Spliceosomal introns are parts of primary transcripts that are removed by RNA splicing. Although introns apparently do not contribute to the function of the mature transcript, in vertebrates they comprise the majority of the transcribed region increasing the metabolic cost of transcription. The persistence of long introns across evolutionary time suggests functional roles that can offset this metabolic cost. The teleosts comprise one of the largest vertebrate clades. They have unusually compact and variable genome sizes and provide a suitable system for analysing intron evolution.We have analysed intron lengths in 172 vertebrate genomes and show that teleost intron lengths are relatively short, highly variable and bimodally distributed. Introns that were long in teleosts were also found to be long in mammals and were more likely to be found in regulatory genes and to contain conserved sequences. Our results argue that intron length has decreased in parallel in a non-random manner throughout teleost evolution and represent a deviation from the ancestral state.Our observations indicate an accelerated rate of intron size evolution in the teleosts and that teleost introns can be divided into two classes by their length. Teleost intron sizes have evolved primarily as a side-effect of genome size evolution and small genomes are dominated by short introns (<256 base pairs). However, a non-random subset of introns has resisted this process across the teleosts and these are more likely have functional roles in all vertebrate clades.The online version contains supplementary material available at (10.1186/s12864-022-08760-w). The presence of spliceosomal introns within both coding and non-coding transcripts is a fundamental property of eukaryotes that separates them from eubacteria and archaea , 2. It iArabidopsis than between human and Drosophila [Nematostella vectensis [Oikopleura are unique with only 17% found at ancestral positions [Conservation of intron positions within orthologous transcripts has been observed across all eukaryotic kingdoms , 10. Intosophila . Remarkaectensis . In contositions . Hence, Drosophila species it seems that long and very short introns are selected against and thus likely to be detrimental to evolutionary success [The presence of splice sites in a transcript has a range of beneficial effects leading to more effective and better regulated translation .In mammals, 3 to 6% of transcribed sequence is exonic, while the vast majority is composed of introns. Consistent with their smaller genome sizes, teleosts have much shorter introns with the transcribed regions being around 10-20% exonic . During the annotation of the genome of In order to determine whether a bimodal intron size distribution is a general and or specific property of teleosts we extended our analysis to include the majority of vertebrate genomes present in the Ensembl database . Our obs8) bp separating short introns peaking at around 76 bp from a more broadly distributed population of longer introns.We made use of the Ensembl database to obtain gene coordinates from 172 different vertebrate species and calculated the distributions of intron sizes for all canonical transcripts from each species . Interestingly, this typical distribution was not observed in Hucho hucho and its most closely related non-salmonid species Esox lucius, as well as some other species . In smaller genomes the short intron peak (< 256bp) was almost completely dominant obscuring the much smaller peak of long introns  and others  of introns shorter than 150 bp Fig.  as previIn addition the intron size distributions in the Sauria could be clearly divided into two groups corresponding to Aves and non-Aves species. Aves genomes contained shorter introns consistent with their generally smaller genome sizes Fig. C.Lepisosteus oculatus, had a distribution more similar to mammals and birds then to teleosts. We also observed a very clear bimodal distribution in the jawless vertebrate Eptatretus burgeri (hagfish), but this distribution was different to that observed in teleosts  introns varied across the vertebrates in a manner unrelated to phylogeny. Genomes with higher proportions of such introns were usually from highly fragmented assemblies with short scaffold lengths . Hence, intron sizes reported to be smaller than this lower-bound are likely to represent annotation errors.Since the short intron peak seemed to dominate the small teleost genomes, we plotted the proportion of introns shorter than 256 bp against genome size to determine whether this represents a general property Fig.  D. IndeeThe first intron of a transcript is generally both longer and more likely to contain regulatory elements –25 and wDanio rerio transcripts as these are derived from the most complete teleost genome annotation. This intron orthology contains a total of 63,068 introns from 5,752 genes. Although we did not find orthologues in all species for all Danio rerio introns, the majority of introns were represented by at least 156 species and 90% (154/172) of species had orthologues to at least 47,609 Danio rerio introns .To investigate how intron size has evolved across the vertebrates we constructed an intron orthology by pairwise alignments of orthologous genes. To simplify the orthology we concentrated on gene families that mostly have only a single orthologue in each species. Since we are primarily interested in intron evolution in teleosts we made use of an orthology based on alignments to 10) bp  and 20% were longer than the 95th percentile  since they obscured otherwise clear general trends. We observed clearly significant levels of mutual information for all pairs of species indicating some conservation of intron sizes across all vertebrates . The amount of mutual information between species was clearly a function of taxonomic grouping with high mutual information observed within both the mammalian and Sauria clades  orthologues. The mutual information for a given Kimura distance was lowest for pairs of teleost species and highest for pairs of mammalian species Fig. B. This aLepisosteus oculatus.Teleost genomes and intron sizes are in general smaller than those of other vertebrates, suggesting a decrease in genome size that is specific to teleosts (i.e. a synapomorphy). However, it is also possible that the common vertebrate ancestor possessed a small genome with small introns and that this expanded in size in non-teleost clades. To evaluate the likelihood of these alternatives we used Sankoff maximum parsimony on a neighbour-joining tree  created from pairwise Kimura two factor distances  [Three teleost species have unusually large genomes and corresponding introns; ts (TEs) .Sphenodon punctatus having one of the largest genomes in the data set.Perhaps most striking is the apparent intron size reduction in avian species; avians are members of the Sauria clade, but compared to other Sauria species they have much smaller intron sizes. The majority of this intron size reduction appears to have occurred in the evolution of their common ancestor with smaller changes after divergence which is consistent with an inference of small genomes in the extinct pterosaur lineage  and in eWe also asked to what extent intron size minimisation is an ongoing process by inspecting the difference in intron size between extant species and their most recent inferred ancestor. Most species appear to have experienced recent increases in intron size . However, the teleost species with the smallest genomes had shorter introns than their most recent ancestors, suggesting that introns in these species are likely to become shorter in their descendants.Importantly, there is relatively little conservation in intron size within the teleosts suggesting an ongoing process of intron size diversification. Although the majority of decrease in intron size seems to have occurred prior to the most recent common teleost ancestor, most teleost species have introns considerably smaller than this. This suggests a subsequent parallel decrease in intron size across the teleost clade.Danio rerio, with two peaks at around 76 and 2000 bp . Introns inferred as shorter than ca. 2000 bp in the JVA were bimodally distributed in Takifugu rubripes, with longer introns having broad distributions peaking at less than half of the JVA size  across the teleosts and performed gene ontology (GO) enrichment analysis using human annotation. Sets of genes containing long introns were strongly enriched for specific biological process (BP), molecular function (MF) and cellular compartment (CC) terms. The enriched BP and MF terms were related to system development, cell signalling and transcriptional regulation (data not shown). This is consistent with genes containing long introns being more regulated than other genes and previous observations of short introns in highly expressed and house-keeping genes [Retention of intron sequence or length across the vertebrates suggests the presence of some regulatory function. If this is the case, then genes which do not contain any long introns should be less likely to be highly regulated and this ought to be reflected in their biological and molecular function. To test this we selected nested sets of genes containing at least one intron that is long , these terms did not overlap with those found by selecting genes with long introns.8-29 bp  and calculated depletion statistics Fig. . For mosDanio rerio chosen by their lengths across the teleosts to their orthologues in other species. Because regulatory function may be present in several regions across the intron we performed local alignments and extracted all non-overlapping alignments to Danio rerio sequences. High scores were more easily found from alignments of introns that were consistently long across the teleosts  relating the frequency of maximally scoring alignments to the total search space  expansion specifically in the Danio lineage. Our results, based on a large number of recently available vertebrate genomes, suggest instead that bimodality is the norm across the teleosts and may have arisen as a result of loss of ancestral sequences rather than expansion.Moss Caenorhabditis elegans) optimal intron sizes [Clear bimodal distributions are rare in genomic data and indicate the division of the underlying entities into distinct classes. Our observations argue that in most teleosts introns can be divided into two classes; short (< 256 bp) and long (> 256 bp) introns. The simplest explanation for our observations is that introns shorter than the antimode contain only sequences necessary for efficient spliceosome association  whereas on sizes . Hence, Danio rerio) this peak represents the most common intron size suggesting either selection for minimal intron size or molecular mechanisms favouring intron size reduction. The general lack of conservation of minimised introns indicate that any such process must be balanced by other processes that enlarge introns. Nevertheless, the preponderance of minimally sized introns in teleosts suggests that the balance of these competing processes has favoured intron size minimisation. The variance in the proportion of minimised introns, and the correlation of this minimisation with genome size  were small would indicate a reduced speed of intron growth in teleosts compared to other vertebrates. This seems less likely given that intron sizes are more variable within the teleosts