Phenotypic characterization and candidate gene analysis of a short kernel and brassinosteroid insensitive mutant from hexaploid oat (Avena sativa).

In an ethyl methanesulfonate oat (Avena sativa) mutant population we have found a mutant with striking differences to the wild-type (WT) cv. Belinda. We phenotyped the mutant and compared it to the WT. The mutant was crossed to the WT and mapping-by-sequencing was performed on a pool of F2 individuals sharing the mutant phenotype, and variants were called. The impacts of the variants on genes present in the reference genome annotation were estimated. The mutant allele frequency distribution was combined with expression data to identify which among the affected genes was likely to cause the observed phenotype. A brassinosteroid sensitivity assay was performed to validate one of the identified candidates. A literature search was performed to identify homologs of genes known to be involved in seed shape from other species. The mutant had short kernels, compact spikelets, altered plant architecture, and was found to be insensitive to brassinosteroids when compared to the WT. The segregation of WT and mutant phenotypes in the F2 population was indicative of a recessive mutation of a single locus. The causal mutation was found to be one of 123 single-nucleotide polymorphisms (SNPs) spanning the entire chromosome 3A, with further filtering narrowing this down to six candidate genes. In-depth analysis of these candidate genes and the brassinosteroid sensitivity assay suggest that a Pro303Leu substitution in AVESA.00010b.r2.3AG0419820.1 could be the causal mutation of the short kernel mutant phenotype. We identified 298 oat proteins belonging to orthogroups of previously published seed shape genes, with AVESA.00010b.r2.3AG0419820.1 being the only of these affected by a SNP in the mutant. The AVESA.00010b.r2.3AG0419820.1 candidate is functionally annotated as a GSK3/SHAGGY-like kinase with homologs in Arabidopsis, wheat, barley, rice, and maize, with several of these proteins having known mutants giving rise to brassinosteroid insensitivity and shorter seeds. The substitution in AVESA.00010b.r2.3AG0419820.1 affects a residue with a known gain-of function substitution in Arabidopsis BRASSINOSTEROID-INSENSITIVE2. We propose a gain-of-function mutation in AVESA.00010b.r2.3AG0419820.1 as the most likely cause of the observed phenotype, and name the gene AsGSK2.1. The findings presented here provide potential targets for oat breeders, and a step on the way towards understanding brassinosteroid signaling, seed shape and nutrition in oats.

Single-cell genomics of a bloom-forming phytoplankton species reveals population genetic structure across continents.

The study of microbial diversity over time and space is fundamental to the understanding of their ecology and evolution. The underlying processes driving these patterns are not fully resolved but can be studied using population genetic approaches. Here we investigated the population genetic structure of Gonyostomum semen, a bloom-forming phytoplankton species, across two continents. The species appears to be expanding in Europe, whereas similar trends are not observed in the USA. Our aim was to investigate if populations of Gonyostomum semen in Europe and in the USA are genetically differentiated, if there is population genetic structure within the continents, and what the potential drivers of differentiation are. To this end, we used a novel method based on single-amplified genomes combined with Restriction-site Associated DNA sequencing that allows de novo genotyping of natural single-cell isolates without the need for culturing. We amplified over 900 single-cell genomes from 25 lake populations across Europe and the USA and identified two distinct population clusters, one in Europe and another in the USA. Low genetic diversity in European populations supports the hypothesized recent expansion of Gonyostomum semen on this continent. Geographic population structure within each continent was associated with differences in environmental variables that may have led to ecological divergence of population clusters. Overall, our results show that single-amplified genomes combined with Restriction-site Associated DNA sequencing can be used to analyze microalgal population structure and differentiation based on single-cell isolates from natural, uncultured samples.

Nigral transcriptomic profiles in Engrailed-1 hemizygous mouse models of Parkinson's disease reveal upregulation of oxidative phosphorylation-related genes associated with delayed dopaminergic neurodegeneration.

