Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs), hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons. Intronic sub-sequence retention within IL1-β mRNA in anucleate platelets has been implicated in activity-dependent splicing and translation. In a recent study, we showed CIRTs harbor functional SINE ID elements which are hypothesized to mediate dendritic localization in neurons. Based on these studies and others, we hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation. We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions. We sequenced two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons. After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species (44-60% of the expressed transcripts). The sequence patterns, including stereotypical length, biased inclusion of specific introns, and intron-intron junctions, suggested CIRT-specific nuclear processing. Our analysis also suggested that these cytoplasmic intron-sequence retaining transcripts may serve as a primary transcript for ncRNAs. Our results show that retaining intronic sequences is not isolated to a few loci but may be a genome-wide phenomenon for embedding functional signals within certain mRNA. The results hypothesize a novel source of cis-sequences for post-transcriptional regulation. Our results hypothesize two potentially novel splicing pathways: one, within the nucleus for CIRT biogenesis; and another, within the cytoplasm for removing CIRT sequences before translation. We also speculate that release of CIRT sequences prior to translation may form RNA-based signals within the cell potentially comprising a novel class of signaling pathways. Post-transcriptional regulatory function arises through the retention of specific intronic, nonprotein-coding sequences within the cytoplasmic mRNA. We call such sequences Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs). For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons and proper channel protein localization to dendrites [Recently, several studies have revealed a new dimension to the diversity of transcripts , for insendrites . Intron endrites . A retaiendrites . In a reendrites . These sHere we performed a transcriptome-wide survey for intronic sequence retention using subcellular RNA sequencing of dissected neurites from rat and mouse hippocampal neurons. Neurons are highly polarized cells with spatially distinct dendrites and axons that allow mechanical isolation of cytoplasmic RNA through microdissection. Strictly speaking, our subcellular dissections do not distinguish dendrites and axons but in these neurons the axonal compartment is less than 5% of the volume of dendritic compartments; therefore, we will call our samples dendritic samples from hereon. In addition to cytoplasmic RNA from dendrites, for contrast we also carried out single cell RNA sequencing of individual cell somas, which also contains some heterogeneous nuclear RNA (hnRNA). These datasets were augmented by single cell sequencing from mouse Brown adipose tissue (BAT)  and mousWe performed a comprehensive transcriptome wide survey of the prevalence of CIRTs in rat and mouse dendritic population. Two batches of dendritic mRNA from primary rat hippocampal neurons and three batches of dendritic mRNA from primary mouse hippocampal neurons, each consisting of 150–300 individually dissected dendrites were independently aRNA amplified [Of the reads mapping to intronic regions, only the uniquely mappable reads by paired-end Bowtie alignment  were couGuttman et al [ab inito reconstruction of these transcriptomes [First, each intron with putative cytoplasmically retained sequence was also filtered for overlap with predicted genes using the tools GENSCAN  and TWINan et al  reportedan et al  for overan et al . Since triptomes . We founAfter processing through these steps to minimize other origins of intronic sequences in the mRNA sequences, we found ~44% rat genes and ~59% of the mouse genes in dendritic samples showing intron sequence read evidence consistent with the hypothesis of CIRTs . For the5’ to 3’ introns of a gene model . We chose to have this larger distance threshold in order to account for the reads arising from repetitive sequences that tend to be enriched in intronic regions and may not be uniquely mappable and also to be conservative in the number of independent putative CIRTs. This distance is also roughly equal to two back-to-back paired-end reads. Of all the possible introns within the genes expressed, we found that cytoplasmic retention occurs in only a small fraction of the introns (13-16%)  in rat ane model . We alsone model . It is lne model . Also, ~The retained intronic contigs in rat dendritic samples had a mean length of 363 nt while in mouse dendritic samples the mean length was ~353 nt . The mear = 0.001, P-value = 0.9, Sample size = 3947). Another factor that may affect the resulting length of the contigs is the multiple rounds of aRNA amplification steps in our protocol, which can cause a shortening of the transcripts due to random primer binding for second-strand synthesis in each subsequent cycle. However, in rat dendritic samples, about 3-5% of the contigs had much longer lengths in the range of 1000 bp to 5000 bp and even for these long contigs there was no correlation between the contig length and maximum pA tract contig length  and they arise in almost equal proportions from the 5’ and 3’ introns of the transcript. In sum, the specific characteristic length distribution and selectivity of introns with cytoplasmically retained sequence seem to display distinct signatures of specific processing for CIRT biogenesis independent of the aRNA procedure. A visual representation for few randomly selected CIRTs has been created in UCSC Genome Browser and is provided in the supplementary -T7 primers which recognizes pA-tracts including those from intronic regions. To assess whether the contig length distribution is related to chance occurrence of pA tracts within introns, we first constructed contigs of pA tracts by clustering pA sequences at least 9 bp long and separated by less than 500 bp. Of all the introns with cytoplasmically retained sequence in rat dendritic samples, ~56% had at least one internal pA tract. For these introns, there appeared to be no correlation between the average pA tract contig length and the average intronic contig length  from the transcripts present in the tissue sample were found to give rise to intronic contigs. However, the contig lengths were significantly longer (mean length ~ 1500 bp). . We founAfter having analyzed the properties of putative CIRTs from the carefully dissected dendrites that are free of nuclear hnRNA, we turned our attention to the whole single cell. Single cell transcriptomes are also expected to contain mostly cytoplasmic RNA see  but sincde novo. By considering only the high quality junctions reported by RUM which have known splice signals and are crossed by at least one uniquely mapping read with at least 8 bp on each side of the junctions, we found several novel intron-intron junctions with canonical or known non-canonical splice signals in the both rat and mouse dendritic as well as soma samples . Additionally we also analyzed the genomic DNA from rat hippocampus to verify whether the putative spliced intronic region is in fact present in the genome. The rat strain used for obtaining the mRNA and genomic DNA was Sprague-Dawley which is the same strain from which we got the dendritic samples. Even then there is a potential for copy number variations between individual animals that cannot be ruled out. Of all the novel intron-intron junctions in rat samples, we selected the junctions for experimental validation based on the following criteria: i) junctions for which there was evidence for sufficiently long cytoplasmic retained intronic sequence surrounding the junction with high complexity sequences for primer design, ii) above median expression of the transcript in rat hippocampus tissue, iii) length of the intron containing the junction is shorter than 20,000 bp, and iv) junctions with spliced out length of >= 100 bp. This left us with 78 putative rat intron-intron junctions of which we selected 7 junctions that were detected in either rat soma or dendrites . For twoLastly, we also surveyed the EST database available on UCSC Genome Browser  to assesHaving observed a widespread presence of CIRTs in our neuronal samples, we were curious about their presence in other cells. To do so we analyzed some non-neuronal single cell datasets, namely five independently collected single cells from mouse brown adipose tissue (BAT)  and eighFor the whole single cell samples: mouse soma, BAT and cardiomyocyte samples, we found the average pair-wise overlap among the genes giving rise to CIRTs to be ~44%. On average, when two cells have retained intronic sequences in the same gene, in ~23% of the cases the retained sequence