than other vertebrate clades. An active process of intron and associated genome size reduction within the teleosts also seems more likely since the teleosts descend from an ancestor that underwent an additional round of genome duplication  and as sA similar decrease in genome and intron sizes has previously been reported in birds (Aves) and in flying vertebrates generally , 32, 33.Why some introns retain length across the vertebrates even in species where there is an apparent evolutionary pressure for genome size minimisation is unclear, but argues for function, either of the length itself, or encoded in sequence. Introns can contain regulatory regions  and indeThe observation that introns that are long across the teleost clade are also likely to be long in other vertebrates supports the notion that these introns have specifically retained length for functional reasons. This is consistent with vertebrate introns being divided into two classes; those containing longer functional regions and those that do not. Although both classes would be subject to minimisation by evolutionary processes, the minimal size of the former would be larger than the latter. In teleosts, which have evolved small genomes, loss of sequence from the latter class of introns would have been more prevalent than from the former class.Our observations argue that long and short introns have different modes of evolution with short introns being less likely to have conserved length Fig.  across sIt is also possible that sequence has been lost from a specific set of introns rather than there being a selective retention of sequence in introns with function. This could conceivably occur as a result of increased recombination rates at such introns, or because of an increased evolutionary selection against length at such loci (eg. as a result of high gene expression ). UntangThe small sizes of teleost genomes and introns are intrinsically linked, with intron size reflecting genome size. Since a reduction in intron size cannot markedly reduce genome size  it is likely that the short introns are a side effect of the evolution of small genomes in the teleost lineage.e) in metazoans [Why teleosts specifically should have small genome sizes remains unclear, but may be related to the high fecundity and associated developmental strategies of the teleosts. Most teleosts produce large numbers of small eggs giving rise to extreme number of offspring, and within the teleosts  and manyetazoans .Takifugu rubripes, Tetraodon nigroviridis, Betta splendens) is reminiscent of those in Drosophila species [Drosophila, large introns are found preferentially at sites of low recombination leading to the idea that an amelioration of Hill-Robertson interference (the competition between two linked loci for fixation in the absence of recombination) may be a driver for increased intron size and that species with lower N e will have larger introns [e supports more effective purifying selection [e and increased genome sizes of groundwater living asellid isopods [e, but smaller genomes than freshwater species [The distribution of intron sizes in teleosts with smaller genomes  species using locally installed Ensembl  core 98 lgorithm  implemenhttps://CRAN.R-project.org/package=entropy[https://cran.r-project.org/web/packages/ape/index.html[log2 values and transition costs being the difference in intron length state. The Sankoff maximum parsimony was implemented as an R plug-in in C and Kimura two factor distances calculated in R.Mutual information estimates were obtained using the entropy package e=entropy. Kimura e=entropy were basndex.html. The ancndex.html with intlog2 sizes. The resulting two-dimensional distributions were scaled column-wise such that the column means and standard deviations were 0 and 1 respectively and then smoothened by a serial convolution in both dimensions by a normal distribution with a mean of 0 and standard deviation of 6 (Gaussian blur). Peaks in columns representing ancestral sizes were joined and smoothened with a normal kernel to facilitate visualisation of the behaviour across the vertebrates.The major modes of ancestor to extant intron size evolution were derived from the joint distribution of introns longer than 75 bp discretised to integral 10×Size discretised correlation coefficients between two species were calculated for the set of introns larger than or equal to 76 bp in both species by dividing the subset of introns lying within the 99th percentile (to remove the effects of outliers) in one species into two sets Fig. H containInitial GO statistics were obtained using human annotation from the org.Hs.eg.db  and GOstD. rerio for all analyses shown). To allow comparisons between introns with different types of length distributions across the teleosts we selected five sets of introns, ‘long’: 1594 introns with a min teleost length of 1024 bp, ‘med’: 2000 introns with a min teleost length between 256 and 1024 bases, two sets of 2000 ‘short’ introns with min teleost lengths between 90 and 256 bases, and a set of ‘ctl’ introns with a median intron length less than 256 bp but with a D. rerio length of more than 1024 bp. The ‘ctl’ and one of the ‘short’ sets were sampled randomly from the allowable subset, the ‘long’ set included all introns satisfying the criteria. All other sets used the 2000 introns with the smallest variance in teleost intron length. Alignments where the individual search space (l1×l2 where l1 and l2 are the lengths of the two sequences) was larger than 1e8 where excluded to reduce memory requirements.We used a recursive Smith-Waterman  implemenD. rerio intron. To compensate for the effect of the differences in search space on the observed top scores we performed alignments for the ‘ctl’, ‘med’ and ‘long’ intron sets but using a non-orthologous D. rerio sequence of similar length. The resulting alignment scores were used to obtain a linear model  representing the likelihood of observing a given score for a given alignment search space . In order to remove the effect of simple repeats and other repetitive sequences from the effects we screened all aligned sequences  by BLASTn of D. rerio sequences against the D. rerio genome. Any aligned D. rerio sequence which had more than 2 sequences aligned to 50% of its length or no sequences at all (indicating simple repeats) were removed from further consideration. Not surprisingly, far more introns were removed from the non-orthologous alignments . The divergence from this model was calculated for both orthologous and non-orthologous sets and considered as a measure of effective sequence conservation.To simplify the statistical assessment we focused on the top scoring alignment for each Additional file 1 Vertebrate intron size distributions. log2 transformed intron size distributions for vertebrates. Blue and red lines indicate the distributions of all and first introns respectively. Panels are ordered by genome size from small to large. The major vertebrate clade  are indicated for each plot. Dashed vertical lines indicates the inferred mammalian small peak (105 bp) and the mammal and teleost antimodes at approximately 150 and 256 bp respectively. All intron sizes are from Ensembl version 98.Additional file 2 Vertebrate intron size distributions. log2 transformed intron size distributions for vertebrates taken from Ensembl version 104. The species taxonomic identifcation taken from the NCBI taxonomy is shown on the right for each species. The two vertical red lines indicate the positions of the antimode and peak of larger intron sizes in D. rerio.Additional file 3 Gene ontology depletion statistics. Individual panels show statistics associated with an under-representation of members of gene ontology groups in nested sets of human genes ordered by the minimum size of their largest orthologous teleost intron. The statistics are shown plotted against the size (log2 transformed) of the largest teleost intron of the nested set (x-axis). The statistics shown are: log2(observed/expected) (black), −log10 hypergeometric probabilities associated with the observations (red), q the number of members of the gene ontology set (blue) and k, the total number of genes in the nested set (purple). The dashed vertical lines show the position of the minimal hypergeometric p-values. The minimal p-values are associated with an inflexion in the observed / expected ratios and occur close to the antimode of the typical teleost intron length distribution. Pages 1-7: Biological Process (BP), 8: Molecular Function (MF), 9-11 Cellular Compartment (CC).Additional file 4 Local alignments of D. rerio intron sequences to vertebrate orthologues for a set of introns that are long (>1024 bp) across the teleosts. Alignments were performed recursively to identify all non-overlapping alignments to the D. rerio sequence.Additional file 5 Local alignments of D. rerio intron sequences to vertebrate orthologues for a set of introns with variable teleost lengths; introns were long (>1024 bp) in D. rerio but had a median teleost length less than 256 bp. Alignments were performed recursively to identify all non-overlapping alignments to the D. rerio sequence.Additional file 6–15D. rerio and teleost intron orthologues (lower) and transcript alignment (upper) used to establish the intron orthology. Grey, white and red parts indicate aligned exonic sequence, gaps and positions of intron meta-characters respectively. Colours in intron alignment represent bases . Curves lying between sequence representations show a normal kernel density smoothed estimate of local similarity ; vertical lines indicate matches. Region between exon and intron alignments indicates the location of the maximally scoring alignment in the introns. Upper sequence D. rerio. Files 6–10 and 11–15 contain alignments to teleost and mammalian sequences respectively. Each file corresponds to one panel in Fig. S17 and to one specific teleost size class: Files 6,11: long , 7,12: medium , 8,13: short.2 , 9,14 short  and 10,15 ctl .Transcript and intron alignments for points in Fig. S14. Each panel shows the maximally scoring alignment between


Nanocomposites of poly (vinylidene fluoride) PVDF with graphene nanoflakes (GNF) were prepared using two different routes. Initially, a mix-melting method was used to prepare composites, and their thermal and mechanical properties were evaluated to choose the better method for future experiment and properties investigation. Then, nanocomposite films were prepared by a simple solution-casting technique using a PVDF/graphene solution. In both cases, the amount of graphene was varied to observe and to compare their thermal and mechanical properties. The addition of graphene to the PVDF matrix resulted in changes in the crystallization and melting behaviors as confirmed by DSC analyses. Increasing the graphene content led to improved thermal stability of the PVDF nanocomposites prepared using both methods. Improvements in mechanical properties by the addition of graphene were also observed. Better performance was observed by the nanocomposites prepared by a mix-melting technique suggesting better dispersion and strong interface bonding between PVDF and graphene particles. Thermal and electrical conductivity were measured and compared. Microstructure and morphology were characterized using FTIR, XRD, and SEM analyses. N-methyl-2-pyrrolidone (NMP), which allows fillers to be dispersed in nanocomposites using a solution-casting method. PVDF is a semicrystalline polymer having a polymorph structure with pyroelectric and piezoelectric characteristics [Among the various polymers, poly(vinyl difluoride) (PVDF) has attracted much attention in various applications because of its excellent stability toward the environment, chemicals, and ultraviolet radiation and its relatively higher temperature and oxidation reactions ,2. Moreoeristics . Moreoveeristics , nylon  (99.9% pure) were purchased from Sigma Aldrich, St. Louis, MO 63118, USA and were used directly. Graphene  was purchased from Grafen Chemical Industries, Turkey, Ankara and was used without further treatment or purification. PVDF  and PVDF composites were prepared using two methods: (1) solution casting, and (2) melt mixing. N,N-Dimethyl formamide) at 90 °C under constant stirring for 4 h. Then, this was mixed with a previously prepared stable dispersion of graphene in 40 mL DMF using ultra sonication for 10 min. The stirring was continued for an additional 1 h using a magnetic stirrer @ 400 rpm. The whole mixture was degassed for 10 min in a vacuum oven, then poured into a stainless sheet Petri dish placed on a leveled flat surface and allowed to dry at 50 °C for 3–4 days. The dried films were peeled off carefully.When PVDF/graphene composites were prepared using a solution-casting method, and different weight percentages of graphene  with respect to the polymer were prepared. The required amount of PVDF was dissolved in 50 mL of DMF  with a counter rotating screw speed of 100 rpm at 190 °C for 10 min. Finally, a test sheet was prepared using a Carver hydraulic press at 165 °C for 12 min. The nomenclature of the samples is explained in TGA analysis was performed using a TA instruments Thermogravimetric analyzer, SDT Q600. Samples (6 mg) were heated in nitrogen atmosphere with purge flow of 100 mL/min and heating rate of 10 °C/min, from 30 to 850 °C.DSC was conducted for the samples at heating and cooling rates of 10 °C/min from −80 to 250 °C under a nitrogen atmosphere using TA instrument DSC Q1000, TA Instruments, 159 Lukens Drive, New Castle, DE 19720, USA. Here, 6 mg samples were again used for analysis. The melting and crystallization behavior were calculated by DSC.A Discovery DMA 850 from TA instruments, 159 Lukens Drive, New Castle, DE 19720, USA was used for the analysis. Dynamic mechanical analysis (DMA) and properties of the samples were determined in a temperature range from −80 °C to 130 °C using an oscillation temperature ramp with a constant frequency of 1 Hz. In all cases, rectangular samples were taken from the sheet with average dimensions of 17.5 × 13 × 0.6 mm.Thermal conductivities of the composites were measured using a TA instruments Fox 50 Heat flow meter, TA Instruments, 159 Lukens Drive, New Castle, DE 19720, USA. The circular samples were taken from the sheet with diameters of 2 inches.δ of the prepared samples as a function of temperature from −80 °C. Nanocomposites made using melt mixing generally showed poor dispersion in the matrix and poor interfacial bonding between the polymer and graphene because of the graphene’s high-aspect ratio, very high specific surface area, and high surface energy [Dynamic mechanical analysis was performed to determine the various dynamic mechanical properties, such as storage modulus E′, loss modulus E″, and damping coefficient tan e energy . In contTwo phenomena were clearly observed. First, the storage moduli of both composites prepared by two different techniques increased with increasing the graphene content. First, 5% dispersed graphene composite exhibited the highest value, followed by 2.0% and 0.5%, respectively. Second, melt-mixed nanocomposites showed much higher storage moduli, i.e., above 30 k MPa, whereas solution-casting nanocomposites exhibited values of only 3–6 k MPa. All samples showed a gradual decrease in storage modulus with increasing the temperature. Storage moduli of the nanocomposites increased with increasing graphene content because of polymer–graphene interactions, a reduction in polymer mobility due to the presence of graphene, increasing viscoelastic properties, and the stiffness of the composites . Grapheng) of both composites were clearly observed. In the case of melt-mix composites, the Tg peak at around −40 °C was very sharp for all samples with different loadings of graphene, and loss modulus increased with increasing graphene content from 0.5 wt% to 5 wt%. The loss modulus peak indicates interactions between the filler and matrix. It is also associated with changes in internal frictions between the filler and the matrix, molecular motions, morphology, and the dispersion between the filler and the matrix. Single and strong peaks indicate the good interfacial interactions and no-phase separation. The drop in loss modulus after the maximum peak is suggested by the free movement of the polymer chain present in the system [g, around −40 °C to −43 °C. The shift in Tg with increasing graphene content indicated the stiffness of composites achieved by adding 5% graphene nanoparticles, which reduced the mobility of the polymer chains. The beta relaxation of composites with 0.5% added graphene was clearly observed; however, it was reduced by the further addition of graphene nanoparticles.e system . The betg of the composites. The Tg of the polymer can be altered by the addition of rigid filler particles. The addition of graphene can alter the Tg of composites, which is explained by [ained by : (i) decg of the melt-mixing nanocomposites indicates better dispersion of graphene particles in the PVDF polymer. Although there is a little higher shift in the Tg peak by the addition of graphene particles, a sharp peak was observed in the melt-mix nanocomposites. Dispersibility of graphene was later confirmed and analyzed by SEM analysis. A wide Tg was observed for solution-cast samples. The Tg became wider after the addition of graphene. At higher graphene content, the aggregation and poor dispersion of graphene was reported with a lower and wider Tg transition peak [The Tg of pure PVDF and their composites by mix-melting method were found at around −30 °C; however, it was above −40 °C and more when fabricated by solution-casting method. The glass transition temperatures of nanocomposites mainly depend on the dispersion of filler particles. The very sharp Tion peak ,28. Grapion peak .Composites prepared by different methods were characterized by measuring the crystallization and melting properties. Crystallization and melting properties of neat PVDF and graphene-dispersed nanocomposites were studied as a function of graphene content. It is well known that the mechanical properties of semicrystalline polymers depend on their crystallinity and the internal microstructure . Figure c onset and Tc peaks), crystallization of enthalpy (ΔHc), and percentage of crystallization (Xc) as a function of graphene content.The DSC crystallization curves in c) of virgin PVDF films prepared by melt-mixing and with added graphene also are shown in c% of PVDF was about 50%. After the addition of only 0.5% graphene, it increased to 58%, which is about a 15% increase. The addition of 5% graphene showed more than a 20% increase in crystallinity. No changes in the onset temperature were observed for the mix-melting composites.It is evident from the DSC curves that the addition of graphene particles has marginal effect on peak melting temperature of virgin PVDF polymer. The peak melting temperature of the melt-mix PVDF sample was 158.77 °C. However, it increased to 159.46 by the addition of 0.5% graphene. It reached a maximum at 159.78 °C when 5% graphene was added. Similar behavior was also observed in nanocomposites prepared by the solution-casting technique. The peak melting temperature of the solution-cast film PVDF sample was 156.46 °C. However, it increased to 158.70 °C after the addition of 0.5% graphene. It reached a maximum at 159.27 °C when 5% graphene was added. The degree of crystallinity (Xc) of virgin PVDF film prepared by solution casting and that of graphene added nanocomposites also are shown in c% of PVDF was about 53.38%. After the addition of only 0.5%, it increased to 57.81%, which is a 12% increase. The addition of 5% graphene showed more than a 20% increase in crystallinity. Overall, there was an increase in crystallinity of the polymer composites (both synthesized by melt blending and solution casting) with an increase in graphene content or crystallinity that remained in very close range. However, particularly for MDFG-0.2, the value is an artifact attributed to possible agglomeration [The degree of crystallinity  bridging of the spherulites enhances heat flow much more efficiently than the neat PVDF. The small increase in thermal conductivity in the mid-range of the GS concentration was attributed to difficulty transferring thermal energy from sheet to sheet.According to reports, fillers with platelet shapes show advantages in terms of the morphology because of the large contact area, which allows closer contact, leading to reduced phonon scattering ,54,55. Aα phase crystals for 100, 020, 100, and 021 reflections, respectively [β phase was assigned for virgin PVDF at 27.5°. The presence of various amount of graphene in PVDF was also confirmed in the composites. Higher graphene content showed a higher peak intensity at 28.0°. The intensity of the peaks increased with increasing amounts of graphene [ectively . Howevergraphene ,56,57,58Similar XRD patterns were observed in solution-cast nanocomposites with slight variations in peak intensity and angle. D-spacing values are shown in −1 (-CF2 wagging) γ-phase, 530 cm−1 (-CF2 bending) α-phase, 840 cm−1 β or γ-phase (-CH2 rocking). Similar peaks were also observed by various investigators [−1 are proposed for spontaneous polarization [−1. Peak intensities of both nanocomposites increased with increasing graphene content. However, the graphene peak at 2978 shifted to lower values at 2916 and 2848 cm−1, which might be due to phase transformation of PVDF or good melt-mixing behavior that hindered the –CH stretching within the graphene structure [e et al. ,66,67,68tigators ,59,60. Arization ,70. Chartructure . The theThe difference in the morphology of MDF and SDF composites is due to different processing techniques. In the melt blending, the polymer is subjected to shear stress and higher temperature, which affect the crystal growth reflected by the melt-flow observation in the morphology. On the other side, the solution-cast sample showed spherulites formation, because spherulites growth is better in viscous solution .Highly dispersed graphene PVDF nanocomposites were successfully fabricated using two different methods. Mechanical properties, such as storage modulus and loss modulus of those nanocomposites, were investigated, and the melt-mixed composites showed better performance compared to the solution-cast method. SEM investigation showed the better dispersion of graphene in the melt-mixing method. Furthermore, a DSC study and SEM images suggested that the graphene behaves as a nucleating agent during the PVDF crystallization and results in an increase in crystallization temperature with increasing graphene content. Thermal properties, such as TGA and DMA results, confirmed the above findings and confirmed that the addition of graphene improved the thermal properties by increasing the graphene content.