Introduction: Parkinson's disease (PD) is the second most common neurodegenerative disorder, increasing both in terms of prevalence and incidence. To date, only symptomatic treatment is available, highlighting the need to increase knowledge on disease etiology in order to develop new therapeutic strategies. Hemizygosity for the gene Engrailed-1 (En1), encoding a conserved transcription factor essential for the programming, survival, and maintenance of midbrain dopaminergic neurons, leads to progressive nigrostriatal degeneration, motor impairment and depressive-like behavior in SwissOF1 (OF1-En1+/-). The neurodegenerative phenotype is, however, absent in C57Bl/6j (C57-En1+/-) mice. En1+/- mice are thus highly relevant tools to identify genetic factors underlying PD susceptibility. Methods: Transcriptome profiles were defined by RNAseq in microdissected substantia nigra from 1-week old OF1, OF1- En1+/-, C57 and C57- En1+/- male mice. Differentially expressed genes (DEGs) were analyzed for functional enrichment. Neurodegeneration was assessed in 4- and 16-week old mice by histology. Results: Nigrostriatal neurodegeneration was manifested in OF1- En1+/- mice by increased dopaminergic striatal axonal swellings from 4 to 16 weeks and decreased number of dopaminergic neurons in the SNpc at 16 weeks compared to OF1. In contrast, C57- En1+/- mice had no significant increase in axonal swellings or cell loss in SNpc at 16 weeks. Transcriptomic analyses identified 198 DEGs between OF1- En1+/- and OF1 mice but only 52 DEGs between C57- En1+/- and C57 mice. Enrichment analysis of DEGs revealed that the neuroprotective phenotype of C57- En1+/- mice was associated with a higher expression of oxidative phosphorylation-related genes compared to both C57 and OF1- En1+/- mice. Discussion: Our results suggest that increased expression of genes encoding mitochondrial proteins before the onset of neurodegeneration is associated with increased resistance to PD-like nigrostriatal neurodegeneration. This highlights the importance of genetic background in PD models, how different strains can be used to model clinical and sub-clinical pathologies and provides insights to gene expression mechanisms associated with PD susceptibility and progression.

Population genomic analyses reveal that salinity and geographic isolation drive diversification in a free-living protist.

Protists make up the vast diversity of eukaryotic life and play a critical role in biogeochemical cycling and in food webs. Because of their small size, cryptic life cycles, and large population sizes, our understanding of speciation in these organisms is very limited. We performed population genomic analyses on 153 strains isolated from eight populations of the recently radiated dinoflagellate genus Apocalathium, to explore the drivers and mechanisms of speciation processes. Species of this genus inhabit both freshwater and saline habitats, lakes and seas, and are found in cold temperate environments across the world. RAD sequencing analyses revealed that the populations were overall highly differentiated, but morphological similarity was not congruent with genetic similarity. While geographic isolation was to some extent coupled to genetic distance, this pattern was not consistent. Instead, we found evidence that the environment, specifically salinity, is a major factor in driving ecological speciation in Apocalathium. While saline populations were unique in loci coupled to genes involved in osmoregulation, freshwater populations appear to lack these. Our study highlights that adaptation to freshwater through loss of osmoregulatory genes may be an important speciation mechanism in free-living aquatic protists.

CGG toolkit: Software components for computational genomics.

Public-domain availability for bioinformatics software resources is a key requirement that ensures long-term permanence and methodological reproducibility for research and development across the life sciences. These issues are particularly critical for widely used, efficient, and well-proven methods, especially those developed in research settings that often face funding discontinuities. We re-launch a range of established software components for computational genomics, as legacy version 1.0.1, suitable for sequence matching, masking, searching, clustering and visualization for protein family discovery, annotation and functional characterization on a genome scale. These applications are made available online as open source and include MagicMatch, GeneCAST, support scripts for CoGenT-like sequence collections, GeneRAGE and DifFuse, supported by centrally administered bioinformatics infrastructure funding. The toolkit may also be conceived as a flexible genome comparison software pipeline that supports research in this domain. We illustrate basic use by examples and pictorial representations of the registered tools, which are further described with appropriate documentation files in the corresponding GitHub release.

Geography and environmental pressure are predictive of class-specific radioresistance in black fungi.

Black fungi are among the most resistant organisms to ionizing radiation on Earth. However, our current knowledge is based on studies on a few isolates, while the overall radioresistance limits across this microbial group and the relationship with local environmental conditions remain largely undetermined. To address this knowledge gap, we assessed the survival of 101 strains of black fungi isolated across a worldwide spatial distribution to gamma radiation doses up to 100 kGy. We found that intra and inter-specific taxonomy, UV radiation, and precipitation levels primarily influence the radioresistance in black fungi. Altogether, this study provides insights into the adaptive mechanisms of black fungi to extreme environments and highlights the role of local adaptation in shaping the survival capabilities of these extreme-tolerant organisms.

