mRNA can direct dose-dependent protein expression in cardiac muscle without genome integration, but to date has not been shown to improve cardiac function in a safe, clinically applicable way. Herein, we report that a purified and optimized mRNA in a biocompatible citrate-saline formulation is tissue specific, long acting, and does not stimulate an immune response. In small- and large-animal, permanent occlusion myocardial infarction models, VEGF-A 165 mRNA improves systolic ventricular function and limits myocardial damage. Following a single administration a week post-infarction in mini pigs, left ventricular ejection fraction, inotropy, and ventricular compliance improved, border zone arteriolar and capillary density increased, and myocardial fibrosis decreased at 2 months post-treatment. Purified VEGF-A mRNA establishes the feasibility of improving cardiac function in the sub-acute therapeutic window and may represent a new class of therapies for ischemic injury.

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Aims/hypothesis: AMP-activated protein kinase (AMPK) regulates metabolic adaptations in skeletal muscle. The aim of this study was to investigate whether AMPK modulates the expression of skeletal muscle genes that have been implicated in lipid and glucose metabolism under fed or fasting conditions. Methods: Two genetically modified animal models were used: AMPK γ3 subunit knockout mice (Prkag3 −/− ) and skeletal musclespecific transgenic mice (Tg-Prkag3225Q ) that express a mutant (R225Q) γ3 subunit. Levels of mRNA transcripts of genes involved in lipid and glucose metabolism in white gastrocnemius muscles of these mice (under fed or 16-h fasting conditions) were assessed by quantitative real-time PCR. Results: Wild-type mice displayed a coordinated increase in the transcription of skeletal muscle genes encoding proteins involved in lipid/oxidative metabolism (lipoprotein lipase, fatty acid transporter, carnitine palmitoyl transferase-1 and citrate synthase) and glucose metabolism (glycogen synthase and lactate dehydrogenase) in response to fasting. In contrast, these fasting-induced responses were impaired in Prkag3 −/− mice. The transcription of genes involved in lipid and oxidative metabolism was increased in the skeletal muscle of Tg-Prkag3 225Q mice compared with that in wild-type mice. Moreover, the expression of the genes encoding hexokinase II and 6-phosphofrucktokinase was decreased in Tg-Prkag3 225Q mice after fasting. Conclusions/ interpretation: AMPK is involved in the coordinated transcription of genes critical for lipid and glucose metabolism in white glycolytic skeletal muscle.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved heterotrimer important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory ␥-subunit of the kinase, AMPK ␥3, has an important role in glucose uptake, glycogen synthesis, and fat oxidation in white skeletal muscle, as previously demonstrated by physiological characterization of AMPK ␥3 mutant (R225Q) transgenic (TgPrkag3 225Q ) and ␥3 knock-out (Prkag3 ؊/؊ ) mice. We determined AMPK ␥3-dependent regulation of gene expression by analyzing global transcription profiles in glycolytic skeletal muscle from ␥3 mutant transgenic and knock-out mice using oligonucleotide microarray technology. Evidence is provided for coordinated and reciprocal regulation of multiple key components in glucose and fat metabolism, as well as skeletal muscle ergogenics in TgPrkag3 225Q and Prkag3 ؊/؊ mice. The differential gene expression profile was consistent with the physiological differences between the models, providing a molecular mechanism for the observed phenotype. The striking pattern of opposing transcriptional changes between TgPrkag3 225Q and Prkag3 ؊/؊ mice identifies differentially expressed targets being truly regulated by AMPK and is consistent with the view that R225Q is an activating mutation, in terms of its downstream effects. Additionally, we identified a wide array of novel targets and regulatory pathways for AMPK in skeletal muscle.AMP-activated protein kinase (AMPK) 2 is a critical regulator of carbohydrate and fat metabolism in eukaryotic cells (reviewed in Refs. 1 and 2). AMPK is a heterotrimer that consists of ␣-, ␤-, and ␥-subunits, all of which are required for its activity. The catalytic ␣-subunit contains a conventional serine/threonine protein kinase domain, and phosphorylation of Thr-172 residue within the activation loop of the ␣-subunit by upstream kinases is essential for the activity of the heterotrimer (3-6). Once phosphorylated at Thr-172, AMPK can be further activated by allosteric binding of AMP to the evolutionary conserved cystathionine ␤-synthase domains in the regulatory ␥-subunit (7). The AMPK ␤-subunit acts as a scaffold for binding of the ␣-and ␥-subunits (8). The ␤-subunit also contains a glycogen-binding domain, and recent findings provide evidence that this motif is involved in targeting the AMPK complex to cellular glycogen stores (9, 10). The mammalian genome contains seven AMPK genes encoding for two ␣-, two ␤-, and three ␥-isoforms. Thus, there are 12 possible combinations of heterotrimeric AMPK, and the physiological function of the AMPK holoenzyme depends on the particular isoforms present in the complex.We have provided evidence that AMPK ␥3 is the predominant ␥-isoform expressed in glycolytic (white, fast-twitch type II) skeletal muscle (11). In contrast, it is expressed at low levels in oxidative (red, slowtwitch type I) skeletal muscle and is undetectable in brain, liver, heart, or white adipose tissue (11). Thus, the AMPK ␥3-subunit is the only isoform exhibiting tissue-specific ...

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Phylogeny and species delimitation of North European Lumbricillus (Clitellata, Enchytraeidae). -Zoologica Scripta, 00, 000-000. The enchytraeid genus Lumbricillus comprises about 80 described species of clitellate worms, which are up to a few centimetres long, and they mostly inhabit the littoral zone of nontropical marine and brackish waters world-wide. The phylogeny of this genus is poorly studied, but previous work has suggested that Lumbricillus is a non-monophyletic group. In this study, species boundaries and the phylogeny of this genus is re-assessed using more than 300 DNA-barcoded specimens (using COI mtDNA), part of which was also sequenced for two additional mitochondrial and four nuclear molecular markers. Statistical and coalescent based applications were used for the delimitation of a total of 24 species, of which 20 were identified as belonging to 17 described morphospecies; one morphospecies was found to be a complex of four delimited species, and another four delimited species could not be matched with any described species. Furthermore, gene trees, concatenation and multispecies coalescent based species trees were estimated using Bayesian inference. The estimated phylogenies confirm a non-monophyletic Lumbricillus as L. semifuscus is clearly excluded from the genus. Furthermore, the placement of a monophyletic clade consisting of L. arenarius, L. dubius, and an unidentified species varies between analyses; they are either found as the sister-group to the genus Grania or as sister-group to the remaining Lumbricillus, where the latter relationship is supported by the multispecies coalescent, which we consider as the most reliable method.

Cardiac progenitor cells (CPCs), capable of differentiating into multiple cardiac cell types including cardiomyocytes (CMs), endothelial cells, and smooth muscle cells, are promising candidates for cardiac repair/regeneration. In vitro model systems where cells are grown in a more in vivo-like environment, such as 3D cultures, have been shown to be more predictive than 2D culture for studying cell biology and disease pathophysiology. In this report, we focused on using Wnt inhibitors to study the differentiation of human iPSC-CPCs under 2D or 3D culture conditions by measuring marker protein and gene expression as well as intracellular Ca 2+ oscillation. Our results show that the 3D culture with aligned nanofiber scaffolds, mimicing the architecture of the extracellular matrix of the heart, improve the differentiation of iPSC-CPCs to functional cardiomyocytes induced by Wnt inhibition, as shown with increased number of cardiac Troponin T (cTnT)-positive cells and synchronized intracellular Ca 2+ oscillation. In addition, we studied if 3D nanofiber culture can be used as an in vitro model for compound screening by testing a number of other differentiation factors including a ALK5 inhibitor and inhibitors of BMP signaling. This work highlights the importance of using a more relevant in vitro model and measuring not only the expression of marker proteins but also the functional readout in a screen in order to identify the best compounds and to investigate the resulting biology.

The oligochaete worm, Cognettia sphagnetorum (Vejdovsk y, 1878), is widely used as a model organism in soil biology, and therefore it is important that its taxonomy is firmly established. A previous study based on both mitochondrial and nuclear genetic markers showed that this taxon is an assemblage of at least four species that do not form a monophyletic group. Also the validity of the genus Cognettia Nielsen and Christensen, 1959 is subject to debate, since the existence of two putative senior synonyms, Euenchytraeus Bretscher, 1906 and Chamaedrilus Friend, 1913 has been pointed out. Herein we revise the generic assignment of the species currently placed in Cognettia: two species, C. clarae Bauer, 1993 and C. piperi Christensen and D ozsa-Farkas, 1999, are transferred to Euenchytraeus, together with its type Eu. bisetosus Bretscher, 1906, whereas the remaining species, including Cognettia sphagnetorum, are being transferred to Chamaedrilus. Five species within the Chamaedrilus sphagnetorum complex are revised: the type species of Chamaedrilus, Ch. chlorophilus Friend, 1913, as well as the type species of Cognettia, Ch. sphagnetorum s. str. are redescribed, and a neotype is designated for the latter; and the cryptic species Ch. pseudosphagnetorum sp. nov. and Ch. chalupskyi sp. nov. are described as new to science and discussed against a paratype of Ch. valeriae (Dumnicka, 2010) comb. nov. DNA-barcodes are provided for all the named species in the complex except Ch. valeriae. A key to the species in the complex is given and the value of different somatic characters for separating and identifying species of Chamaedrilus is discussed. No morphological feature seems to distinguish Ch. sphagnetorum from Ch. pseudosphagnetorum. Thus, for a reliable identification of these species, molecular methods, e.g. DNA barcoding, are recommended. http://zoobank.org/urn:lsid:zoobank.org:pub:F840CD92-F784-429E-B4BF-3E61F6632A8D