ANXA11 gene polymorphism rs1049550 is exclusively associated with sarcoidosis, making it a key gene of interest for sarcoidosis disease pathogenesis. However, sarcoidosis is a heterogeneous disease and contradictory findings for ANXA11 have been reported for disease phenotypes. We performed a case–control association study to investigate if ANXA11 associates with benign (Löfgren’s syndrome (LS)) or chronic sarcoidosis and performed a meta-analysis on previously reported findings. A total of 262 sarcoidosis patients, of which 149 had LS and 113 chronic sarcoidosis, and 363 controls were genotyped for rs1049550. Meta-analysis included allele findings for rs1049550 from 6 additional studies. We found a significantly lower T allele frequency in sarcoidosis patients than in healthy controls  0.61, 95% confidence interval (CI) 0.48–0.77, p = 3 × 10−5). In LS the T allele frequency of 0.33, and in chronic sarcoidosis the T allele frequency of 0.26 were significantly lower than in healthy controls . Meta-analysis including previously published European, African American and Asian cohorts confirmed the association of rs1049550 with sarcoidosis and resulted in a pooled OR of 0.70 . Presence of the T allele of rs1049550 in ANXA11 is protective for sarcoidosis, including benign and chronic phenotypes of the disease.Sarcoidosis is an immune mediated granulomatous disease commonly affecting the lungs. Genome wide association studies identified many genomic regions that are shared among multiple immune mediated diseases. However,  Sarcoidosis is a systemic inflammatory disorder of unknown cause with a wide clinical spectrum. The disease is characterized by the formation of non-caseating epithelioid cell granulomas. It commonly affects the lungs and intrathoracic lymph nodes, but any organ can be involved. The clinical course and prognosis is heterogeneous. Löfgren’s syndrome (LS) is a well-defined phenotype of sarcoidosis manifesting as acute onset, bilateral hilair lymphadenopathy (BHL), erythema nodosum, and/or bilateral ankle arthritis or periarticular inflammation . PatientANXA11 and sarcoidosis, which was for the first time described in a German cohort by Hofmann et al. [ANXA11 Single Nucleotide Polymorphism (SNP) rs1049550 and sarcoidosis [Sarcoidosis is a complex disease, suggested to be the result of an interaction between an environmental trigger and a patient’s genetic make-up. Genome wide association studies (GWAS) showed that multiple genes associate with sarcoidosis, and most of these gene regions are shared among multiple immune mediated diseases. However, the association between n et al.  is uniqucoidosis ,7,8,9. AFurthermore, two studies described findings in a sarcoidosis subgroup consisting of LS patients. Mrazek et al.  found a ANXA11 is currently the only gene identified by GWAS that predisposes to sarcoidosis but not to other immune mediated diseases [ANXA11 may therefore provide the key to understanding sarcoidosis-specific disease processes. However, while the association between sarcoidosis in general and rs1049550 has been confirmed in several studies, the protective role of the rs1049550 T allele for clinical phenotypes of sarcoidosis remains to be determined. Prior to further experimental studies into the role of ANXA11 in sarcoidosis, it is essential to determine for which phenotypes the T allele may protect. The aim of this study was therefore, first, to investigate the association of the ANXA11 SNP rs1049550 with sarcoidosis in general and corroborate previous findings by adding new results in a meta-analysis. Second, to analyze clinical sub-phenotypes LS and chronic sarcoidosis to provide new evidence about the effect of rs1049550 T allele. These data will provide insights on the role of ANXA11 polymorphism in sarcoidosis in general and specific disease phenotypes and may influence experimental design and choice of therapy in the future.Most genes identified by GWAS do not only associate with sarcoidosis, but have also been identified in studies for immune-mediated disorders, such as inflammatory bowel disease and Crohn’s disease . ANXA11 diseases . ANXA11 A total of 262 Dutch sarcoidosis patients from St. Antonius Hospital, Nieuwegein, the Netherlands, were included in the study. All patients were diagnosed in accordance with the consensus of the ATS/ERS/WASOG statement on sarcoidosis .LS was defined as presenting with the classic symptoms: acute onset with bilateral hilar lymphadenopathy, fever, erythema nodosum (EN), and/or bilateral ankle arthritis . Thoracic involvement was classified using the Scadding criteria : stage 0Our group consisted of 149 patients with LS and 113 patients with chronic sarcoidosis. Patients with chronic disease had evidence of disease after at least 4 years of follow-up. Three hundred and sixty-three healthy Caucasian subjects were included as controls in this study.Written consent was obtained from all subjects, and authorization was given by the Medical research Ethics Committees United (MEC-U) of the St. Antonius Hospital, Nieuwegein .ANXA11 rs1049550 was genotyped with a pre-designed taqman SNP genotyping assay (Assay ID C_7881261_1) and the Quantstudio® 5 real-time PCR system .Genomic DNA was extracted from peripheral blood of each individual using standard methods. For meta-analysis, we included studies with (a) a case–control study design, (b) diagnosis of sarcoidosis according to internationally accepted criteria, and (c) reporting genotype or allele frequencies. For the study by Levin et al. , we switANXA11 rs1049500 are presented in Most of the studies included in the first meta-analysis also analyzed phenotypes of sarcoidosis. We also performed a meta-analysis for the phenotypes of sarcoidosis, resolving and chronic sarcoidosis, LS and non-LS, by using the data provided in the studies. In our LS patients, there were 7 patients with chronic disease. By excluding these 7 patients from our LS patients, we had a group of patients with resolving disease, which we included for the meta-analysis. We did not perform a meta-analysis with the Scadding stage data due to the variation in selected Scadding stages. However, the data about Scadding stages and p value < 0.05 was considered statistically significant. Allele and genotype frequencies were calculated for SNP rs1049550 and tested for Hardy–Weinberg equilibrium (HWE). Differences in allele frequencies were calculated with the Pearson’s goodness-of-fit Chi-square test. Three genetic models  were assessed with a logistic regression analysis on genotype results using SNPStats . The oddp-value were calculated from the data described in the original publication; when these data were not available the OR, 95% CI, and p-values were copied from the original article. Furthermore, when presenting data from previous studies, the OR, 95% CI, and 2 statistics and Cochran’s Q test was low for the allele contrast. The fixed-effect estimate method inverse variance was used. Publication bias was investigated by Egger’s regression test. Meta-analyses were performed using the allele contrast in the web tool META-Genyo . HeterogIn total, 262 sarcoidosis patients and 363 healthy controls were included. The patient cohorts consisted of 149 patients with LS and 113 non-LS patients with chronic sarcoidosis and Scadding stage II–IV . Patientp > 0.05). A significantly decreased minor T allele frequency was observed in the total group of sarcoidosis patients compared with controls . Genotyping results are shown in p = 0.01). Additionally, comparison of chronic sarcoidosis patients with controls also yielded a significantly lower minor T allele frequency of 0.26 with an OR 0.51 . There was no significant heterogeneity across the studies, and no significant publication bias was detected by Egger’s test, p = 0.52. As the T allele frequency of the control cohort in Feng and co-authors [p = 2.03 × 10−24).For meta-analysis, we included six previous studies ,5,6,7,8 In Combining previous studies and new data, we performed meta-analyses for the following phenotypes of sarcoidosis: resolving disease and chronic disease, LS and non-LS. Levin et al.  and Mrazp = 4.7 × 10−6), an OR of 0.62 , an OR of 0.69 , and an OR of 0.54 , respectively ; p = 3.58 × 10−29. Furthermore, we show that this association is independent of sarcoidosis phenotype, because in both LS and chronic sarcoidosis a similar association was present.In the present study, we investigated if The association between rs1049550 and sarcoidosis was described in a German cohort for the first time by Hofmann et al. , who perFurthermore, to determine if control cohorts conferred to established allele frequencies in ethnic cohorts, we checked rs1049550 in the genome aggregation database (gnomAD) . We obseIn several studies, associations between rs1049550 and phenotypes of sarcoidosis were investigated . These pANXA11 in LS are scarce; only two studies mention patients with LS; Mrazek et al. [n = 55) and non-LS patients in T allele frequency. We therefore analyzed rs1049550 in a large cohort of 149 Dutch LS patients, all with Scadding stage 0 or I except for nine patients with Scadding stage II. Our results demonstrated a similar association of rs1049550 with LS, as seen in sarcoidosis patients, which is a protective effect of the minor T allele. Furthermore, we showed that there is no difference between LS and chronic sarcoidosis. A meta-analysis with the pooled data from Morais et al. [ANXA11 rs1049550 applies for sarcoidosis in general and the clinical phenotypes of LS and chronic disease. Presumably, the association may be found in all sarcoidosis cohorts, regardless of phenotype.LS is one of the best studied phenotypes of sarcoidosis. This phenotype is characterized by specific genetic associations, like with HLA-DR3 , CCR5 2, and CCRk et al.  did not s et al.  and our ANXA11, no associations with other immune modulated diseases are published. The ANXA11 rs1049550 polymorphism leads to an amino-acid substitution of arginine to cysteine, at position 230 (p.(R230C)) of the Annexin A11 protein, within a highly conserved domain responsible for Ca2+-binding properties [ANXA11 mRNA expression in CD8+T and CD19+B cells after stimulation. Furthermore, ANXA11 mRNA expression in bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMC) was not associated with rs1049550 genotype [In the past years, several studies found genetic commonalities between sarcoidosis and other immune modulated diseases, such as Crohn’s disease . Howeveroperties . Annexinoperties ,26. The genotype  databasegenotype . HLA SNP. This may indicate an interplay between HLA molecules, by presenting antigens, and ANXA11 by influencing the inflammatory response. Both ANXA11 and HLA molecules are among the few proteins detectable in human B cell exosomes. In BAL fluid of sarcoidosis patients, an increased level of exosomes was shown, especially expressing major histocompatibility complex class I and II proteins [Levin et al.  found a proteins . It is hproteins . It has proteins . FurtherANXA11 rs1049550 associates with sarcoidosis in general, regardless of disease phenotype, more research is needed to elucidate the effects of the gene variant on Annexin A11 function and how this effects granuloma formation and maintenance in sarcoidosis patients. On the subject level, we found that the genetic additive or recessive (CC+CT vs. TT) model may underlie the association with sarcoidosis and its phenotypes, suggesting that future experimental studies should study differences between TT and CC genotypes. It would be of great interest to investigate ANXA11 in cell types known to be involved in granuloma formation, such as macrophages and monocytes. Other interesting cells would be CD4+, CD8+, and CD19+ T cells, which are also known to play an important role in sarcoidosis.Now that we established that


Herein, we report a novel nanofiber as a humidity sensor applied to autonomous vehicles. We prepared copper oxide nanofibers by electrospinning, characterized the obtained materials by XRD, SEM, and TEM, and fabricated MEMS sensors based on copper oxide nanofibers. The humidity sensitivity performance of the sensor was tested in different humidity environments. We found that the MEMS humidity sensor based on copper oxide nanofibers can detect the change of humidity in the environment over a large humidity range. Its fast response/mixing speed (1 s), good stability, and sensitivity make it to fully adapt to the high speed of the car. As one of the basic substances that constitute life, water plays an inaccessible but important role in human production and life. In the environment of our daily life, water can be said to be innumerable, and it affects every bit of our lives all the time . The terA humidity sensor is an important device used to detect environmental humidity, which is widely used in medical, biological, construction, and other fields . The perAmong them, the humidity sensor based on semiconductor metal oxide has excellent sensitivity, can maintain good humidity sensitivity at room temperature, and can effectively detect humidity values in a wide humidity range, gradually becoming a research hotspot in the field of humidity sensing , 9. For Copper oxide (CuO) is a typical p-type semiconductor metal oxide with a band gap of only 1.2 eV , which nCopper oxide is a typical chemical resistance sensing material, and its sensing mechanism is inseparable from the change in the conductivity of the material itself caused by the adsorption of water molecules on the surface . As showIt is worth mentioning that the operating temperature is always an important factor affecting the performance of the sensor , 19. TheIn addition to playing a huge role in medical, biological, and other fields, humidity sensors can also be applied to a new generation of automotive autonomous driving technology, as shown in In this work, we fabricated a novel MEMS humidity sensor using copper oxide nanofibers for environmental humidity detection in autonomous vehicles. The material was synthesized by electrospinning technology. Our work aims to provide a guideline for mass production at an industrial level. The obtained sample was characterized by XRD/SEM and fabricated into a MEMS sensor. The MEMS sensor integrates a Pt wire for temperature measurement. We installed the sensor on the body behind the car tire. When the vehicle is running, the updraft rolled up by the tire will bring the water molecules on the ground to the sensor position, so as to feedback the humidity of the ground, and the sensor can be used for automatic driving braking.2·6H2O) were purchased from Merck Company. Vinyl alcohol  and vinylpyrrolidone  were obtained from Sigma Aldrich. All chemicals were of analytical grade and were used as received without further purification.Ethanol (>99%) and copper chloride (CuCl2·6H2O) and polyvinyl alcohol (PVA). The specific method is to first dissolve the polyvinyl alcohol powder in deionized water, stir at 60°C for 5 h to obtain a 10 wt% PVA solution, and then add 20 wt% copper chloride solution and polyvinylpyrrolidone powder to the solution; after fully stirring for 12 h, inject the resulting solution into a 20 ml syringe equipped with a metal needle tip in a controlled electrospinning device. The applied voltage for electrospinning was 20 kV, the feeding rate of the mixed solution was 0.2 mL/h, and the distance between the glass substrate and the needle tip was 10 cm. Finally, the fibers obtained by electrospinning were peeled off with tweezers and placed in a crucible. The material was calcined at a temperature of 400 °C for two hours. Simultaneously remove organic components (PVP/PVA) in nanofibers.We used electrospinning to prepare copper oxide nanofibers by electrospinning a mixture of copper chloride (CuClKα (Kα = 1.540 Å and 2θ = 10–80 radiation as the X-ray source)). Scanning electron microscopy (SEM) images of calcined nanofibers were obtained using a LEO 1450 VP (Germany) instrument.The structure of the calcined CuO nanofibers was verified using the X-ray diffraction (XRD) technique  is used to control the different humidity and then evaporated into the well-defined concentration of the target gas in the reaction chamber. We used dry air through pure water to control the humidity level inside the test chamber by adjusting the flow rates of dry air and humid air at room temperature. During these measurements, the humidity level in the room is also monitored by a standard hygrometer.The schematic structure of the sensor is shown in θ = 35/55° suggests that CuO grows with a preferential orientation of (0 0 2) on the glass plate and illustrates the formation of the single-phase of monoclinic CuO. The resulting XRD spectrum is in good agreement with the 2θ values reported in JCPDS Card Nos. 48–1548.43.The X-ray diffraction pattern  of CuO nanofibers placed at 400°C for 2 h is shown in Figures Then, in the humidity range of 0–100%, we detected the change in the impedance of the MEMS humidity sensor during the two changes of humidity rising and falling. The results are shown in In this work, we prepared copper oxide nanofibers by electrospinning and fabricated MEMS sensors based on copper oxide nanofibers by traditional methods. The humidity sensitivity performance of the sensor was tested in different humidity environments. We found that the MEMS humidity sensor based on copper oxide nanofibers can detect the change of humidity in the environment over a large humidity range. Its fast response/mixing speed (1 s), good stability, and sensitivity make it to fully adapt to the high speed of the car. In addition, based on the significant changes in the humidity and temperature of the sensor during acceleration and deceleration, the MEMS sensor can also monitor the speed of the car, which can realize braking adjustment for autonomous driving.


Trim24 short hair RNA (shTrim24) suppressed hypoxia-induced increase in Ki-67 positive PASMCs and wound-healing rate. Furthermore, hypoxia caused enhanced phosphorylation of AKT and two major effectors of mammalian target of rapamycin complex 1 (mTORC1), S6 and 4EBP1 in PASMCs. The enhancement was then attenuated after silencing TRIM24. Both restoring mTORC1 activity and AKT reactivation abolished silencing TRIM24-mediated inhibition of proliferation and migration of PASMCs. Additionally, AKT reactivation also reversed the declined phosphorylation of S6 and 4EBP1 induced by shTrim24. In conclusion, TRIM24 is involved in the excessive proliferation and migration of PASMCs after hypoxic stimulus during PAH. The mechanism of TRIM24-mediated regulation of PASMCs is partly illustrated by promoting activity of AKT/mTORC1 signaling pathway.Tripartite Motif Containing 24 (TRIM24) is an oncogenic protein and promotes proliferation in several cancer cell lines. Nevertheless, the role of TRIM24 in proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) remains to be clarified. The current study was aimed to reveal the role of TRIM24 in proliferation and migration of PASMCs during the development of pulmonary arterial hypertension (PAH). In pulmonary arteries (PAs) of chronic hypoxia-PAH (CH-PAH) mice and PASMCs challenged with hypoxia, the expression of TRIM24 was increased. Silencing TRIM24 by   TRIM24 is increased in hypoxia-treated PASMCs and PAs of CH-PAH mice.Silencing TRIM24 suppresses hypoxia-induced proliferation and migration of PASMCs.The activation of AKT/mTORC1 is responsible for TRIM24-mediated regulation on PASMCs.Chronic pulmonary arterial hypertension (PAH) is a disease characterized by pathological pulmonary vascular remodeling and progressive pathological pulmonary artery (PA) contraction, resulting in high vascular resistance, right ventricular hypertrophy, and failure . PAH is + channel, Ca2+ channel, transforming growth factor-β receptors and membrane receptors, etc [Pulmonary artery smooth muscle cells (PASMCs) are the main component of PA, thereby exerting a crucial role in physiological regulation of pulmonary vessel. Previous research demonstrates that chronic hypoxia-induced abnormal proliferation and migration of PASMCs is involved in the development of PAH and also associated with vascular stenosis and occlusion . A varieors, etc . HoweverTripartite Motif Containing 24 (TRIM24), also called TIF1α, belongs to the member of Transcription Intermediary Factor family. It consists of a terminal plant homeodomain (PHD)-bromodomain and a RING (E3 ubiquitin ligase) domain . TRIM24 AKT /mammalian target of rapamycin (mTOR) is an important intracellular transduction system regulating cell proliferation, differentiation, migration, and protein synthesis . mTOR exWe hypothesized that TRIM24 might exert a regulatory role in the development of PAH. In the current study, we found TRIM24 was increased in PAs of chronic hypoxia-pulmonary arterial hypertension (CH-PAH) mice and hypoxia-challenged PASMCs. Silencing TRIM24 inhibited proliferation and migration of PASMCs via suppressing AKT/mTORC1 activity. This study aimed to search novel target for preventing PAH.2) for 3 weeks. Normoxic control mice were subcutaneously injected with equivalent DMSO and kept in room air. All animal experiments in the study were performed in accordance with the Institutional Animal Care and Use Committee and the Ethic Committee of The General Hospital of Western Theater Command . Mice were finally anesthetized with pentobarbital (100 mg/kg) and sacrificed for other experiments.C57BL/6 J mice obtained from Dashuo Animal Science and Technology . Mice were housed at room temperature in a 12-h light–dark cycle and have free access to water and food. CH-PAH mice model were established by using SU5416-hypoxia as previously described . BrieflyRVSP and RVHI were measured as previously described . BrieflyThr308 , AKT , p-S6Ser235/236 , S6 , p-4EBP1Thr37/46 , 4EBP1 , phosphoinositide 3-kinase (PI3K) p110α , cyclin D1  and GAPDH  overnight at 4°C. The membranes were next incubated with secondary antibodies.Immunoblotting was performed as previously described . The lys2, 92% N2 and 5% CO2 for 6, 12 and 24 hours (h), while normoxic PASMCs were cultured at 37°C under the condition of 21% O2, 74% N2 and 5% CO2 for the same time points. The adenovirus carrying Trim24 short hairpin RNA (shTrim24) and control viruses (shCon) were constructed according to the manufacturer’s instructions . For transfection in PASMCs, cells were transfected with relative adenovirus (3 pfu/cell) for 72 hours. For siRNA transfection, PASMCs were incubated with the mixture of control siRNA or Tsc1 siRNA (siTsc1) and 10 μL X-tremeGENE siRNA transfection reagent for 8 h. Then, PASMCs were cultured for another 48 h before experiments. The sequence of siTsc1: GCUUUGACUCUCCCUUCUA [PASMCs were isolated and cultured as previously described . BrieflyCCCUUCUA . SC-79  with an appropriate post hoc test. P < 0.05 was considered to be statistically significant. All data are expressed as mean ± standard deviation (SD.).Comparisons between two groups were conducted by Student’s In our current study, we explored the role of TRIM24 in proliferation and migration of PASMCs by utilizing the adenovirus carrying shTrim24. We demonstrated that silencing Trim24 suppressed hypoxia-induced proliferation and migration in PASMCs in an AKT/mTORC1-dependent manner.To investigate whether TRIM24 is implicated in the modulation of PAHs, we first determined the expression of TRIM24 in PAs of CH-PAH mice. The model of CH-PAH mice was confirmed by increased RVSP ) and RVHin vitro scratch assay. Hypoxia treatment accelerated the rate of wound-healing compared to normoxic condition. Simultaneously, the increased wound-healing rate of PASMCs was slowed down once TRIM24 was silenced  positive PASMCs compared to normoxic treatment. However, the elevation of Ki-67 positive cell number was obviously reversed after silencing TRIM24 ). Subseqsilenced ). These We next tried to reveal the molecular mechanism by which TRIM24 promoted proliferation and migration of PASMCs. It was shown that both excessive proliferation and migration of PASMCs and the development of PAH were associated with a significant increase in mTORC1 activity . WhetherS6 and 4EBP1 are two major downstream effectors of mTORC1. Therefore, the phosphorylation levels of the two proteins reflect the activity of mTORC1. As shown in our data, the phosphorylation levels of S6 and 4EBP1 in PASMCs were both increased after hypoxic stimulus . MoreoveWe next identified the signaling pathways that underlie mTORC1 inactivation in PASMCs after shTrim24 transfection. Serving as a sensor for cellular stress and energy metabolism, mTORC1 is affected by varies of stress-associated kinases such as PI3K/AKT . TRIM24 As illustrated in Published observations confirm the important role of AKT/mTORC1 axis in proliferation and migration of PASMCs . Since rin vitro reduced proliferation and migration of PASMCs challenged with hypoxia. In PASMCs, TRIM24 inhibition reduced AKT phosphorylation and mTORC1 activity. AKT reactivation and restoring mTORC1 activity both reversed silencing TRIM24-mediated regulation of PASMCs, which provided novel insight into the regulatory role and mechanism of TRIM24 in proliferation and migration of PASMCs and revealed a potential therapeutic target for PAH.The current study was aimed to reveal the regulatory role of TRIM24 in the proliferation and migration of PASMCs and the underlying mechanism. The protein level of TRIM24 was elevated in both PAs of CH-PAH mice and PASMCs after hypoxic treatment. Silencing TRIM24 TRIM24 is a member of the transcription intermediary factor family and plays important roles in T cell differentiation, innate immune regulation, and tumor development . TRIM24 mTORC1 is a crucial serine/threonine kinase and implements regulatory roles in cell growth, proliferation, and differentiation, etc . mTORC1 The PI3K/AKT is a classical signaling pathway that regulates proliferation, migration of PASMCs and PAH . mTORC1 The current study also has some limitations. For example, how TRIM24 affects AKT phosphorylation is still unclear. In addition, though we have confirmed the role of TRIM24/AKT/mTORC1 axis in proliferation and migration of PASMCs, whether this signaling pathway works in humans remains unknown. These doubts need further investigation in our future study.In summary, our findings identify TRIM24 as a potential target for the prevention and treatment of PAH. We first demonstrated that TRIM24 was increased in hypoxia-challenged PASMCs and PAs of CH-PAH mice. Decrease in TRIM24 abolished hypoxia-associated proliferation and migration of PASMCs, indicating hypoxia-induced upregulation of TRIM24 was responsible for excessive proliferation and migration of PASMCs. Next, we found mTORC1 signaling was activated by TRIM24 and acted downstream of TRIM24 to promote proliferation and migration of PASMCs. Our data also revealed a mechanism by which TRIM24 activated mTORC1 in PASMCs through phosphorylating AKT. The newly elucidated roles of TRIM24 in PASMCs also help us better understand the pathological mechanisms of PAH development.Click here for additional data file.


Exechonella vieirai, and five, 17 and 18 introns were found in the partial mitogenomes of Parantropora penelope, Discoporella cookae and Cupuladria biporosa, respectively. Intron-encoded protein domains reverse transcriptase and intron maturase (RVT-IM) were identified in all species. Introns in E. vieirai and P. penelope had conserved Group II intron ribozyme domains V and VI. Conserved domains were lacking from introns in D. cookae and C. biporosa, preventing their further categorization. Putative origins of metazoan introns were explored in a phylogenetic context, using an up-to-date alignment of mitochondrial RVT-IM domains. Results confirmed previous findings of multiple origins of annelid, placozoan and sponge RVT-IM domains and provided evidence for common intron donor sources across metazoan phyla. Our results corroborate growing evidence that some metazoans with regenerative abilities  are susceptible to intron integration, most likely via horizontal gene transfer.Animal mitogenomes are typically devoid of introns. Here, we report the largest number of mitochondrial introns ever recorded from bilaterian animals. Mitochondrial introns were identified for the first time from the phylum Bryozoa. They were found in four species from three families (Order Cheilostomatida). A total of eight introns were found in the complete mitogenome of  Since this origin, the various evolutionary trajectories of eukaryotes have produced mitochondrial (mt) genomes (mitogenomes) that vary substantially in their sizes, gene content, genetic code, organization and physical shape2. Plant, fungal and other non-metazoan eukaryote mitogenomes range in size from 12 to 236 Kb, 66–11.3 Mb and 34–113 Kb, respectively and harbour a large proportion of non-coding DNA, including introns. In contrast, bilaterian mitogenomes are typically circular, streamlined molecules of approximately 16 kb (range: 11–50 Kb), which are typically devoid of introns3. The lack of introns in bilaterian mitogenomes has been attributed to the relatively high mutation rate of animal mtDNA, which can be ~ 9 to 25 times faster than that of corresponding nuclear DNA4. Introns, like all non-coding DNA, provide a substrate for potentially harmful mutations in the presence of high mutation rates, and are therefore hypothesised to have been purged from most animal mitogenomes5.A crucial step in early eukaryote evolution was the origin of mitochondria, which arose by incorporating α Proteobacteria endosymbionts as cellular organelles9. Group II introns occur in bacterial, archaeal and organellar genomes , but are not found in nuclear genomes11. Both types are self-splicing, mobile ribozymes, but they are distinguished by their splicing mechanisms. Group I introns use external guanosine nucleotides as cofactors, whereas Group II introns use a mechanism more akin to that found in spliceosomal introns8 and are believed to be precursors of eukaryote spliceosomal introns, retroelements and spliceosome components12. Both types frequently harbour intron-encoded proteins (IEPs) that facilitate intron mobility; Group I introns typically encode homing endonucleases, whereas Group II IEPs include reverse transcriptase (RVT) and intron maturase (IM) domains12. In terms of their secondary structures, Group II introns form six domains (DI—DVI) that radiate out from a central loop. Based on the intricacies of their secondary structures, five Group II introns have been identified 14. Conversely, Group I introns typically form secondary structures of P1–P10 stems15.In addition to spliceosomal introns, which occur in the nuclear genomes of eukaryotes and require ribonucleoprotein complexes called spliceosomes to facilitate their excision from precursor mRNA, there are two other types of introns. Group I introns occur in bacterial, archaeal, viral and organellar genomes as well as some eukaryote nuclear genomes22 and Placozoa26, whereas Cnidaria are only known to harbour Group I introns29. Conversely, in bilaterians, mitogenome introns are apparently very rare, but Group II introns have been recognised in the cox1 gene of annelids Nephtys (Phyllodocida)30, Glycera fallax, G. unicornis (Phyllodocida)31, Decemunciger sp. (Terebellida)32 and the myzostomid Endomyzostoma sp.33. Furthermore, high-throughput DNA sequencing has revealed additional introns of unknown type(s) in other protein-coding genes (PCGs) of Decemunciger sp. (nad1 and nad432) and the mollusc Cucullaea labiata (Arcoida) (cox135).Although typically not found in metazoans, both intron types have been recorded in the mitogenomes of non-bilaterian phyla Porifera36, are modular invertebrates, found worldwide in aquatic habitats, including freshwater bodies, shallow coastal waters, and the deep sea38. Almost exclusively colonial, they form encrustations on a variety of substrates, construct erect, three-dimensional structures or produce free living disks40. There are three classes of bryozoans. The least diverse of these is the exclusively freshwater class Phylactolaemata with ~ 86 Recent species41. The Phylactolaemata forms the sister group to all remaining bryozoans42. The class Stenolaemata, whose only surviving order is the exclusively marine Cyclostomatida, constitutes ~ 543 Recent species41 and they form the sister group to the class Gymnolaemata42. Although predominantly marine, the Gymnolaemata also includes a few brackish and freshwater species. It is the most speciose class with their ~ 5240 Recent species, of which ~ 319 belong to the order Ctenostomatida and ~ 4921 belong to the order Cheilostomatida41; the Cheilostomatida nest within a paraphyletic Ctenostomatida42. Apart from phylactolaemates, which can disperse via asexually produced propagules called statoblasts, and some gymnolaemates which can form resting structures called hibernacula43, bryozoan colonies are typically established by sexually produced larvae, which settle and metamorphose into the founding module (zooid) of the colony, the ancestrula37. The ancestrula then buds the adjacent zooids, which in turn continue the colony growth via asexual budding of further zooids44. Reparative growth of damaged colony parts frequently occurs45, and some species propagate largely via colony fragmentation and subsequent regeneration46.Here, we reveal the widespread occurrence of introns in the mitogenomes of taxa of the lophotrochozoan phylum Bryozoa. Bryozoans, whose fossil record goes back as far as the Cambrianhttps://www.ncbi.nlm.nih.gov/), none of which contain introns . As part of a wider study sequencing hundreds of mitogenomes from species of the bryozoan order Cheilostomatida, genome-skimming has revealed the occurrence of introns in multiple PCGs in single mitogenomes, multiple introns within single genes, as well as in intergenic regions. Here, we document introns discovered in the mitogenomes of four cheilostome bryozoans: two encrusting species – Parantropora penelope  and Exechonella vieirai  and two free-living species from the family Cupuladriidae , Cupuladria biporosa (AW817) and Discoporella cookae (AW3739)  and Contig B . Missing genes were: PCGs atp6 and nad4 and tRNAs trnA, trnC, trnE, trnH, trnR, trnT, trnV and trnW; genes nad2 and nad6 were incomplete at their 5’ ends. For diagrammatic representation of gene order of mt fragments, see Fig. Numbers of raw Illumina reads per species, contig sizes and coverage, and GenBank accession numbers are given in Table A large number of introns were found within several PCGs, sometimes multiple introns per gene, across multiple species of cheilostome bryozoans Fig. . We elimE. vieirai harboured single introns in three genes , cytb (PP-cytb) and nad5 (PP-nad5) and two introns in cox1 (PP-cox1-i-ii)  Fig. . FurtherC. biporosa and D. cookae Although there are substantial similarities in the intron distribution across these two species , D. cookae harboured three introns in nad5, whereas C. biporosa harboured only two introns in this gene. A further two introns were found in nad4 in C. biporosa, but due the partial nature of the D. cookae mitogenome, nad4 was missing from its contigs, thus, no inferences can be made about shared introns for this gene. Furthermore, both species have one intergenic region that harbours an open reading frame containing RVT and IM domains  IEP with RVT-IM domains, b) conserved Group II ribozyme domains V and VI, c) Group II intron-typical start and stop motifs (except the non-standard start motif in PP-cox1-ii). All introns in these two species finish on YAY, which is a typical Group IIA end motif, versus RAY, which is typical for Group IIB introns13. However, the secondary structures of the two IEP-containing introns 50. Although the secondary structure drawing of EV-cox1 , AU-rich with a base bias of U > A > G > C, have degenerate Group II intron-like boundaries , and lack any consistent secondary structure54, although they have been shown to have a structure resembling Group II intron domain VI55. Regarding the cupuladriid introns, their lengths, although shorter than introns in the non-cupuladriids, exceed the typical Group III intron size. Intron sizes range from 221–519 bp (average 272 bp) and 240–397 bp (average 273 bp) in C. biporosa and D. cookae, respectively . This was found toward the 5’ end of intron CB-cox3-ii (AACUAAG). Similarly, only two of the 15 introns in D. cookae for which the 5’ ends were available, had the degenerate Group III intron motif towards their 5’ ends: DC-nad5-i (UGACUUUUG) and DC-nad4L (GUAUG); nucleotides not emboldened are currently considered parts of the introns as they are the nucleotides immediately following the preceding exons. The degeneracy of the 3’ end motif means that it is present too frequently to make any meaningful inferences. Lastly, the secondary structure drawings frequently show a stem towards the 3’ end  of bryozoan mitochondrial introns in an evolutionary context, a phylogeny of RVT-IM domains with representatives from across the tree of life was constructed. The following description of phylogenetic interrelationships of RVT-IM domains Fig.  focussesCupuladria biporosa and Discoporella cookae is strongly supported (91% bootstrap support [bs]). They formed strongly supported Clade I together with RVT-IM domains from polychaete taxa Glycera unicornis , Glycera fallax  and Nephtys sp. (94% bs); this close association of annelid terminals and the non-monophyly of the two tandemly repeated Glycera fallax FS14 intron copies I1 and I2 (see Clade IV description below for copy I1 position) was also found by Richter et al.31. This clade was sister to a paraphyletic assemblage of fungal sequences that also included the diatom Halamphora calidilacuna, and a strongly supported clade (99% bs) composed of RVT-IM domains of rhodophyte genera Pyropia, Bangia and Neoporphyra and the sponge Axinella verrucosa (copy 96620), which formed the moderately supported sister (63% bs) to a clade of five placozoan sequences . As in Signorovitch et al.24 and Huchon et al.20, an independent origin of Placozoa sp. strains BZ49  and BZ2423 + Trichoplax adhaerens  was found in our analysis. Furthermore, this close association of the abovementioned rhodophyte genera Bangia and Pyropia, placozoans and sponges is in agreement with findings by Huchon et al.20.The common origin of RVT-IM domains in the two cupuladriids 26, Placozoa sp. strain BZ242324 and Trichoplax adhaerens23 nested in a moderately supported clade (67% bs) with charophyte Coleochaete, chlorophyte Ulva and fungi Auricularia and Termitomyces. In the wider context of a well-supported node (80% bs) that included members of Rhizaria, Fungi, Chlorophyta, Charophyta, Marchantiophyta and Bryophyta as well as placozoan Clade III, our results, although using a more elaborate taxon sampling, broadly agrees with results obtained by Huchon et al.20. All genera recovered as close relatives of Clade III in Huchon et al.20, i.e. Marchantia, Treubia, Klebsormidium, Chlorokybus and Schizosaccharomyces were also found to be part of this clade. A close association  of Trichoplax adhaerens with Marchantia, Chlorokybus and Schizosaccharomyces was also found by Vallès et al.30. A novel finding of the present study, as a result of a broader taxon sampling, is the strongly supported (89%) sister-group relationship of placozoan Clade III with Ulva compressa.A second clade of placozoan sequences composed of Placozoa sp. haplotypes H2, H17Exechonella vieirai was placed with strong nodal support (82% bs) in a clade with polychaete taxa Glycera fallax  and Decemunciger sp. The sister to Clade IV was formed by the tracheophyte Selaginella, but support for this was weak (26% bs). Similarly, neither Richter et al.31 nor Bernardino et al.32 were able to determine a supported position for Glycera fallax , although it formed a weakly supported clade with RVT-IM domains from Marchantia and land plants  in both of their analyses. Considering that both used the alignment by Zimmerly et al.56 as a starting point , this is to be expected. In contrast to the present analysis, in Bernardino et al.32 the RVT-IM copies of Decemunciger sp. formed a strongly supported clade with Nephtys sp. (91% bs), nesting in a weakly supported clade with Glycera unicornis (FS15 isolate31) and Glycera fallax . This difference in topology might be explained by the high sequence divergence in Decemunciger sp. copies, as evidenced by the long branch leading to the clade of the three Decemunciger sp. copies in Bernardino et al.32 and in Fig. Exechonella vieirai copy, might have led to this difference in topology.Clades I-IV, there were two terminals that did not group with any other metazoan RVT-IM copies. The first, Axinella verrucosa (copy 114120), formed a weakly supported clade (40% bs) with rhodophyte genera Hildenbrandia, Ahnfeltia, Pyropia, Bangia, Neoporphyra , all of which also formed the sister group in Huchon et al.20. In addition, in the present analysis, this rhodophyte clade also included Paralemanea, Grateloupia. This A. verrucosa + Rhodophyta clade formed the sister group with strong support (95% bs) to a strongly supported clade (99% bs) composed of eustigmatophycean Nannochloropsis, a gamma-proteobacterium, and diatom genera Psammoneis, Pseudo-nitschia, Thalassiosira and Cylindrotheca; in Huchon et al.20 this corresponding sister group was formed of Thalassiosira and Chattonella. Thus, there is broad agreement with our analysis and that by Huchon et al.20, except that our broader taxon sampling added additional genera. We also observed, judging by the associated taxa in our analysis and that by Huchon et al.20, the two intron copies of Axinella verrucosa  came out in switched positions in ours and their topologies. We suspect that this could either be due to a mislabelling of Fig. 7 in Huchon et al.20 or a mislabelling of their GenBank accession records. The position of the second terminal, Parantropora penelope, could not be unambiguously resolved as it nested on a very long branch in a poorly supported clade (16% bs) together with representatives of Fungi, Haptophyta, Bacillariophyceae, Chlorophyta, Charophyta and Marchantiophyta.In addition to Exechonella vieirai and Parantropora penelope. Furthermore, we infer a separate but possibly shared origin in the two cupuladriid taxa Cupuladria biporosa and Discoporella cookae . Moreover, the monophyly of bryozoan and annelid RVT-IM domains  and vertical transmission58. However, much evidence points to intron insertion via HGT from microbial or algal donors. In sponges, putative intron donors include fungi22, rhodophytes, diatoms and raphidophytes20. Additionally, placozoans have been proposed as possible intron donors in sponges21. Our analysis confirmed a close association of RVT-IM domains in sponges and placozoans (Clade II), as well as a close relationship of both with red algae  which is consistent with their particular reproductive life history strategy. The cupuladriid family are all free-living bryozoans that rely heavily on clonal propagation by fragmentation and regeneration of their disc-shaped colonies (e.g.46). This mode of reproduction often results in zooids being split open60 which could provide intron donors easy access to undifferentiated somatic cells. Discoporella cookae has undergone rates of clonal propagation exceeding 95% for at least 8 million years61 and clonal propagation in free-living bryozoans extends into the Cretaceous62. More generally, bryozoans are a rich source of bioactive compounds, many of which are likely produced by microbial symbionts64. Although the associated microbial species are mostly unknown, detailed studies on some bryozoan species have shown their colonies to harbour symbiotic bacteria on the surfaces of rhizoids65, intercellularly in the pallial sinus of their larvae and across larval surfaces66, as well as in tissue strands (funicular cords) that connect colony modules to one another67. Furthermore, bryozoans are often associated with microbial films68 and have been found to be infected by fungal species69. These close relationships between bryozoan hosts and microbes may have facilitated the acquisition of introns in this group.A commonality of the metazoan taxa harbouring Group II introns is their ability to bud and regenerate. The idea that budding and regeneration ability may favour intron transmission via somatic cells/tissues was already formulated by Szitenberg et alE. vieirai and P. penelope showed that the five copies of the latter formed a monophyly with strong nodal support . Thus, there is only evidence for a species-specific common ancestry of IEP-less Group II introns in P. penelope. This suggests that these introns may have either been inserted multiple times from the same type of source organism or that the introns self-propagated within the mitogenomes post initial insertion. Conversely, phylogenetic analyses of the IEP-less introns of the two cupuladriid taxa was completely unresolved and showed no grouping by species  and five, 18 and 17 in the incomplete mitogenomes of P. penelope, C. biporosa and D. cookae, respectively. This is the largest number of introns ever recorded in bilaterians . Furthermore, RVT-IM domains have only ever been found in cox1 in metazoans . Although this was also the case in our E. vieirai and P. penelope mitogenomes, open-reading frames with RVT-IM domains were found in intergenic regions in C. biporosa and D. cookae. Thus, this is the first time that these domains have been found residing outside of cox1 introns in metazoans.There are several features in the bryozoan mitogenomes investigated here that distinguish them from other bilaterian and metazoan intron-harbouring mitogenomes. In the context of bilaterians, our observed intron frequency in the four species of Bryozoa is unprecedented: eight in 31, increased genome sequencing has revealed more Group II introns within the Bilateria, with more likely to be uncovered in the future. Nevertheless, for now at least, the frequency of mitochondrial introns in bryozoans is exceptional when compared to other bilaterians. This provides an unparalleled opportunity for bryozoans to perhaps become not only a model for studying introns but also bilaterian mitogenome architecture overall, challenging our understanding of their function and evolution. Initial observations of intron absence versus presence in a broad phylogenetic context point towards a random acquisition process, rather than one guided by shared common ancestry (unpublished data). However, future work that dissects the process of intron gain and loss amongst closely related species ought to be explored. Furthermore, seeing that HGT from microbial donors is a likely mechanism for intron integration, studying the intraspecific distribution and variability of introns could provide interesting insights into their heritability and persistence.As foreseen by Richter et al.Parantropora penelope  for E. vieirai, P. penelope and D. cookae. Sequencing was performed by Novogene (HK) Company Limited on the Illumina HiSeq 4000 platform  using 2 × 150 bp paired-end sequencing.Total genomic DNA (gDNA) was extracted using the DNeasy Blood & Tissue Kit (Qiagen) following manufacturer’s instructions. Double stranded (ds) DNA concentration was quantified using a Qubit™ fluorometer using either the Qubit™ dsDNA BR (Broad Range) or dsDNA HS (High Sensitivity) assay kits. Dual-indexed libraries were prepared using the TruSeq DNA Nano Library Prep Kit  for 70 and assembled de novo using SPAdes 3.13.071 with k-mer sizes of 33, 55, 77, 99 and 127. Contigs of putative mitogenomes were identified by conducting blast searches72 against a local custom database of reference bryozoan mitogenomes in Geneious 11.1.4 (https://www.geneious.com). Candidate mt contigs were further verified by blastn searches against the NCBI database (https://blast.ncbi.nlm.nih.gov).Paired-end reads were trimmed using Trimmomatic Version 0.39http://mitos.bioinf.uni-leipzig.de/)73. PCG boundaries (start/stop codons) were refined manually in Geneious following alignment with curated sets of reference bryozoan sequences using TranslatorX (http://translatorx.co.uk/)74. Mitogenomes were circularised where possible using the Repeat Finder plug-in in Geneious. Assembly quality and read coverage were assessed by reference mapping of trimmed reads to mitogenome contigs under strict settings in Geneious. The settings used were as follows: allow gaps = maximum 1% per read, maximum gap size = 3, minimum overlap = 50, minimum overlap identity = 95%, word length = 18, index word length = 13, ignore words repeated more than 12 times, maximum mismatches per read 2%, maximum ambiguity = 4.Mitogenome contigs were annotated using MITOS (https://www.ebi.ac.uk/Tools/st/emboss_transeq) and searched using blastp (https://blast.ncbi.nlm.nih.gov) to identify additional exons. In cases where blastp failed to identify matches, the amino acid translation was scanned for conserved motifs as gleaned from reference alignments.Introns were identified through the observation of fragmented exons. Exons were knitted together in light of alignments with non-intron containing reference data. In cases where exons had not been identified by MITOS, nucleotide data in between exons were translated into amino acids using EMBOSS Transeq (http://pfam.xfam.org/75) and blastp. No RVT-IM domains were initially found for Cupuladria and Discoporella. However, blastx searches of the complete mt fragments against the ucalgary.ca database  identified intergenic regions that were subsequently confirmed as RVT-IM domains by blastp and Pfam searches. Significant as well as insignificant Pfam results were considered. Conserved Group II ribozyme domains were identified using Rfam (https://rfam.xfam.org76). Intron boundaries were refined by searching for conserved 5’ (GUGYG) and 3’ terminals ([Y]AY)13.In order to identify intron-encoded RVT-IM domains, intron sequences were translated into amino acids using EMBOSS Transeq from all six reading frames. Translated sequences were subjected to searches using Pfam 33.1 , with a final extension step at 72 °C for 10 min (see Supplementary Table ann). Two PCRs (EV-cox1 and PP-cox1-i) failed and were repeated with Takara Long-range PCR kit (Takara Bio Inc.). Total reaction volume was 50 μl, using 0.5 μl enzyme, 5 μl buffer, 8 μl dNTPS, 2 μl of 10 μM of each primer and 4 μl gDNA. PCR cycling conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 40 cycles at 94 °C for 20 s, 51 °C for 30 s, 68 °C for 3 min, with a final extension at 68 °C for 10 min. Sequencing using the PCR primers was performed using an Applied Biosystems 3730 DNA Analyser, using BigDye version 3.1. Sanger reads were edited in Geneious. Sequence identity was verified by mapping to the reference contigs in Geneious.The E. vieirai and P. penelope were subjected to secondary structure folding at 20 °C using mfold (www.unafold.org)79. The consensus Group II secondary structures on the Zimmerly website 50 were taken as guides for subsequent manual secondary structure manipulation using forna (http://rna.tbi.univie.ac.at/forna/)80 in conjunction with iterative folding of parts of the sequences in mfold. Regarding intron sequences of C. biporosa and D. cookae, secondary structure folding was conducted at 20 °C using mfold, but no manual manipulations were performed.Following the excision of IEP ORFs, intron sequences of Trichoplax adhaerens (DQ112541), Placozoa sp. , Hoilungia sp. , Axinella verrucosa , Nephtys sp. (EU293739), Glycera unicornis , Glycera fallax  and Decemunciger sp. ; this is the first time that RVT-IM domains of placozoan haplotypes H2, H9, H11, H17, H19 and of the previously unsampled genus Hoilungia (H24)26, have been put into a ‘tree-of-life’ phylogenetic context. These ORFs were trimmed following Pfam analyses. No Pfam match for RVT-IM could be obtained for Endomyzostoma sp. In order to maximise our chances of identifying metazoan Group II intron origins, we also included blast matches of the abovementioned accessions and the bryozoans RVT-IM domains in our analyses. Duplicate accessions were removed in Mesquite v.3.5181. Amino acid sequence duplicates were removed using the web server for FASTA tools unique sequences (https://www.ncbi.nlm.nih.gov/CBBresearch/Spouge/html_ncbi/html/fasta/uniqueseq.cgi).Phylogenetic analyses of Group II intron RVT-IM domains were carried out as follows: Genbank searches of the protein database were conducted using terms ‘reverse transcriptase AND mitochondrion’  and ‘maturase AND mitochondrion’ . Furthermore, RVT-IM domains of the following metazoans were added: 82 using the --auto setting. Alignments were examined by eye in Geneious and obvious outliers were removed. Initial neighbour-joining trees, produced in PAUP* v.4.0a83, identified a large clade composed of plant RVT-IM sequences. As none of the focus sequences were nesting in this clade, it was removed from further analyses. The final alignment was carried out using the MAFFT settings --amino --bl 30 --genafpair --maxiterate 100. Ambiguously aligned positions were excluded using the stand-alone version of Gblocks v. 0.91b85 using the following settings: minimum number of sequences for a conserved position = lowest; minimum number of sequences or a flank position = lowest; maximum number of contiguous non-conserved positions = 10; minimum length of a block = 5; allowed gap positions = with half. The final alignment consisted of 1059 terminals and 3947 positions, of which 367 remained included. ModelTest-NG v.0.1.686 was used to determine the best model of amino acid substitution. Maximum likelihood (ML) phylogenetic analysis with 1000 fast bootstrap replicates was carried out using RAxML v.8.2.1287 under the LG + G4 + F model. Uncorrected p-distances were calculated using PAUP* v.4.0a83.Initial alignments were constructed in MAFFT v.7.453Exechonella and Parantropora, and (b) IEP-less introns of Cupuladria and Discoporella were carried out. The outgroup for analysis (a) was the well-annotated Group II intron Nephtys sp. (EU29373930) from which the IEP ORF had been excised according to their secondary structure drawing ; no suitable outgroup was available for analysis (b). Alignments were constructed in MAFFT with settings --genafpair --maxiterate 1000. Ambiguously aligned positions were excluded using the stand-alone version of Gblocks as outlined above. ModelTest-NG was used to determine the best model of nucleotide substitution. ML with 1000 fast bootstrap replicates and BI analyses were conducted in RAxML and MrBayes v.3.2.688 under the HKY + G model. Two parallel MrBayes runs were performed for 5 million generations. The burn-in was defined as the point at which the average standard deviation of split frequencies was < 0.01.To assess common ancestry of introns within species, phylogenetic analyses of (a) Group II IEP-less introns of Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6.Supplementary Figure S7.Supplementary Figure S8.Supplementary Figure S9.Supplementary Legends.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.