was from identical introns. A gene may show shared evidence of retained introns in various different cell types, but the particular retained introns may differ. For example, one such gene was the MLH3 gene which encodes the DNA mismatch repair protein Mlh3. This gene is associated with mammalian microsatellite instability . This geGiven the widespread prevalence of CIRTs as detected by our analysis we investigated the functionality of the genes giving rise to CIRTs. It is possible that for majority of genes with CIRTs, the CIRTs comprise a minor transcript form while majority of the transcripts do not contain CIRTs as shown in . These m There were 707 genes in rat dendritic samples with intronic read count >= 256 from at least one retained intron. We performed GO analysis on this set using DAVID Bioinformatics Resources 6.7 ,31. AmonAs reported in the above, as part of our filtering step we analyzed for overlap of CIRTs with ncRNAs. We found <2% of the retained introns in mouse and rat dendrite samples to have the Rfam  annotateIn this paper, we use RNA sequencing data from micro-dissected cytoplasmic compartments to show evidence supporting genome-wide prevalence of cytoplasmic intron-sequence retaining transcripts (CIRTs). Surprisingly, up to 59% of expressed genes in rodent neurons showed evidence of sequence reads from intronic sequences. Similar proportions were also seen in mouse Brown Adipose Tissue (BAT) cells and mouse cardiomyocytes. Support for this genome-wide presence of CIRTs come from several sources. First, our RNA populations for the neuronal cells were derived from carefully dissected non-nuclear dendritic segments with little potential for contamination from hnRNA. This fact was supported also by distinct distribution of intronic RNA sequences in contrast to the distributions seen from single cell soma sequencing and whole tissue sequencing. Second, we used multiple computational filters to remove potential effects of cryptic exons, ncRNA, etc. Third, previous work from our lab showed extensive evidence for CIRTs using PCR cloning, in situ hybridization, and RNA sequencing leading to the hypothesis that CIRTs were prevalent across the transcriptome . In addiIf CIRTs play a role in post-transcriptional cell physiological processes, we hypothesize that non-random subsequences of introns are specifically retained rather than retaining any introns of a given gene. Many mammalian introns are extremely large and the median size of mouse and rat introns is ~1300 bp . The contigs assembled from our RNA sequencing suggest typical CIRT size in the range of 350-400 bp, suggesting that the CIRT region of a transcript is processed in the nucleus to a specific subsequence. This size range data is supported by existence of a sharp second mode around 300 bp in intronic sequences from normal tissue RNA sample. In addition, when we find evidence of cytoplasmic intronic sequences for a given gene, typically the sequence evidence is present for only a specific intron rather than all of the gene’s introns. Finally, we found numerous examples of intron-intron junctions surrounding the CIRTs and carried out experiment validation for seven putative intron-intron junctions. We were able to show evidence of spliced product for five of these junctions. We note that our annotation of the intron-intron junctions were limited to known canonical and non-canonical splice signals and if we were to expand the definition to novel junction types, we may have considerably larger annotation of intron-intron processing. Thus from these data we hypothesize that at least some proportion of transcripts for many genes in the genome are alternatively processed during splicing to retain specific subsequences of intronic sequences. Recently, there have been other findings that also lead to the speculation of alternate splicing mechanisms giving rise to a diversity of transcripts not thought to exist previously. For instance, the detection of chimeric RNAs comprising of sequences from different genic boundaries, even those that are on different chromosomes, suggest the involvement of an alternate splicing machinery [The functional roles of CIRTs are uncertain at this stage. In Buckley et al. , we demoIn conclusion, we hypothesize that retention of subsequences of introns involves specific alternative splicing pathways and such sequence retention is wide spread for genes in the mammalian genome. We speculate that these CIRTs play a role in post-transcriptional regulation and possible RNA-based signaling after cytoplasmic processing. Overall, the presence of CIRTs increases the complexity of the transcriptome and CIRT modulation may present another pathway for epigenesis of cellular complexity and play a role in neuronal plasticity.Hippocampi were harvested from embryonic day 18 rat (Sprague-Dawley) and mouse (C57BL/6) pups and dispersed and plated at 100,000 cells per ml of neurobasal medium and B27 (Invitrogen). Neurons are grown in culture for 14 days on 12 mm round German Spiegelglas coverslips (Bellco) coated with poly-L lysine (Peptide Institute).Independent samples of two sets of approximately 150-300 dendrites and 3 cell soma from primary rat hippocampal neurons and three sets of approximately 150-300 dendrites and 3 cell soma from mouse hippocampal neurons were harvested by mechanical isolation and used as template material for aRNA amplification (as described in ).de novo junctions. The number of reads in each library and alignment statistics are provided in Paired-end libraries were constructed from fragmented dendritic and soma aRNA material and sequenced by using Illumina II short-read technology. Paired-end libraries were also constructed for total RNA from whole hippocampus tissue and sequenced using Illumina II short-read technology. The read length is 50 bp. Reads were trimmed for adapter sequence and poly-A, poly-T and then aligned to the Rat (rn4) and Mouse (mm9) genome by using the RUM pipeline . The RUMThe BAT cells were collected in the lab of J. Eberwine . BrieflyThe cardiomyocytes were collected in the lab of B. Kuhn. Briefly, embryonic cardiomyocytes were isolated from the ventricles of 14.5 day old of a hemizygous Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator) embryo . The carRNA from these single cells were collected, amplified and sequenced as described above.The collection of the primary cultured cells utilized animal by-products protocol, "Genome Biology of Single Neuron Funciton and its Modulation with Age" under University of Pennsylvania IACUC protocol #803321 . Animals were sacrified under University of Pennsylavania IACUC protocol #804867, "Molecular Biology of Single Aging Neurons and Glia" , but the sacrifice of the animals were independent of the work reported in this paper. The cardiomyocytes were collected under Harvard University IACUC protocol 12-05-2169R, "Mechanisms of myocardial regeneration" . All protocols were approved by University of Pennsylvania and Harvard Office of Regulatory Affairs and IACUC committee.Primers were designed from the intronic region surrounding the putative intron-intron junction for which we had sequence evidence for cytoplasmic retention . cDNA waFor every intron in the dendritic samples that showed evidence for cytoplasmic intron sequence retention, we selected another intron randomly such that it had a similar length and GC content (within a range of 5%) as well as an identical internal pA-tract length and belonged to a gene that had no cytoplasmic intron sequence retention. In this way we were able to construct a background intron set size matched with the set of retained introns for both mouse as well as rat. File S1Figure S1, Fraction of CIRTs as a function of reads mapping to the retained intronic contigs. Figure S2, Fraction of retained introns/gene as a function of total number of intronic reads/gene. Figure S3, Fraction of intronic contigs falling within each one-third region of the introns. Figure S4. Visual representation of the reads mapping to randomly picked genes that give rise to putative CIRTs. Figure S5, Experimental validation by RT-PCR of seven putative intron-intron junctions from rat dendrites and soma. Table S1, Position of Rat and Mouse dendrites retained introns within the gene (for genes with at least 5 introns). Table S2, Retained introns in Rat and Mouse dendrites with internal pA-tract of varying lengths.(DOC)Click here for additional data file.Table S3List of novel intron-intron splice junctions.(XLS)Click here for additional data file.Table S4Experimentally validated intron-intron splice junctions.(XLS)Click here for additional data file.Table S5Gene ontology analysis results.(XLSX)Click here for additional data file.Table S6Next generation sequencing alignment statistics.(XLS)Click here for additional data file.Table S7List of primers used for experimental validations.(XLS)Click here for additional data file.


Although intron loss in evolution has been described, the mechanism involved is still unclear. Three models have been proposed, the reverse transcriptase (RT) model, genomic deletion model and double-strand-break repair model. The RT model, also termed mRNA-mediated intron loss, suggests that cDNA molecules reverse transcribed from spliced mRNA recombine with genomic DNA causing intron loss. Many studies have attempted to test this model based on its predictions, such as simultaneous loss of adjacent introns, 3'-side bias of intron loss, and germline expression of intron-lost genes. Evidence either supporting or opposing the model has been reported. The mechanism of intron loss proposed in the RT model shares the process of reverse transcription with the formation of processed pseudogenes. If the RT model is correct, genes that have produced more processed pseudogenes are more likely to undergo intron loss.In the present study, we observed that the frequency of intron loss is correlated with processed pseudogene abundance by analyzing a new dataset of intron loss obtained in mice and rats. Furthermore, we found that mRNA molecules of intron-lost genes are mostly translated on free cytoplasmic ribosomes, a feature shared by mRNA molecules of the parental genes of processed pseudogenes and long interspersed elements. This feature is likely convenient for intron-lost gene mRNA molecules to be reverse transcribed. Analyses of adjacent intron loss, 3'-side bias of intron loss, and germline expression of intron-lost genes also support the RT model.Compared with previous evidence, the correlation between the abundance of processed pseudogenes and intron loss frequency more directly supports the RT model of intron loss. Exploring such a correlation is a new strategy to test the RT model in organisms with abundant processed pseudogenes. The loss of spliceosomal introns in eukaryotic evolution has been well documented -11. HoweSaccharomyces cerevisiae, recombination between a reverse transcript and its chromosomal homologue was detected [Drosophila and mammals showed that genes expressed in the germline have a higher frequency of intron loss [Arabidopsis [Although the RT model of intron loss is widely cited, it is still controversial and far from being universally accepted. In the yeast detected ,19. Howedetected -26. By cdetected -29. Secodetected ,25,30-33detected ,27,34-36detected ,38. In tdetected ,24. ThirIn this paper, we describe a new approach to test the RT model. In the RT model, the frequency of intron loss depends on the abundance of reverse transcripts, which are thought to be determined by RT activity and the affinity of mRNA templates for RT. Processed pseudogenes (PPs) are the byproducts of RT encoded by retrotransposons . The actAmong the 16,241 intron-containing orthologs between mice and rats, we identified 148,176 conserved introns and 937 unique intron positions that might refer to possible intron losses or gains. By consulting the orthologous genes in seven outgroup mammal species .In rats, we found that 27.7% IL genes were parental genes of PPs. By contrast, among 8,410 NIL genes, only 7.4% were parental genes of PPs. Fisher's exact test showed that the proportion of parental genes of PPs in IL genes was significantly higher than that of NIL genes . These results showed that intron loss frequency in rats is positively correlated with the abundance of PPs.Also in rats, we found that the parental genes of PPs had a higher rate of intron loss than other genes. On average, each parental gene of PPs lost 0.041 introns whereas each non-parental gene lost only 0.008 introns (Mann-Whitney The PPs produced by genes with short mRNAs are more likely to be detected  and geneP < 10-7). Genes with larger numbers of PPs had fewer extant introns.If genes with more PPs have more introns, they would be more likely to lose introns just by statistical chance. However, we observed a significant negative correlation between the number of extant introns and the abundance of PPs . Similar results were obtained by comparing the IL genes and NIL genes of mice . Further analyses using a larger sample size or high coverage data of mRNA decay rate are required to reach a definite conclusion.Pavlicek et al.  also fouet al.  and the -4 indicated that adjacent introns tend to be lost simultaneously  . In totaet al. [P = 0.32; rats, 12 out of 86 versus 28,914 out of 148,176, P = 0.24). Therefore, we found evidence of genomic deletion but no evidence of the NHEJ model of intron loss in mice and rats.Besides the evidence supporting the RT model of intron loss, we also searched for evidence of genomic deletion and the NHEJ model. First, in our dataset of intron loss, there were seven imprecise intron losses in mice and rats  and the formation of PPs. IL genes and parental genes of PPs share properties that may facilitate reverse transcription, such as being translated on free cytoplasmic ribosomes. More importantly, we found a positive correlation between the frequency of intron loss and the abundance of PPs. Our results strongly indicate that reverse transcription is a necessary step in intron loss. IL genes in mammals were found to be highly expressed . We alsoBeside direct recombination of cDNA with genomic DNA, the RT model has another sub-model: recombination or gene conversion of genomic DNA by intronless PPs . If a PPThere is another possible but less likely explanation for the correlation between intron loss frequency and PP abundance. Highly expressed genes may be more likely to lose introns to reduce metabolic load and the probability of mis-splicing than lowly transcribed genes. Because highly expressed genes have generated more PPs, the intron loss frequency and PP abundance are linked together superficially by high expression level. The metabolic load of introns was previously supposed to be a selective force to intron length reduction in highly expressed genes -60. HoweDrosophila and Arabidopsis thaliana using the datasets of intron loss previously published [Drosophila or Arabidopsis (0.23% for Drosophila and 0.73% for Arabidopsis). Fisher's exact tests showed that the differences between IL genes and NIL genes are not significant in either Drosophila or Arabidopsis (P > 0.6 for both cases). Considered just from this result, it seems that the RT model is not the major mechanism of intron loss in either Drosophila or Arabidopsis. However, previous studies suggested the RT model might be the major mechanism of intron loss in Drosophila but not in Arabidopsis [Drosophila and Arabidopsis, it is unreliable to reach a conclusion on the RT model based on PP analysis.Using the abundance of PPs to test the RT model has limitations. Mammalian genomes have an especially high content of PPs and abundance of retrotransposons. In some organisms, transposable elements are subject to constant turnover . If PPs ublished ,64. Nonebidopsis ,24,27. CThe RT model, the genomic deletion model and the double-strand-break repair model attempt to describe how intron loss occurs. In evolution, the fate of a new mutation may be fixed, eliminated or randomly lost depending on its effect on the fitness of the host organism and the population size of the organism. Except that intron losses are neutral and thus randomly lost or fixed, the mutational models of intron loss cannot fully account for the pattern of intron loss. The inaccuracy of the mutational models to account for the patterns of intron loss in many studies may also be explained by the selective fixation of some special cases of intron loss -67. MiceReverse transcription is proposed as the main mechanism of intron loss in many species, and PPs are byproducts of RT. Therefore, if RT accounts for most of the intron-loss events, genes undergoing intron loss would produce more PPs, as observed in our study. This novel evidence of RT-mediated intron loss is consistent with previously proposed evidence, such as adjacent intron loss, 3'-biased intron loss and germline-biased expression of IL genes. We also found that both IL genes and parental genes of PPs are more likely to be translated on free cytoplasmic ribosomes where the LINEs are reverse transcribed. This phenomenon indicates that IL genes and parental genes of PPs are closely linked via their same subcellular translation locations. By contrast, in several imprecise IL genes whose introns were unlikely lost via RT process, no related PPs were found. The correlation between PP abundance and intron loss frequency more directly supports the RT model of intron loss than previously reported evidence. It provides a new strategy to test the model in eukaryotes that are rich in PPs.Mus musculus), rat (Rattus norvegicus) and six outgroup species, the guinea pig (Cavia porcellus), European rabbit (Oryctolagus cuniculus), human (Homo sapiens), cattle (Bos taurus), African bush elephant (Loxodonta africana) and gray short-tailed opossum (Monodelphis domestica) from Ensembl (Release 65) [Cricetulus griseus), from National Center for Biotechnology Information (Build 1.1) [C. griseus and C. porcellus, were selected because of their close relatedness with mice and rats whereas the other species were selected as outgroups because of their relatively large genome sizes among sequenced mammal genomes. A larger genome is likely to retain more orthologous genes and introns, which are essential for the identification of intron losses in mice and rats. Genes with obvious annotation errors such as those having coding sequences with non-multiples of three nucleotides or conflicting with their protein sequences were discarded. For genes with alternative splicing isoforms, we retained the longest mRNA for analysis.We downloaded the genome sequences and gene annotations of mouse (ease 65) , and anoild 1.1) . Two rodhttp://Pseudogene.org (Build 60 for mice and build 50 for rats) [Pseudogene annotations for mice and rats were obtained from or rats) ,71. Onlyor rats)  to conveE values < 10-10 and identities > 0.35. Where a protein from one species matched multiple proteins from other species , the genomic context was used to improve orthology assignment. OrthoCluster 2.0 [The best reciprocal BLAST was used to search orthologous proteins between mice and rats, with thresholds of ster 2.0  was usedClustal W 2.0  was usedC. griseus, C. porcellus, O. cuniculus, H. sapiens, B. taurus, L. africana and M. domestica were used to distinguish intron losses from intron gains  Percentage of mouse and rat genes producing processed pseudogenes. (B) Comparison of the abundance of processed pseudogenes and mRNA lengths between intron-lost genes and no-intron-lost genes in mice. (C) Comparison of lost introns and mRNA lengths between parents of processed pseudogenes and other genes in mice.Click here for fileImprecise intron loss events mediated by genomic deletion. (A) Sequence alignments around seven lost introns.  Another case of intron loss that may be mis-recognized as imprecise deletion.Click here for fileComparison of expression levels between intron-lost genes and no-intron-lost genes.Click here for filehttp://pseudogene.orgProcessed pseudogenes obtained from .Click here for file16,241 intron-containing orthologous genes between mice and rats.Click here for fileDetails for lost introns.Click here for fileList of intron-lost genes and no-intron-lost genes in mice and rats.Click here for file


Sarcoidosis is a multisystem inflammatory disease of unknown etiology affecting multiple organs. Earlier reports suggested that sarcoidosis was a disease of the developed world. However, recent reports suggest that the disease is found in the developing countries as well. Clinical, radiological, and histopathological similarities with tuberculosis pose a great challenge in countries endemic for tuberculosis. Mantoux test, high resolution computed tomography, and transbronchial lymph node and lung biopsies are diagnostic modalities, which play an important role in the diagnosis of sarcoid. In this review, we look at the epidemiology of sarcoid in tuberculosis-endemic regions, the sarcoidosis-tuberculosis link, clinical profile, diagnostic modalities, dilemma in the diagnosis, and the treatment of this disease. Sarcoidosis is a chronic, multisystem inflammatory disease of unknown etiology characterized by the presence of non-caseating granulomas in different organs. Although the lungs are the most common sites of inflammation, sarcoidosis can involve other organs such as the eyes , skin, lymph nodes, salivary glands, heart, spleen, liver, and the nervous system. A combiSarcoidosis was considered to be a disease seen more commonly in the west and rarely in the developing world in earlier reports. In a study from eight countries of Asia and Africa in 1976, fewer than 30 patients were reported from India, Malaysia, Thailand, Taiwan, and United Arab Emirates, while none from Singapore and Korea. In the The true burden of sarcoidosis in India is not clearly known, as reliable epidemiological data are not available. It has been estimated that sarcoidosis constituted 10 to 12 cases per 1,000 new registrations in a respiratory unit at Kolkata and 61.2/100,000 new cases at a center in New Delhi in a report published in 2002. The casMTb catalase peroxidase (mKatG), superoxide dismutase, mycolyl transferase, and heat shock proteins have been detected[Mycobacterium tuberculosis as a cause of sarcoidosis has been extensively studied in the literature-25. Repodetected; immune detected-33. Thisdetected.The course of sarcoidosis ranges from asymptomatic to severe disease. Male preponderance with a mean age of 40 years is reported in most Indian reports,4,7,10. The disease affects predominantly the lungs, thoracic lymph nodes, skin, and eyes. Clinically, patients may be asymptomatic or complain of cough or breathlessness. Constitutional symptoms such as fever, malaise, fatigue, and weight loss may be present. The most frequent intrathoracic involvement is the presence of hilar or mediastinal lymph node enlargements with or without radiologic evidence of interstitial involvement in one-third of patients. The miliary pattern can be seen in sarcoidosis and may be misdiagnosed and treated for tuberculosis. High reSkin involvement may include sarcoid granulomas and erythema nodosum. Bilateral parotid gland enlargement, hepatic involvement with granulomas, renal insufficiency, myocardial involvement with heart failure, hypertension and conduction abnormalities, arthropathy, neurological involvement in the form of recurrent laryngeal nerve palsy, Bells' palsy, other cranial neuropathies and spinal chord involvement, generalized lymphadenopathy, and minor hematologic abnormalities have been reported from India,4. SporaPatients may present directly to an ophthalmology clinic with blurred vision, floaters, redness or discomfort, or may be referred by the pulmonologists or rheumatologists for an ocular evaluation. Ocular disease may be the initial manifestation in sarcoidosis. Ocular involvement occurs in 25% to 60% of patients with systemic sarcoidosis presenting to a respiratory center. This waIn contrast to reports from western literature, panuveiAnterior uveitis could be acute iridocyclitis or a chronic granulomatous uveitis with keratic precipitates, which may vary from cellular to large mutton fat type of keratic precipitates. Iris nodules such as the Koeppe and busacca nodules and iris granulomas may be seen. Granulomas may also be seen in the trabecular meshwork causing increase in intraocular pressure at the time of activity. Intermediate uveitis with vitritis, peripheral vasculitis, snowball opacities, and snow banking have been described in reports from India as well. Posterior segment involvement includes periphlebitis with characteristic ‘candle wax dripping appearances’ , multifocal chorioretinitis, peripheral punched out chorioretinal lesions, choroid and optic nerve granulomas, optic disc edema, papilledema, optociliary shunts, and arterial macro aneurysms. Rare manifestations of scleral nodule, vasoproliferative tumors, and retinitis have also been reported from India. Conjunctival granulomas, bilateral lacrimal gland enlargement, and orbital mass lesions have been reported from India,34. IlluA negative Mantoux (PPD) skin test is considered to suggest sarcoidosis and is usually seen in 90% of sarcoid patients. ElevateHigh resolution CT is of great help in making the diagnosis of sarcoidosis by detecting interstitial involvement and lymphadenopathy. Contrast-enhanced CT may help distinguish sarcoid from tubercular lymph node enlargement, which would usually show a central hypodensity corresponding to the caseation and peripheral contrast enhancement. The frequent use of fiberoptic bronchoscopy with transbronchial lymph node and endobronchial lung biopsies with diagnostic yield of about 80% have helped in the recognition of sarcoidosis. Microbiology (smear and culture for MTB) and histopathology are routinely done on the aspirates/biopsy material to rule out TB.Both TB and sarcoid share remarkable similarities in their clinical and radiological presentations. Both diseases present with constitutional symptoms of fever, malaise, weight loss, and fatigue. Respiratory symptoms are common to both diseases. Similar ocular manifestations are seen in both diseases. Serpiginous-like choroiditis in the Indian subcontinent are more likely to be associated with tuberculosis; howeverRadiologic similarities may be seen in both diseases. Discrete, bilateral, symmetrical lymph node enlargement is usually seen in sarcoidosis. Fibrosis and military distribution although common in TB may be seen in both diseases. Both diseases show granulomatous inflammation on histopathology. Caseation is seen in tuberculosis. Fibrinoid necrosis though rare can occur in up to 30% of cases of sarcoidosis. There are no specific features enough to differentiate the two conditions with certainty, except a positive culture of MTb. A positive PCR for MTb may be seen in the biopsy samples of sarcoidosis.Differentiating the two conditions pose a challenge, particularly in countries with high prevalence of TB. A combination of clinical, radiologic, and laboratory tests are used to make a diagnosis of sarcoidosis. Tuberculin skin test plays an important role in the diagnosis. TubercuCorticosteroids are the mainstay of treatment in sarcoidosis. In chronic cases or in those patients were steroids are contraindicated, immunosuppressants like methotrexate, azathioprine, mycophenolate mofetil, cyclosporine, and cyclophosphamide have been used,40. MethDiagnosing sarcoid in countries with high TB burden does pose a significant challenge. However, new cases of sarcoid are increasingly diagnosed in TB endemic areas in recent years due to increased awareness and better availability of diagnostic modalities. Ocular evaluations do contribute in making a diagnosis of systemic sarcoid. Negative Mantoux test is still being considered an important diagnostic test to make a diagnosis. Sarcoidosis does exist in populations with high TB burden, and the association between sarcoid and TB continues to be an enigma. Prospective studies from the Indian continent on the epidemiological data, genotypic and phenotypic variations, and the role of mycobacteria in the development of sarcoidosis would contribute a great deal in understanding this disease.Written informed consent was obtained from the patient for the publication of this report and any accompanying images. The consent to publish has been provided by the patient with the face photograph and has been approved by our Institutional Review Board .The author declares that she has no competing interests.


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.