SAG-RAD: A Method for Single-Cell Population Genomics of Unicellular Eukaryotes.

Sequencing of reduced representation libraries enables genotyping of many individuals for population genomic studies. However, high amounts of DNA are required, and the method cannot be applied directly on single cells, preventing its use on most microbes. We developed and implemented the analysis of single amplified genomes followed by restriction-site-associated DNA sequencing to bypass labor-intensive culturing and to avoid culturing bias in population genomic studies of unicellular eukaryotes. This method thus opens the way for addressing important questions about the genetic diversity, gene flow, adaptation, dispersal, and biogeography of hitherto unexplored species.

Immune gene expression in the mosquito vector Culex quinquefasciatus during an avian malaria infection.

Plasmodium relictum is the most widespread avian malaria parasite in the world. It is listed as one of the 100 most dangerous invasive species, having been responsible for the extinction of several endemic bird species, and the near-demise of several others. Here we present the first transcriptomic study focused on the effect of P. relictum on the immune system of its vector (the mosquito Culex quinquefasciatus) at different times post-infection. We show that over 50% of immune genes identified as being part of the Toll pathway and 30%-40% of the immune genes identified within the Imd pathway are overexpressed during the critical period spanning the parasite's oocyst and sporozoite formation (8-12 days), revealing the crucial role played by both these pathways in this natural mosquito-Plasmodium combination. Comparison of infected mosquitoes with their uninfected counterparts also revealed some unexpected immune RNA expression patterns earlier and later in the infection: significant differences in expression of several immune effectors were observed as early as 30 min after ingestion of the infected blood meal. In addition, in the later stages of the infection (towards the end of the mosquito lifespan), we observed an unexpected increase in immune investment in uninfected, but not in infected, mosquitoes. In conclusion, our work extends the comparative transcriptomic analyses of malaria-infected mosquitoes beyond human and rodent parasites and provides insights into the degree of conservation of immune pathways and into the selective pressures exerted by Plasmodium parasites on their vectors.

Gene regulation of the avian malaria parasite Plasmodium relictum, during the different stages within the mosquito vector.

The malaria parasite Plasmodium relictum is one of the most widespread species of avian malaria. As in the case of its human counterparts, bird Plasmodium undergoes a complex life cycle infecting two hosts: the arthropod vector and the vertebrate host. In this study, we examined transcriptomes of P. relictum (SGS1) during crucial timepoints within its vector, Culex pipiens quinquefasciatus. Differential gene-expression analyses identified genes linked to the parasites life-stages at: i) a few minutes after the blood meal is ingested, ii) during peak oocyst production phase, iii) during peak sporozoite phase and iv) during the late-stages of the infection. A large amount of genes coding for functions linked to host-immune invasion and multifunctional genes was active throughout the infection cycle. One gene associated with a conserved Plasmodium membrane protein with unknown function was upregulated throughout the parasite development in the vector, suggesting an important role in the successful completion of the sporogonic cycle. Gene expression analysis further identified genes, with unknown functions to be significantly differentially expressed during the infection in the vector as well as upregulation of reticulocyte-binding proteins, which raises the possibility of the multifunctionality of these RBPs. We establish the existence of highly stage-specific pathways being overexpressed during the infection. This first study of gene-expression of a non-human Plasmodium species in its vector provides a comprehensive insight into the molecular mechanisms of the common avian malaria parasite P. relictum and provides essential information on the evolutionary diversity in gene regulation of the Plasmodium's vector stages.

Continent-wide genomic signatures of adaptation to urbanisation in a songbird across Europe.

Urbanisation is increasing worldwide, and there is now ample evidence of phenotypic changes in wild organisms in response to this novel environment. Yet, the genetic changes and genomic architecture underlying these adaptations are poorly understood. Here, we genotype 192 great tits (Parus major) from nine European cities, each paired with an adjacent rural site, to address this major knowledge gap in our understanding of wildlife urban adaptation. We find that a combination of polygenic allele frequency shifts and recurrent selective sweeps are associated with the adaptation of great tits to urban environments. While haplotypes under selection are rarely shared across urban populations, selective sweeps occur within the same genes, mostly linked to neural function and development. Collectively, we show that urban adaptation in a widespread songbird occurs through unique and shared selective sweeps in a core-set of behaviour-linked genes.

