Functional Myogenic Engraftment from Mouse iPS Cells

Direct reprogramming of adult fibroblasts to a pluripotent state has opened new possibilities for the generation of patient- and disease-specific stem cells. However the ability of induced pluripotent stem (iPS) cells to generate tissue that mediates functional repair has been demonstrated in very few animal models of disease to date. Here we present the proof of principle that iPS cells may be used effectively for the treatment of muscle disorders. We combine the generation of iPS cells with conditional expression of Pax7, a robust approach to derive myogenic progenitors. Transplantation of Pax7-induced iPS-derived myogenic progenitors into dystrophic mice results in extensive engraftment, which is accompanied by improved contractility of treated muscles. These findings demonstrate the myogenic regenerative potential of iPS cells and provide rationale for their future therapeutic application for muscular dystrophies

Beta-synemin expression in cardiotoxin-injected rat skeletal muscle

Background: β-synemin was originally identified in humans as an α-dystrobrevin-binding protein through a yeast two-hybrid screen using an amino acid sequence derived from exons 1 through 16 of α-dystrobrevin, a region common to both α-dystrobrevin-1 and -2. α-Dystrobrevin-1 and -2 are both expressed in muscle and co-localization experiments have determined which isoform preferentially functions with β-synemin in vivo. The aim of our study is to show whether each α-dystrobrevin isoform has the same affinity for β-synemin or whether one of the isoforms preferentially functions with β-synemin in muscle. Methods: The two α-dystrobrevin isoforms (-1 and -2) and β-synemin were localized in regenerating rat tibialis anterior muscle using immunoprecipitation, immunohistochemical and immunoblot analyses. Immunoprecipitation and co-localization studies for α-dystrobrevin and β-synemin were performed in regenerating muscle following cardiotoxin injection. Protein expression was then compared to that of developing rat muscle using immunoblot analysis.Results: With an anti-α-dystrobrevin antibody, β-synemin co-immunoprecipitated with α-dystrobrevin whereas with an anti-β-synemin antibody, α-dystrobrevin-1 (rather than the -2 isoform) preferentially co-immunoprecipitated with β-synemin. Immunohistochemical experiments show that β-synemin and α-dystrobrevin co-localize in rat skeletal muscle. In regenerating muscle, β-synemin is first expressed at the sarcolemma and in the cytoplasm at day 5 following cardiotoxin injection. Similarly, β-synemin and α-dystrobrevin-1 are detected by immunoblot analysis as weak bands by day 7. In contrast, immunoblot analysis shows that α-dystrobrevin-2 is expressed as early as 1 day post-injection in regenerating muscle. These results are similar to that of developing muscle. For example, in embryonic rats, immunoblot analysis shows that β-synemin and α-dystrobevin-1 are weakly expressed in developing lower limb muscle at 5 days post-birth, while α-dystrobrevin-2 is detectable before birth in 20-day post-fertilization embryos. Conclusion: Our results clearly show that β-synemin expression correlates with that of α-dystrobrevin-1, suggesting that β-synemin preferentially functions with α-dystrobrevin-1 in vivo and that these proteins are likely to function coordinately to play a vital role in developing and regenerating muscle

Poor Regenerative Outcome after Skeletal Muscle Necrosis Induced by Bothrops asper Venom: Alterations in Microvasculature and Nerves

artículo (arbitrado) -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2011Background: Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood. Methodology/Principal Findings: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A2 (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration. Conclusions/Significance: The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model.This study was supported by NeTropica (grant 2-N-2008), by Vicerrectoría de Investigación, Universidad de Costa Rica (project 741-A7-604). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Soil warming accelerates decomposition of fine woody debris

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Plant and Soil 356 (2012): 405-417, doi:10.1007/s11104-012-1130-x.Soil warming from global climate change could increase decomposition of fine woody debris (FWD), but debris size and quality may mitigate this effect. The goal of this study was to investigate the effect of soil warming on decomposition of fine woody debris of differing size and quality. We placed FWD of two size classes (2 × 20 cm and 4 × 40 cm) and four species (Acer saccharum, Betula lenta, Quercus rubra and Tsuga canadensis) in a soil warming and ambient area at Harvard Forest in central Massachusetts. We collected the debris from each area over two years and measured mass loss and lignin concentration. Warming increased mass loss for all species and size classes (by as much as 30%), but larger debris and debris with higher initial lignin content decomposed slower than smaller debris and debris with lower initial lignin content. Lignin degradation did not follow the same trends as mass loss. Lignin loss from the most lignin-rich species, T. canadensis, was the highest despite the fact that it lost mass the slowest. Our results suggest that soil warming will increase decomposition of FWD in temperate forests. It is imperative that future models and policy efforts account for this potential shift in the carbon storage pool

Long-term and realistic global change manipulations had low impact on diversity of soil biota in temperate heathland

In a dry heathland ecosystem we manipulated temperature (warming), precipitation (drought) and atmospheric concentration of CO(2) in a full-factorial experiment in order to investigate changes in below-ground biodiversity as a result of future climate change. We investigated the responses in community diversity of nematodes, enchytraeids, collembolans and oribatid mites at two and eight years of manipulations. We used a structural equation modelling (SEM) approach analyzing the three manipulations, soil moisture and temperature, and seven soil biological and chemical variables. The analysis revealed a persistent and positive effect of elevated CO(2) on litter C:N ratio. After two years of treatment, the fungi to bacteria ratio was increased by warming, and the diversities within oribatid mites, collembolans and nematode groups were all affected by elevated CO(2) mediated through increased litter C:N ratio. After eight years of treatment, however, the CO(2)-increased litter C:N ratio did not influence the diversity in any of the four fauna groups. The number of significant correlations between treatments, food source quality, and soil biota diversities was reduced from six to three after two and eight years, respectively. These results suggest a remarkable resilience within the soil biota against global climate change treatments in the long term

Protist diversity on a nature reserve in NW England − with particular reference to their role in soil biogenic silicon pools

Soil protists play fundamental roles in many earth system processes, yet we are only beginning to understand the true diversity of the organisms involved. In this study we used conventional (microscopy-based) methods to characterise the diversity and estimate protist population sizes in soils from a variety of distinct habitats within Mere Sands Wood nature reserve in NW England. We produced population size data for over ninety soil protists belonging to two major eukaryotic functional groups: testate amoebae (TA) and diatoms, adding substantial ‘cryptic diversity’ to the nature reserves recorded biota. From these population size data we estimated relative contributions of TA and diatoms to soil biogenic silicon (BSi) pools and found significant correlations between taxon richness and the TA and diatom Si pool. This could indicate that protist functional diversity can influence terrestrial BSi pools, especially in early successional plant communities where TA and diatoms can potentially increase Si mineralisation and/or create Si ‘hot spots’ and hence, the biological availability of this element for subsequent plant uptake. TA were particularly abundant in mor humus type soils further supporting the idea that they could be important players in nutrient cycling in such soils. Overall, we demonstrate this is a useful approach in order to start to attempt to estimate the role of protists in the Si cycle and other ecological processes

A Novel Conserved Isoform of the Ubiquitin Ligase UFD2a/UBE4B Is Expressed Exclusively in Mature Striated Muscle Cells

Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish), UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3′ introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis