Inhibition of microRNA-92a protects against ischemia/reperfusion injury in a large-animal model.

BACKGROUND: MicroRNAs (miRs) are small noncoding RNAs that posttranscriptionally control gene expression. Small-animal studies suggest that miRs might offer novel therapeutic targets in cardiovascular diseases such as cardioprotection of murine hearts after myocardial infarction via miR-92a inhibitors. Because the functional benefits of miR-92a inhibitors in larger preclinical models are not known, we assessed the therapeutic efficacy of miR-92a inhibition in a porcine model of ischemia and reperfusion. METHODS AND RESULTS: Pigs (n=5 per group) underwent percutaneous ischemia/reperfusion (60 min/72 h or 7 days, respectively). Locked nucleic acid-modified antisense miR-92a (LNA-92a) was applied either regionally (antegrade or retrograde) with a catheter or systemically (intravenously). LNA-92a significantly (P<0.01) reduced miR-92a expression in the infarct zone regardless of the application venue. However, catheter-based delivery, but not intravenous infusion, of LNA-92a significantly (P<0.05) reduced the infarct size compared with control LNA-treated pigs, which correlated with an improved ejection fraction and left ventricular end-diastolic pressure (P<0.05). Histochemistry revealed that LNA-92a increased capillary density but decreased leukocyte influx and cardiac cell death. Complete loss of miR-92a in mice attenuated the infarct-related myocardial dysfunction to a larger extent than cardiomyocyte-specific miR-92a deletion. In vitro, LNA-92a protected against hypoxia/reoxygenation-induced cardiomyocyte cell death. CONCLUSIONS: Regional LNA-92a delivery reduces miR-92a levels and infarct size and postischemic loss of function. LNA-92a exerts cell-protective, proangiogenic, and anti-inflammatory effects. miR-92a inhibition might be a novel therapeutic tool to preserve cardiac function after ischemia.

Mazaedium evolution in the Ascomycota (Fungi) and the classification of mazaediate groups of formerly unclear relationship.

Calicioid or mazaediate fungi constitute a heterogeneous assemblage of fungi sharing the presence of a mazaedium. These fungi were once treated as an order (Caliciales) of the Ascomycota but many are now known to be nested within the Arthoniomycetes, Eurotiomycetes, Lecanoromycetes and Leotiomycetes. In this study we employ multigene phylogenetic analyses of main mazaediate groups (based on nuclear 18S, 28S, 5.8S rDNA, mitochondrial 16S, and the protein coding RPB1 and Mcm7) of 116 taxa corresponding to most major groups of the inoperculate ascomycetes ("Leotiomyceta") and a selection of Pezizomycetes, to trace the evolution of the mazaedium in the Pezizomycotina (the "Euascomycetes"). In particular, we studied the placement of three calicioid groups of uncertain position, Calycidiaceae, Coniocybaceae and Microcaliciaceae. Here, we show that the Calycidiaceae is closely related to the Sphaerophoraceae in the Lecanoromycetidae (Lecanoromycetes), as supported by overall morphology and the production of sphaerophorin. The Coniocybaceae constitute an early divergent line in the inoperculate ascomycetes and here we propose to recognize this group formally as the new class and order Coniocybomycetes, Coniocybales. The Microcaliciaceae is nested within the Ostropomycetidae (Lecanoromycetes). Both Coniocybaceae and Microcaliciaceae, although highly distinctive, lack morphological similarities to related main fungal groups. Ancestral state reconstruction suggests that the ancestor of all inoperculate ascomycetes and the ancestor of all main inoperculate ascomycete groups, with the exception of the Coniocybomycetes, was non-mazediate, and thus confirms the large amount of parallel evolution and independent gains of the mazaedium in the history of the Ascomycota.

The relationships of Odontotrema (Odontotremataceae) and the resurrected Sphaeropezia (Stictidaceae)--new combinations and three new Sphaeropezia species.

Odontotremataceae is polyphyletic and constitutes two distantly related clades, the true Odontotremataceae and a segregate group within Stictidaceae including "Odontotrema" cassiopes, "O." diffindens, lichenicolous "Odontotrema" species and "Bryodiscus" arctoalpinus. Sphaeropezia here is accepted as the name for this latter group. An updated phylogeny of the Stictidaceae based on mtSSU, nuLSU and the protein coding gene RPB2 is presented together with a taxonomic revision of Swedish taxa of Odontotrema and Sphaeropezia. Bryodiscus and Lethariicola are synonymized under Sphaeropezia, and three new Sphaeropezia species are described: the lignicolous S. capreae, the fungicolous S. lyckselensis and the lichenicolous S. mycoblasti. The new species are distinguished from other species by molecular and morphological characters, and substrate preferences. The new combinations Sphaeropezia arctoalpina, S. cassiopes, S. grimmiae, S. hepaticarum, S. melaneliae, S. ochrolechiae and S. thamnoliae are proposed. The morphology of these species was studied in detail. We further propose to combine the remaining lichenicolous Odontotrema species, exept O. stereocaulicola, in Sphaeropezia based on their close relationship to the studied lichenicolous taxa. These additional new combinations include Sphaeropezia bryoriae, S. cucularis, S. figulina, S. intermedia, S. japewiae, S. lecanorae, S. navarinoi, S. pertusariae, S. rhizocarpicola, S. santessonii, and S. sipei. A lectotype is designated for the name Odontotrema diffindens Nyl.

Characterization of a porcine model of atrial arrhythmogenicity in the context of ischaemic heart failure.

Atrial fibrillation (AF) is a major healthcare challenge contributing to high morbidity and mortality. Treatment options are still limited, mainly due to insufficient understanding of the underlying pathophysiology. Further research and the development of reliable animal models resembling the human disease phenotype is therefore necessary to develop novel, innovative and ideally causal therapies. Since ischaemic heart failure (IHF) is a major cause for AF in patients we investigated AF in the context of IHF in a close-to-human porcine ischaemia-reperfusion model. Myocardial infarction (AMI) was induced in propofol/fentanyl/midazolam-anaesthetized pigs by occluding the left anterior descending artery for 90 minutes to model ischaemia with reperfusion. After 30 days ejection fraction (EF) was significantly reduced and haemodynamic parameters (pulmonary capillary wedge pressure (PCWP), right atrial pressure (RAP), left ventricular enddiastolic pressure (LVEDP)) were significantly elevated compared to age/weight matched control pigs without AMI, demonstrating an IHF phenotype. Electrophysiological properties (sinus node recovery time (SNRT), atrial/AV nodal refractory periods (AERP, AVERP)) did not differ between groups. Atrial burst pacing at 1200 bpm, however, revealed a significantly higher inducibility of atrial arrhythmia episodes including AF in IHF pigs (3/15 vs. 10/16, p = 0.029). Histological analysis showed pronounced left atrial and left ventricular fibrosis demonstrating a structural substrate underlying the increased arrhythmogenicity. Consequently, selective ventricular infarction via LAD occlusion causes haemodynamic alterations inducing structural atrial remodeling which results in increased atrial fibrosis as the arrhythmogenic atrial substrate in pigs with IHF.

Pronounced genetic diversity in tropical epiphyllous lichen fungi.

Lowland tropical habitats harbour an unexplored genetic diversity of epiphyllous fungi. In the shade of rainforest understoreys, lichenized fungi are specialized to an ephemeral habitat where they produce little vegetative biomass and develop reproductive structures early. In a first population genetic study of epiphyllous lichen fungi, we analysed the intraspecific genetic diversity of five leaf-colonizing lichen mycobiont species. Sampling focused on a lowland perhumid forest plot in Costa Rica, with additional collections from other localities throughout the country. In all species we detected sympatric occurrence of highly diverged haplotypes. Haplotypes belonging to distinct clades in networks were also found on the same leaf, clearly indicating multiple independent colonization events on single leaves. Despite the unusually high genetic diversity of these leaf-colonizing tropical fungi, we did not detect pronounced spatial structure of the haplotype distribution between geographical regions. The observed patterns suggest that the diversity of foliicolous lichens could be much higher than expected, with several cryptic genetic lineages within each morphologically characterized species.

Gymnosperms on the EDGE.

Driven by limited resources and a sense of urgency, the prioritization of species for conservation has been a persistent concern in conservation science. Gymnosperms (comprising ginkgo, conifers, cycads, and gnetophytes) are one of the most threatened groups of living organisms, with 40% of the species at high risk of extinction, about twice as many as the most recent estimates for all plants (i.e. 21.4%). This high proportion of species facing extinction highlights the urgent action required to secure their future through an objective prioritization approach. The Evolutionary Distinct and Globally Endangered (EDGE) method rapidly ranks species based on their evolutionary distinctiveness and the extinction risks they face. EDGE is applied to gymnosperms using a phylogenetic tree comprising DNA sequence data for 85% of gymnosperm species (923 out of 1090 species), to which the 167 missing species were added, and IUCN Red List assessments available for 92% of species. The effect of different extinction probability transformations and the handling of IUCN data deficient species on the resulting rankings is investigated. Although top entries in our ranking comprise species that were expected to score well (e.g. Wollemia nobilis, Ginkgo biloba), many were unexpected (e.g. Araucaria araucana). These results highlight the necessity of using approaches that integrate evolutionary information in conservation science.

Green Plants in the Red: A Baseline Global Assessment for the IUCN Sampled Red List Index for Plants.

Plants provide fundamental support systems for life on Earth and are the basis for all terrestrial ecosystems; a decline in plant diversity will be detrimental to all other groups of organisms including humans. Decline in plant diversity has been hard to quantify, due to the huge numbers of known and yet to be discovered species and the lack of an adequate baseline assessment of extinction risk against which to track changes. The biodiversity of many remote parts of the world remains poorly known, and the rate of new assessments of extinction risk for individual plant species approximates the rate at which new plant species are described. Thus the question 'How threatened are plants?' is still very difficult to answer accurately. While completing assessments for each species of plant remains a distant prospect, by assessing a randomly selected sample of species the Sampled Red List Index for Plants gives, for the first time, an accurate view of how threatened plants are across the world. It represents the first key phase of ongoing efforts to monitor the status of the world's plants. More than 20% of plant species assessed are threatened with extinction, and the habitat with the most threatened species is overwhelmingly tropical rain forest, where the greatest threat to plants is anthropogenic habitat conversion, for arable and livestock agriculture, and harvesting of natural resources. Gymnosperms (e.g. conifers and cycads) are the most threatened group, while a third of plant species included in this study have yet to receive an assessment or are so poorly known that we cannot yet ascertain whether they are threatened or not. This study provides a baseline assessment from which trends in the status of plant biodiversity can be measured and periodically reassessed.

Feasibility of dynamic CT-based adenosine stress myocardial perfusion imaging to detect and differentiate ischemic and infarcted myocardium in an large experimental porcine animal model.

The purpose of the study is feasibility of dynamic CT perfusion imaging to detect and differentiate ischemic and infarcted myocardium in a large porcine model. 12 Country pigs completed either implantation of a 75 % luminal coronary stenosis in the left anterior descending coronary artery simulating ischemia or balloon-occlusion inducing infarction. Dynamic CT-perfusion imaging (100 kV, 300 mAs), fluorescent microspheres, and histopathology were performed in all models. CT based myocardial blood flow (MBFCT), blood volume (MBVCT) and transit constant (Ktrans), as well as microsphere's based myocardial blood flow (MBFMic) were derived for each myocardial segment. According to histopathology or microsphere measurements, 20 myocardial segments were classified as infarcted and 23 were ischemic (12 and 14 %, respectively). Across all perfusion states, MBFCT strongly predicted MBFMic (ß 0.88 ± 0.12, p < 0.0001). MBFCT, MBVCT, and Ktrans were significantly lower in ischemic/infarcted when compared to reference myocardium (all p < 0.01). Relative differences of all CT parameters between affected and non-affected myocardium were higher for infarcted when compared to ischemic segments under rest (48.4 vs. 22.6 % and 46.1 vs. 22.9 % for MBFCT, MBVCT, respectively). Under stress, MBFCT was significantly lower in infarcted than in ischemic myocardium (67.8 ± 26 vs. 88.2 ± 22 ml/100 ml/min, p = 0.002). In a large animal model, CT-derived parameters of myocardial perfusion may enable detection and differentiation of ischemic and infarcted myocardium.

Myocardial CT perfusion imaging in a large animal model: comparison of dynamic versus single-phase acquisitions.

OBJECTIVES: This study sought to compare dynamic versus single-phase high-pitch computed tomography (CT) acquisitions for the assessment of myocardial perfusion in a porcine model with adjustable degrees of coronary stenosis. BACKGROUND: The incremental value of the 2 different approaches to CT-based myocardial perfusion imaging remains unclear. METHODS: Country pigs received stent implantation in the left anterior descending coronary artery, in which an adjustable narrowing (50% and 75% stenoses) was created using a balloon catheter. All animals underwent CT-based rest and adenosine-stress myocardial perfusion imaging using dynamic and single-phase high-pitch acquisitions at both degrees of stenosis. Fluorescent microspheres served as a reference standard for myocardial blood flow. Segmental CT-based myocardial blood flow (MBFCT) was derived from dynamic acquisitions. Segmental single-phase enhancement (SPE) was recorded from high-pitch, single-phase examinations. MBFCT and SPE were compared between post-stenotic and reference segments, and receiver-operating characteristic curve analysis was performed. RESULTS: Among 6 animals (28 ± 2 kg), there were significant differences of MBFCT and SPE between post-stenotic and reference segments for all acquisitions at 75% stenosis. By contrast, although for 50% stenosis at rest, MBFCT was lower in post-stenotic than in reference segments (0.65 ± 0.10 ml/g/min vs. 0.75 ± 0.16 ml/g/min, p < 0.05), there was no difference for SPE (128 ± 27 Hounsfield units vs. 137 ± 35 Hounsfield units, p = 0.17), which also did not significantly change under adenosine stress. In receiver-operating characteristic curve analyses, segmental MBFCT showed significantly better performance for ischemia prediction at 75% stenosis and stress (area under the curve: 0.99 vs. 0.89, p < 0.05) as well as for 50% stenosis, regardless of adenosine administration (area under the curve: 0.74 vs. 0.57 and 0.88 vs. 0.61, respectively, both p < 0.05). CONCLUSIONS: At higher degrees of coronary stenosis, both MBFCT and SPE permit an accurate prediction of segmental myocardial hypoperfusion. However, accuracy of MBFCT is higher than that of SPE at 50% stenosis and can be increased by adenosine stress at both degrees of stenosis.

Accuracy of dynamic computed tomography adenosine stress myocardial perfusion imaging in estimating myocardial blood flow at various degrees of coronary artery stenosis using a porcine animal model.

OBJECTIVE: To determine the accuracy of computed tomography (CT) dynamic stress myocardial perfusion imaging to estimate myocardial blood flow (MBF) in a porcine animal model with variable degrees of induced coronary artery stenosis in comparison with microsphere-derived MBF. METHODS AND MATERIALS: Seven domestic pigs (36 ± 4 kg) received stents (confirmed 3.0 mm diameter) in the left anterior descending coronary artery distal to first diagonal branch. A balloon catheter was placed within the stent and inflated to various degrees to obtain a defined luminal narrowing (50% and 75% diameter stenosis) as confirmed by intra-arterial flow wire measurement. All models underwent adenosine-mediated (140 µg/kg/min) dynamic stress and rest myocardial perfusion CT imaging using a dual-source CT scanner (shuttle-mode with 100 kV/300 mAs, 20 mL iopromide) with prospective acquisitions every second heartbeat for 30 seconds. CT-estimated MBF (MBFCT) was calculated using a model-based parametric deconvolution method and correlated to that of fluorescent microspheres (MBFmic) injected at each perfusion state. RESULTS: All study procedures were performed without complications, and all animals completed the study protocol. Among 448 myocardial segments, 31 (7%) were considered nonevaluable because of motion artifacts. With stress, MBFCT increased significantly (1.10 ± 0.25 vs. 0.80 ± 0.28 mL/g/min, P < 0.001; at stress and rest, respectively) in all myocardial segments and correlated with MBFmic (r = 0.67, P < 0.001). MBFCT overestimated MBFmic, independently of adenosine-stress and degree of coronary stenosis (ß = 2.3, 95% confidence interval: 1.81-2.79 mL/g/min, P < 0.001). Although there were no differences in MBFCT between 50% and 75% coronary stenosis at rest (0.01 ± 0.08 mL/g/min, P = 0.86), MBFCT was significantly lower at 75% than at 50% under stress conditions (0.53 ± 0.19 vs. 0.71 ± 0.24 mL/g/min, P = 0.002). CONCLUSIONS: CT-derived MBF measurements at rest and stress with varying degrees of coronary stenosis show a valid difference but an underestimated correlation with microsphere-derived MBF in a porcine animal model.

Photobiont association and genetic diversity of the optionally lichenized fungus Schizoxylon albescens.

The fungus Schizoxylon albescens occurs both as lichen and as saprobe. Lichenized colonies grow on the bark of Populus tremula; saprotrophic morphs grow on dead Populus branches. We wanted to (1) test whether lichenized and saprotrophic S. albescens are genetically distinct, (2) investigate photobiont association and diversity, (3) investigate the interactions between fungi and algae that occur during co-cultivation and (4) test whether Schizoxylon shows algal selectivity during lichenization. Fungal and algal genetic diversity were investigated for three markers. Algae from lichenized thalli were isolated in axenic cultures, and isolate sequence diversity was compared with algae amplified directly from thallus fragments. Co-culture experiments of fungi and algae were performed to study the morphological interaction patterns. Two distinct phylogenetic units are revealed in S. albescens, which are interpreted as phenotypically cryptic species. The algae are related to Coccomyxa and Pseudococcomyxa, and form two distinct sister clades separating samples isolated in cultures from those amplified directly from thallus fragments, indicating that more easily cultured strains of algae are not necessarily major components of the lichens. Schizoxylon albescens interacts with isolated algal strains, similar to fungal-Coccomyxa symbioses in nature. As the system is maintained without difficulty in culture, it can potentially be an easily controlled lichen symbiosis study system under laboratory conditions.

Evolution and phylogenetic relationships within Porinaceae (Ostropomycetidae), focusing on foliicolous species.

A phylogeny of the lichen family Porinaceae using mitochondrial SSU rDNA sequences is presented, with special focus on foliicolous taxa. Fifty specimens of 28 mostly tropical species, representing eight species groups of Porina as well as the genus Trichothelium, were analysed together with species of other members of Ostropomycetidae, and using Agyriaceae as outgroup. We performed the phylogenetic analyses with a Bayesian approach and under the criterion of maximum parsimony. Four main clades can be distinguished: the P. nitidula-group s. lat. (including Trichothelium, P. papillifera and P. rubescens), the Porina epiphylla-group s. lat. (including the P. radiata-, the P. nucula-, the P. imitatrix- and the P. epiphylla-group s. str.) and two clades of the P. rufula-group. The genus Porina as understood by all recent concepts is paraphyletic, and Trichothelium is nested within the Porina nitidula-group. The non-setose P. repanda forms a monophyletic clade with Trichothelium. The tree does not support a monophyletic origin of substrate preferences or photobiont selection. Species-specific associations with morphologically different trentepohlioid photobionts mapped on the tree suggest that closely related mycobiont species switch between different types of algae.

A phylogenetic study of the Lecanora rupicola group (Lecanoraceae, Ascomycota).

A molecular phylogeny of the Lecanora rupicola group is presented, based on ITS sequence analyses. The study includes saxicolous and corticolous members of the Lecanora rupicola group as well as other Lecanora species with pruinose apothecia. A phylogenetic hypothesis for species in Lecanora s. lat. and various other genera in Lecanoraceae, based on an alignment-free distance estimation technique, shows that the Lecanora rupicola group forms a monophyletic clade within Lecanoraceae. Affinities to the core group of Lecanora are not well supported, likewise the monophyly of Lecanora s. str. with other species groups in Lecanora, such as the lobate taxa (and Rhizoplaca) is not supported. A more detailed analysis involving Lecanora species with pruinose apothecial discs was carried out with model-based Bayesian Markov chain Monte Carlo (B/MCMC) tree sampling. The results suggest the monophyly of the Lecanora species that are characterized by the presence of chromones. Corticolous as well as saxicolous species are included. Lepraria flavescens is closely related to the Lecanora swartzii subgroup, and the new name Lecanora rouxii nom. nov. is introduced for that species. Other Lecanora species with pruinose discs are not closely related to the Lecanora rupicola group.

The phylogeny of Porinaceae (Ostropomycetidae) suggests a neotenic origin of perithecia in Lecanoromycetes.

The family Porinaceae (Trichotheliales) is characterized by perithecial ascomata of ascohymenial origin. The phylogenetic position of this family in the system of ascomycetes has been uncertain and is investigated using mtSSU rDNA sequences. The dataset consists of lichenized representatives of major ascomycete lineages, including those that were previously suspected as relatives of Porinaceae, such as Pyrenulaceae. The dataset was subjected to a Bayesian phylogenetic analysis implementing a Metropolis Coupled Markov Chain Monte Carlo method. The analysis confirms previous classification of the apothecial Gomphillaceae near to Graphidaceae, and suggests that the pyrenocarpous Porinaceae are also close to Graphidaceae, Gyalectaceae, and Stictidaceae. The subclass Ostropomycetidae is here suggested to include the families Gomphillaceae, Gyalectaceae, Graphidaceae (incl. Thelotremataceae), Porinaceae, and Stictidaceae. A special type of hemiangiocarpous ontogeny of the ascomata is shared throughout the Ostropomycetidae, and the closed fruit bodies of Porinaceae are apparently a result of a neotenic ontogeny. This is associated with special hymenial characters: rather thin-walled narrow asci, and a different consistency of the hymenial gels.

Assembling the fungal tree of life: progress, classification, and evolution of subcellular traits.

Based on an overview of progress in molecular systematics of the true fungi (Fungi/Eumycota) since 1990, little overlap was found among single-locus data matrices, which explains why no large-scale multilocus phylogenetic analysis had been undertaken to reveal deep relationships among fungi. As part of the project "Assembling the Fungal Tree of Life" (AFTOL), results of four Bayesian analyses are reported with complementary bootstrap assessment of phylogenetic confidence based on (1) a combined two-locus data set (nucSSU and nucLSU rDNA) with 558 species representing all traditionally recognized fungal phyla (Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota) and the Glomeromycota, (2) a combined three-locus data set (nucSSU, nucLSU, and mitSSU rDNA) with 236 species, (3) a combined three-locus data set (nucSSU, nucLSU rDNA, and RPB2) with 157 species, and (4) a combined four-locus data set (nucSSU, nucLSU, mitSSU rDNA, and RPB2) with 103 species. Because of the lack of complementarity among single-locus data sets, the last three analyses included only members of the Ascomycota and Basidiomycota. The four-locus analysis resolved multiple deep relationships within the Ascomycota and Basidiomycota that were not revealed previously or that received only weak support in previous studies. The impact of this newly discovered phylogenetic structure on supraordinal classifications is discussed. Based on these results and reanalysis of subcellular data, current knowledge of the evolution of septal features of fungal hyphae is synthesized, and a preliminary reassessment of ascomal evolution is presented. Based on previously unpublished data and sequences from GenBank, this study provides a phylogenetic synthesis for the Fungi and a framework for future phylogenetic studies on fungi.