Shifts in gene expression variability in the blood-stage of Plasmodium relictum.

Avian malaria is a common and widespread disease of birds caused by a diverse group of pathogens of the genera Plasmodium. We investigated the transcriptomal profiles of one of the most common species, Plasmodium relictum, lineage SGS1, at multiple timepoints during the blood stages of the infection under experimental settings. The parasite showed well separated overall transcriptome profiles between day 8 and 20 after the infection, shown by well separated PCA profiles. Moreover, gene expression becomes more heterogenous within the experimental group late in the infection, either due to adaptations to individual differences between the experimental hosts, or due to desynchronisation of the life-cycle of the parasite. Overall, this study shows how the avian malaria system can be used to study gene expression of the avian Plasmodium parasite under controlled experimental settings, thus allowing for future comparative analysis of gene responses of parasite with different life-history traits and host effects.

Genome-wide single nucleotide polymorphism markers reveal population structure and dispersal direction of an expanding nuisance algal bloom species.

Species invasion and range expansion are currently under scrutiny due to increasing anthropogenic impact on the natural environment. This is also true for harmful algal blooms, which have been reported to have increased in frequency. However, this research is challenging due to the ephemeral nature, small size and mostly low concentrations of microalgae in the environment. One such species is the nuisance microalga Gonyostomum semen (Raphidophyceae), which has increased in occurrence in northern Europe in recent decades. The question of whether the species has expanded its habitat range or if it was already present in the lakes but was too rare to be detected remains unanswered. The aim of the present study was to determine the genetic structure and dispersal pathways of G. semen using RAD (restriction-site-associated DNA) tag sequencing. For G. semen, which has a huge genome (32 Gbp), we faced particular challenges, but were nevertheless able to recover over 1000 single nucleotide polymorphisms at high coverage. Our data revealed a distinct population genetic structure, demonstrating a divide of western and eastern populations that probably represent different lineages. Despite significant genetic differentiation among lakes, we found only limited isolation-by-distance. While we had expected a pattern of recent expansion northwards, the data demonstrated gene flow from the northeast/east towards the southwest/west. This genetic signature suggests that the observed gene flow may be due to dispersal by autumn migratory birds, which act as dispersal vectors of resistant resting propagules that form at the end of the G. semen blooms.

Uncovering the hidden diversity of litter-decomposition mechanisms in mushroom-forming fungi.

Litter decomposing Agaricales play key role in terrestrial carbon cycling, but little is known about their decomposition mechanisms. We assembled datasets of 42 gene families involved in plant-cell-wall decomposition from seven newly sequenced litter decomposers and 35 other Agaricomycotina members, mostly white-rot and brown-rot species. Using sequence similarity and phylogenetics, we split the families into phylogroups and compared their gene composition across nutritional strategies. Subsequently, we used Raman spectroscopy to examine the ability of litter decomposers, white-rot fungi, and brown-rot fungi to decompose crystalline cellulose. Both litter decomposers and white-rot fungi share the enzymatic cellulose decomposition, whereas brown-rot fungi possess a distinct mechanism that disrupts cellulose crystallinity. However, litter decomposers and white-rot fungi differ with respect to hemicellulose and lignin degradation phylogroups, suggesting adaptation of the former group to the litter environment. Litter decomposers show high phylogroup diversity, which is indicative of high functional versatility within the group, whereas a set of white-rot species shows adaptation to bulk-wood decomposition. In both groups, we detected species that have unique characteristics associated with hitherto unknown adaptations to diverse wood and litter substrates. Our results suggest that the terms white-rot fungi and litter decomposers mask a much larger functional diversity.

Physiology, Metabolism, and Fossilization of Hot-Spring Filamentous Microbial Mats.

