding and the humidity sensitive film in order to increase the total effective refractive index of the coating. With this intermediate design, a three-fold improvement in the sensitivity was obtained. Wavelength shifts up to 15 nm against 5 nm were achieved in a humidity range from 20% to 80%.This work addresses a new configuration that improves the sensitivity of a humidity sensor based on a long-period fiber grating coated with a SiO Several sensing fiber architectures such as hollow core fibers, tapered optical fibers, side-polished fiber, U-bends, nano-Fabry-Perot cavities etc., have been reported for humidity measurements induced by refractive index changes of the external medium –4. Also,In previous works, humidity sensors based on polymeric overlays have been developed –8. Howev2 nanospheres, as a porous hydrophilic material, can be used as a humidity sensitivity film. In previous work [2 nanospheres coated on a LPG could provide a fast response humidity sensor. However, due to its low index of refraction, lower than the fiber itself, the sensitivity is quite low and should be improved in order to avoid expensive equipment. This can be done by increasing the total effective refractive index of the sensitive coating using an intermediate overlay with higher refractive index.It was proved that SiOous work  it was a2.01β and 0jβ are the propagation constants of the core and the j cladding modes respectively, and Λ is the period of the grating. Results obtained using this approximation present appreciable variation when compared with those values calculated with rigorous coupled mode differential equations [01,01ζ and 0j,0jζ are the self-coupling coefficients of the core and the j cladding modes, 0s is the coefficient of the first Fourier component of the grating and N is the refraction order. If the grating function is sinusoidal, 0s simplifies to unity. In the case of arc-induced LPG, this last approximation can be used without lacking rigor because the margin of error is compared with the fabrication tolerances of the own LPG.In order to analyze the spectrum evolution in structures based on LPG with a sensing overlay when the aim is only to detect the displacement of the resonance wavelengths, it is possible to use either the Bragg or the Bragg modified conditions . The firquations . Howeverquations :(2)β01 is halfway between its minimum value and the wavelength of the next lower cladding mode before deposition. More theoretical details can be seen in .3.The LPG used in this study was an arc-induced LPG  with Λ =® SM-30 SiO2-water colloid. In this case, the PAH and PDDA acted as polycations, and PolyR-478 and SM-30 were the anionic species. The number of overlays of PDDA/PolyR-478 was 14 layers and the number of PAH/SM30 overlays was 14 layers, which confer a total film thickness of less than 300 nanometers. Specifically, although the film thickness is difficult to measure due to the fibre geometry, in previous works it has been demonstrated that each PDDA/PolyR-478 and PAH/SM30 layers had thicknesses of approximately 12 nm and 7 nm, respectively [The polymeric layers were deposited using the ESA layer-by-layer method . In thisThe thickness overlay is chosen to guarantee that the attenuation band is located where there is good sensitivity and where it does not vanish at the same time. In this work, the good sensitivity band corresponds to ∼1,520 nm. In fact, the same effect noticeable when the ambient humidity increases, can be appreciated when the thickness of the coating is getting increased see . If the In 4.HR in percentual values):HR is the Relative Humidity and Δλ the wavelength shift. These relations allow obtaining the sensing heads sensitivity at different relative humidity levels. For example, in the range 20–50%, the sensitivity is ≈ 0.04 nm/%RH and 0.06 nm/%RH for the PAH/SM30 coating and the PDDA/PolyR + PAH/SM30 structures, respectively, while around a relative humidity level of 70% those values are improved to ≈ 0.13 nm/%HR and ≈ 0.48 nm/%HR for the PAH/SM30 coating and the PDDA/PolyR + PAH/SM30 structures, respectively. These results indicate that the configuration using an intermediate overlay of higher refractive index improves the sensitivity of the sensor by a factor of 1.5 at a HR ≈ 30%, which is improved to a value of 3.5 when dealing with HR around 70%.HR time response of the sensing head in the range 100–200 ms, far better than what is achieved using standard electrical humidity sensorsPreliminary experiments performed indicate a 5.2-nanospheres thin film was experimentally demonstrated. An intermediate higher refractive index overlay is placed between the fiber cladding and the humidity sensitive SiO2-nanospheres film in order to increase the total effective refractive index of the coating. In this way, compared with the situation of using only the layer sensitive to humidity, a sensitivity improvement factor larger than three was obtained. This result indicates a high potential of utilization of this sensing structure in the context of chemical and biochemical applications.In summary, a new configuration that improves the sensitivity of a humidity sensor based on a LPG coated with a SiO


Most bacterial group II introns harbour an open reading frame (ORF), coding for a protein with reverse transcriptase, maturase and occasionally DNA binding and endonuclease activities. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA target sites. From an evolutionary perspective, group II introns are hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs  that are important functional elements of the spliceosome machinery. The ability of some group II introns fragmented in two or three pieces to assemble and undergo splicing in Lactococcus lactis to be fragmented into three pieces in vivo. Trans-splicing tripartite variants of Ll.LtrB were selected using a highly efficient and sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support tripartite trans-splicing, showing that this intron is remarkably tolerant to fragmentation.We used a transposon-based genetic screen to explore the ability of the Ll.LtrB group II intron from the Gram-positive bacterium trans-splice their flanking exons in vivo. The selected introns represent the first evidence of functional tripartite group II introns in bacteria and provide experimental support for the proposed evolutionary relationship between group II introns and snRNAs.This work unveils the great versatility of group II intron fragments to assemble and accurately  Self-splicing group II introns were initially discovered interrupting various mitochondrial and chloroplastic genes of higher plants and algae, as well as mitochondrial genes of lower eukaryotes Despite a lack of sequence similarity, group II introns fold into a conserved secondary structure composed of six domains (DI-DVI) radiating from a central wheel in vivo and in vitro. In vivo however, folding of these large ribozymes into their catalytically active structure depends on the maturase activity of their IEP or free-standing maturases. Group II introns self-splice from pre-mRNA transcripts through two consecutive transesterification reactions  that constitute the heart of the nuclear intron splicing machinery called the spliceosome trans-acting RNAs trans . Relaxase initiates conjugation by nicking the conjugative element at the origin of transfer (oriT) and delivers the DNA to recipient cells trans-splicing/conjugation assay in L. lactis, showing that Ll.LtrB is able to splice in trans when fragmented at natural group II intron fragmentation sites trans-splices when fragmented at various locations throughout its structure, revealing its trans-splicing versatility The Ll.LtrB intron from the Gram-positive bacterium trans-splicing Ll.LtrB variants were randomly generated and selected using an in vivo trans-splicing/conjugation assay. We discovered that Ll.LtrB can assemble and accurately trans-splice when fragmented into three pieces at many locations throughout the majority of its structure. Moreover, some of the Ll.LtrB tripartite variants studied were found to trans-splice only 11- to 35-fold less efficiently than their bipartite counterparts. This is the first demonstration of the tripartite trans-splicing ability of Ll.LtrB, as well as the tripartite trans-splicing versatility of a bacterial group II intron. This study further illustrates the great plasticity of group II intron to fragmentation and supports the theory suggesting that spliceosomal snRNAs were derived from group II intron fragments.Here, we took advantage of our previously engineered Tn5-based genetic screen to explore sites throughout Ll.LtrB that would tolerate fragmentation into three pieces. Tripartite trans-splicing/conjugation assays in L. lactis. In one assay, trans-splicing of Ll.LtrB fragmented in two pieces at natural group II intron fragmentation sites was monitored by conjugation of a relaxase-deficient chromosomal sex factor (SF) (107-fold detection range) trans-splicers fragmented at various positions throughout the intron by the conjugation of their carrying pLE12 mobilizable plasmid (104-fold detection range) We previously established two very sensitive group II intron trans-splicing efficiency in three pieces by the conjugative transfer of its carrying pLE1 mobilizable plasmid (oriT) of the L. lactis SF so that it could be mobilized upon relaxase production from tripartite trans-spliced Ll.LtrB transcripts   . PrevioultrB::tet was used as the donor strain for plasmid conjugation  (ltrB::Tn bank (ltrB exon 1 (PvuI) and spans domains I through III of the first intron fragment including a portion of the ltrA gene in DIV (RT domain) (BsaI) (The engineered Tn5 transposon harbors a promoter  [17]. ThTn bank) , step 1 :Tn bank , step 2.ltrB L. lactis strain and the transformants were pooled and used as donor cells for the conjugation assay , only 11- to 35-fold lower than their bipartite counterpart, S3 (4.2×10−5) (−7 range (−9 (ltrA (data not shown). Expression of LtrA from a second plasmid (pDEltrA) led to a significant increase in trans-splicing efficiency, the tripartite intron being as efficient as its bipartite counterpart, S3 (Twelve tripartite introns were chosen for further investigation (.2×10−5) . One iso−7 range , S3-447,ange   and only 3.5- to 11.5-fold over background . However, even though the conjugation efficiencies of these tripartite variants were significantly lower compared to their bipartite counterparts we were able to detect ligated exons by RT-PCR for the majority of them , and the transposon insertion sites were identified by sequence analysis. The Tn5 fragmentation sites were found again scattered throughout the regions subjected to the screen . Next, wn assays . All the.5×10−8) , 100- to of them .trans-splicing conjugation assays we previously demonstrated the bipartite trans-splicing versatility of the Ll.LtrB group II intron. Ll.LtrB was shown to support fragmentation at natural fragmentation sites trans-splicing of various bipartite introns in vivoUsing psaA gene of C. reinhardtiinad5 gene of O. berterianatrans-splicing ability of Ll.LtrB when fragmented into three pieces. The power and reliability of this experimental approach designed to select fragmented introns that can assemble and splice in trans from a bank of fragmented introns was previously demonstrated trans-splicing variants were selected from bipartite introns using sensitive trans-splicing/conjugation assays in L. lactis . Although LtrA is not detectable by Western blot, six potential start codons downstream of the fragmentation site are found to be in the correct reading frame. The use of one of these initiation codons may lead to low levels of expression of a N-terminal truncated LtrA protein with maturase activity. Accordingly, the expression of LtrA in trans from a second plasmid rescued splicing of this tripartite intron inducing conjugation efficiencies at levels similar to the bipartite S3 intron. This shows that the additional fragmentation site within DIV does not significantly affect Ll.LtrB trans-splicing efficiency. This is reminiscent of the previously selected bipartite introns fragmented within ltrA but downstream from the maturase domain. We proposed that these introns expressed C-terminal truncated versions of active maturases. Similarly, the expression of full-length LtrA from a second plasmid significantly increased the trans-splicing efficiency of these introns The lack of fragmentation observed within DIV confirms the importance of LtrA as a co-factor for Ll.LtrB As previously observed in our screens for bipartite introns trans-splicing efficiencies. Although we hypothesize that the location of the fragmentation site determines the assembly and trans-splicing efficiency, the stability of the first two fragments may not be a significant factor, especially if the third fragment is available in limiting amounts. Nevertheless, the three RNA fragments retain the resilience to fold, assemble and trans-splice accurately in vivo.Through Northern blots we clearly detected the three RNA fragments of the twelve Ll.LtrB tripartite variants chosen. All three fragments seem to be expressed to similar levels while some degradation products can be seen. The third fragment appears to be the least stable when compared to the first two fragments. In identifying the first intron fragment of the tripartite variants, we observe an increase in the intensity of the bands when fragmentations occur downstream of DI. Since DI serves as a docking site for the remaining domains, its folding as a complete module possibly contribute to the increase in stability of the fragments extending into DII to DIV. Even though certain tripartite variants appear to have fragments more stable than others this does not seem to significantly affect their trans-splicing respectively 11- and 28-fold less efficiently. In addition, significantly less transconjugants were obtained following the trans-splicing conjugation screens. Accordingly, the analysis of independent