The evolutionarily ancient Aquificales bacterium Sulfurihydrogenibium spp. dominates filamentous microbial mat communities in shallow, fast-flowing, and dysoxic hot-spring drainage systems around the world. In the present study, field observations of these fettuccini-like microbial mats at Mammoth Hot Springs in Yellowstone National Park are integrated with geology, geochemistry, hydrology, microscopy, and multi-omic molecular biology analyses. Strategic sampling of living filamentous mats along with the hot-spring CaCO3 (travertine) in which they are actively being entombed and fossilized has permitted the first direct linkage of Sulfurihydrogenibium spp. physiology and metabolism with the formation of distinct travertine streamer microbial biomarkers. Results indicate that, during chemoautotrophy and CO2 carbon fixation, the 87-98% Sulfurihydrogenibium-dominated mats utilize chaperons to facilitate enzyme stability and function. High-abundance transcripts and proteins for type IV pili and extracellular polymeric substances (EPSs) are consistent with their strong mucus-rich filaments tens of centimeters long that withstand hydrodynamic shear as they become encrusted by more than 5 mm of travertine per day. Their primary energy source is the oxidation of reduced sulfur (e.g., sulfide, sulfur, or thiosulfate) and the simultaneous uptake of extremely low concentrations of dissolved O2 facilitated by bd-type cytochromes. The formation of elevated travertine ridges permits the Sulfurihydrogenibium-dominated mats to create a shallow platform from which to access low levels of dissolved oxygen at the virtual exclusion of other microorganisms. These ridged travertine streamer microbial biomarkers are well preserved and create a robust fossil record of microbial physiological and metabolic activities in modern and ancient hot-spring ecosystems.

Delineating closely related dinoflagellate lineages using phylotranscriptomics.

Recently radiated dinoflagellates Apocalathium aciculiferum (collected in Lake Erken, Sweden), Apocalathium malmogiense (Baltic Sea) and Apocalathium aff. malmogiense (Highway Lake, Antarctica) represent a lineage with an unresolved phylogeny. We determined their phylogenetic relationships using phylotranscriptomics based on 792 amino acid sequences. Our results showed that A. aciculiferum diverged from the other two closely related lineages, consistent with their different morphologies in cell size, relative cell length and presence of spines. We hypothesized that A. aff. malmogiense and A. malmogiense, which inhabit different hemispheres, are evolutionarily more closely related because they diverged from a marine common ancestor, adapting to a wide salinity range, while A. aciculiferum colonized a freshwater habitat, by acquiring adaptations to this environment, in particular, salinity intolerance. We show that phylotranscriptomics can resolve the phylogeny of recently diverged protists. This has broad relevance, given that many phytoplankton species are morphologically very similar, and single genes sometimes lack the information to determine species' relationships.

The discovery of the virulence gene ToxA in the wheat and barley pathogen Bipolaris sorokiniana.

Bipolaris sorokiniana is the causal agent of multiple diseases on wheat and barley and is the primary constraint to cereal production throughout South Asia. Despite its significance, the molecular basis of disease is poorly understood. To address this, the genomes of three Australian isolates of B. sorokiniana were sequenced and screened for known pathogenicity genes. Sequence analysis revealed that the isolate BRIP10943 harboured the ToxA gene, which has been associated previously with disease in the wheat pathogens Parastagonospora nodorum and Pyrenophora tritici-repentis. Analysis of the regions flanking ToxA within B. sorokiniana revealed that it was embedded within a 12-kb genomic element nearly identical to the corresponding regions in P. nodorum and P. tritici-repentis. A screen of 35 Australian B. sorokiniana isolates confirmed that ToxA was present in 12 isolates. Sequencing of the ToxA genes within these isolates revealed two haplotypes, which differed by a single non-synonymous nucleotide substitution. Pathogenicity assays showed that a B. sorokiniana isolate harbouring ToxA was more virulent on wheat lines that contained the sensitivity gene when compared with a non-ToxA isolate. This work demonstrates that proteins that confer host-specific virulence can be horizontally acquired across multiple species. This acquisition can dramatically increase the virulence of pathogenic strains on susceptible cultivars, which, in an agricultural setting, can have devastating economic and social impacts.

Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus Malbranchea cinnamomea.

BACKGROUND: Genome and transcriptome sequencing has greatly facilitated the understanding of biomass-degrading mechanisms in a number of fungal species. The information obtained enables the investigation and discovery of genes encoding proteins involved in plant cell wall degradation, which are crucial for saccharification of lignocellulosic biomass in second-generation biorefinery applications. The thermophilic fungus Malbranchea cinnamomea is an efficient producer of many industrially relevant enzymes and a detailed analysis of its genomic content will considerably enhance our understanding of its lignocellulolytic system and promote the discovery of novel proteins. RESULTS: The 25-million-base-pair genome of M. cinnamomea FCH 10.5 was sequenced with 225× coverage. A total of 9437 protein-coding genes were predicted and annotated, among which 301 carbohydrate-active enzyme (CAZyme) domains were found. The putative CAZymes of M. cinnamomea cover cellulases, hemicellulases, chitinases and pectinases, equipping the fungus with the ability to grow on a wide variety of biomass types. Upregulation of 438 and 150 genes during growth on wheat bran and xylan, respectively, in comparison to growth on glucose was revealed. Among the most highly upregulated CAZymes on xylan were glycoside hydrolase family GH10 and GH11 xylanases, as well as a putative glucuronoyl esterase and a putative lytic polysaccharide monooxygenase (LPMO). AA9-domain-containing proteins were also found to be upregulated on wheat bran, as well as a putative cutinase and a protein harbouring a CBM9 domain. Several genes encoding secreted proteins of unknown function were also more abundant on wheat bran and xylan than on glucose. CONCLUSIONS: The comprehensive combined genome and transcriptome analysis of M. cinnamomea provides a detailed insight into its response to growth on different types of biomass. In addition, the study facilitates the further exploration and exploitation of the repertoire of industrially relevant lignocellulolytic enzymes of this fungus.

The transcriptome of the avian malaria parasite Plasmodium ashfordi displays host-specific gene expression.

Malaria parasites (Plasmodium spp.) include some of the world's most widespread and virulent pathogens. Our knowledge of the molecular mechanisms these parasites use to invade and exploit their hosts other than in mice and primates is, however, extremely limited. It is therefore imperative to characterize transcriptome-wide gene expression from nonmodel malaria parasites and how this varies across individual hosts. Here, we used high-throughput Illumina RNA sequencing on blood from wild-caught Eurasian siskins experimentally infected with a clonal strain of the avian malaria parasite Plasmodium ashfordi (lineage GRW2). Using a bioinformatic multistep approach to filter out host transcripts, we successfully assembled the blood-stage transcriptome of P. ashfordi. A total of 11 954 expressed transcripts were identified, and 7860 were annotated with protein information. We quantified gene expression levels of all parasite transcripts across three hosts during two infection stages - peak and decreasing parasitemia. Interestingly, parasites from the same host displayed remarkably similar expression profiles during different infection stages, but showed large differences across hosts, indicating that P. ashfordi may adjust its gene expression to specific host individuals. We further show that the majority of transcripts are most similar to the human parasite Plasmodium falciparum, and a large number of red blood cell invasion genes were discovered, suggesting evolutionary conserved invasion strategies between mammalian and avian Plasmodium. The transcriptome of P. ashfordi and its host-specific gene expression advances our understanding of Plasmodium plasticity and is a valuable resource as it allows for further studies analysing gene evolution and comparisons of parasite gene expression.

DNA Content in Extracellular Vesicles Isolated from Porcine Coronary Venous Blood Directly after Myocardial Ischemic Preconditioning.

BACKGROUND: Extracellular vesicles (EV) are nano-sized membranous structures released from most cells. They have the capacity to carry bioactive molecules and gene expression signals between cells, thus mediating intercellular communication. It is believed that EV confer protection after ischemic preconditioning (IPC). We hypothesize that myocardial ischemic preconditioning will lead to rapid alteration of EV DNA content in EV collected from coronary venous effluent. MATERIALS AND METHODS: In a porcine myocardial ischemic preconditioning model, EV were isolated from coronary venous blood before and after IPC by differential centrifugation steps culminating in preparative ultracentrifugation combined with density gradient ultracentrifugation. The EV preparation was validated, the DNA was extracted and further characterized by DNA sequencing followed by bioinformatics analysis. RESULTS: Porcine genomic DNA fragments representing each chromosome, including mitochondrial DNA sequences, were detected in EV isolated before and after IPC. There was no difference detected in the number of sequenced gene fragments (reads) or in the genomic coverage of the sequenced DNA fragments in EV isolated before and after IPC. Gene ontology analysis showed an enrichment of genes coding for ion channels, enzymes and proteins for basal metabolism and vesicle biogenesis and specific cardiac proteins. CONCLUSIONS: This study demonstrates that porcine EV isolated from coronary venous blood plasma contain fragments of DNA from the entire genome, including the mitochondria. In this model we did not find specific qualitative or quantitative changes of the DNA content in EV collected immediately after an in vivo myocardial IPC provocation. This does not rule out the possibility that EV DNA content changes in response to myocardial IPC which could occur in a later time frame.