transconjugants from the pLES4::Tn and pLES10::Tn screens showed conjugation efficiencies 100- to 600-fold lower than their bipartite counterparts only 11.5- to 3.5-fold over background. This is in contrast to the pLES3::Tn transconjugants that trans-splice only 11- to 35-fold less efficiently than S3 and 2,900- to 920-fold over background. Similarly to what was previously observed following bipartite screens, when the end of DIV was subjected to transposon insertion (S4), introns fragmented within the ltrA gene downstream of the maturase domain were selected suggesting the expression of truncated LtrA proteins with maturase activity trans-splicing introns fragmented in DI are tripartite introns also fragmented in DIV. While the great majority of bipartite introns were found fragmented in DIV, no bipartite introns were ever found fragmented in DI. Taken together, this suggests that the two natural tripartite introns probably underwent fragmentation in DIV before being further fragmented in DI.In a complementary approach to fragmenting the first piece of a bipartite intron (S3), we submitted the second fragment of bipartite introns severed in DI at either position 172 (S4) or 44 (S10) to our fragmentation screen. We first noticed that the S4 and S10 bipartite introns were not as proficient as S3, psaA gene of C. reinhardtiinad5 gene of O. berterianaL. lactis is quite tolerant to tripartite fragmentation as it can be fragmented into three pieces not only in DI and DIV like the two organellar tripartite introns but at various locations throughout its structure. The selected Ll.LtrB tripartite introns retained the ability to fold correctly, assemble and accurately splice in trans. This suggests that following fragmentation, trans-splicing group II introns would be under significant pressure to improve their splicing efficiency in order to restore the expression level of the gene they interrupt. Such adaptation would lead to fragmented introns that trans-splice more efficiently and that can be further fragmented. Since no functional bipartite or tripartite group II introns were found yet in bacterial genomes the selected introns represent the first evidence of functional tripartite group II introns in bacteria. Assembly of three intron RNA fragments during trans-splicing in vivo probably involves a complex combination of base pairing, long-range tertiary interactions and the potential involvement of the splicing co-factor LtrA The discovery of naturally occurring group II introns fragmented into two or three pieces supports the theories proposing that self-splicing group II introns are the progenitors of nuclear introns and that the spliceosomal snRNAs were derived from fragments of group II introns Demonstration that a bacterial group II intron can be fragmented in three pieces at multiple sites throughout its structure and still be functional provides additional experimental support to the theory proposing that the five snRNAs of the eukaryotic spliceosome were derived from group II intron fragments.Escherichia coli DH10β strain was used for both cloning and plasmid amplification, and was grown in LB broth at 37°C with shaking. The methylase-free E. coli GM119 strain was used to isolate non-methylated DNA since methyl-free PvuI digested DNA fragments are ligated more efficiently. L. lactis strains NZ9800ΔltrB::tet (NZΔL-b) (TetR) R) The oriT) from L. lactis SF was PCR-amplified  under the control of P23 right (BssHII) and the second piece (3′-intron–ltrBExon2) under the control of P23 left (NotI). These constructs were shown to be mobilizable by conjugation  resistance, and the P23 constitutive promoter  and 2.8 times in NZ9800ΔltrB following transformation. The Tn5 transposon was therefore inserted between each nucleotide of the PvuI/BsaI fragment of the intron 2.8 times immediately preceding the trans-splicing/conjugation assay. The same procedure was done to subject the second intron fragment to the screen in pLE-S4 (BsrGI/AatII) and pLE-S10 (EagI/BsaI). The statistical representation of the generated banks was calculated by dividing the number of Cam/Spc colonies recuperated following transformation of the Tn5-containing plasmids by twice the size of the fragments . This method of calculation takes into consideration that Tn5 can insert randomly in both orientation within the target plasmid The Tn5 transposon variant used in this study was previously generated promoter . With thConjugations were performed on milk plates as previously described ltrB cells harbouring various selected tripartite introns as previously described 32P labelled oligonucleotide probe (Total RNA was isolated from NZ9800Δde probe  and visu