DE-Cadherin regulates unconventional Myosin ID and Myosin IC in Drosophila left-right asymmetry establishment

In bilateria, positioning and looping of visceral organs requires proper left-right (L/R) asymmetry establishment. Recent work in Drosophila has identified a novel situs inversus gene encoding the unconventional type ID myosin (MyoID). In myoID mutant flies, the L/R axis is inverted, causing reversed looping of organs, such as the gut, spermiduct and genitalia. We have previously shown that MyoID interacts physically with β-Catenin, suggesting a role of the adherens junction in Drosophila L/R asymmetry. Here, we show that DE-Cadherin co-immunoprecipitates with MyoID and is required for MyoID L/R activity. We further demonstrate that MyoIC, a closely related unconventional type I myosin, can antagonize MyoID L/R activity by preventing its binding to adherens junction components, both in vitro and in vivo. Interestingly, DE-Cadherin inhibits MyoIC, providing a protective mechanism to MyoID function. Conditional genetic experiments indicate that DE-Cadherin, MyoIC and MyoID show temporal synchronicity for their function in L/R asymmetry. These data suggest that following MyoID recruitment by β-Catenin at the adherens junction, DE-Cadherin has a twofold effect on Drosophila L/R asymmetry by promoting MyoID activity and repressing that of MyoIC. Interestingly, the product of the vertebrate situs inversus gene inversin also physically interacts with β-Catenin, suggesting that the adherens junction might serve as a conserved platform for determinants to establish L/R asymmetry both in vertebrates and invertebrates.     

Development of polymorphic microsatellite markers issued from pyrosequencing technology for the medicinal mushroom Agaricus subrufescens

The recently described procedure of microsatellite-enriched library pyrosequencing was used to isolate microsatellite loci in the gourmet and medicinal mushroom Agaricus subrufescens. Three hundred and five candidate loci containing at least one simple sequence repeats (SSR) locus and for which primers design was successful, were obtained. From a subset of 95 loci, 35 operational and polymorphic SSR markers were developed and characterized on a sample of 14 A.?subrufescens genotypes from diverse origins. These SubSSR markers each displayed from two to 10 alleles with an average of 4.66 alleles per locus. The observed heterozygosity ranged from 0 to 0.71. Several multiplex combinations can be set up, making it possible to genotype up to six markers easily and simultaneously. Cross-amplification in some closely congeneric species was successful for a subset of loci. The 35 microsatellite markers developed here provide a highly valuable molecular tool to study genetic diversity and reproductive biology of A.?subrufescens.     

From ApoA1 upregulation to BET family bromodomain inhibition: discovery of I-BET151

The discovery, synthesis and biological evaluation of a novel series of 7-isoxazoloquinolines is described. Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepatic cell line HepG2.     

Multi-stemmed trees of Nothofagus pumilio second-growth forest in Patagonia are formed by highly related individuals

Background and Aims Multi-stemmed trees (tree clusters) in Nothofagus pumilio, a dominant tree species in Patagonia, are very uncommon and are restricted to the edge of second-growth forests following human-provoked fires. No vegetative reproduction has been reported so far. The genetic structure of multi-stemmed trees of this species was investigated and it was hypothesized that genets within a cluster were more closely related than average in the population. Methods Fifteen clusters (composed of at least three purported stems) and 15 single trees were sampled at the edge of a second-growth forest and genotyped using two amplified fragment length polymorphism (AFLP) primer pairs. We obtained 119 polymorphic markers that allowed clonality to be determined, together with sibship structure and relatedness among samples. Key Results Clonality was detected in seven clusters but all clusters had at least two different genotypes. Full sibs were found exclusively within clusters and in all clusters. Within a cluster, stems that were not identified as full sibs were often half sibs. Relatedness values for the full sibs and half sibs were higher than the theoretical values of 0·5 and 0·25 but the relatedness between clusters was very low. Conclusions Tree clusters that are merged at the edge of the second-growth forest of N. pumilio are composed of stems of the same genotype and of other genotypes that are highly related (but not always). It is suggested that this peculiar genetic structure results from a combination of several causes, including selection for merging of related individuals.     

A novel synthesis of D-galactofuranosyl,D-glucofuranosyl and D-mannofuranosyl 1-phosphates based on remote activation of new and free hexofuranosyl donors

The selective synthesis of 1,2-cis-hexofuranosyl 1-phosphates was readily accomplished according to a procedure based on the 'Remote Activation Concept'. This approach required (i) the preparation of suitable 1,2-trans-hexofuranosyl donors, so that new heterocyclic thiofuranosides were designed and synthesized, (ii) the stereocontrolled phosphorylation of the corresponding unprotected donors and (iii) the simple and fast purification of the resulting anomeric phosphates. This approach showed to be equally efficient in the galactose, glucose and mannose series.     

Recombinant peroxiredoxin 5 protects against excitotoxic brain lesions in newborn mice

The pathophysiology of brain lesions associated with cerebral palsy is multifactorial and likely involves excess release of glutamate and excess production of free radicals, among other factors. Theoretically, antioxidants could limit the severity of these brain lesions. Peroxiredoxins are a family of peroxidases widely distributed in eukaryotes and prokaryotes. Peroxiredoxin 5 (PRDX5) is a recently discovered mammalian member of this family of antioxidant enzymes that is able to reduce hydrogen peroxide and alkyl hydroperoxides. The present study was designed to examine the neuroprotective effects of recombinant PRDX5 against neonatal excitotoxic challenge in both in vivo and in vitro experiments. For in vivo experiments, mice (postnatal day 5) were injected intraneopallially with ibotenate acting on NMDA and metabotropic receptors, or S-bromowillardiine acting on AMPA-kainate receptors to produce excitotoxic stress and brain lesions. Systemically administered recombinant PRDX5 provided protection against ibotenate-induced excitotoxic stress. Brain lesions of animals given ibotenate and PRDX5 were up to 63% smaller than that given ibotenate alone. However, PRDX5 provided no prevention from lesions induced with S-bromowillardiine. A mutated recombinant PRDX5 that is devoid of peroxidase activity was also tested and showed no protection against lesions induced by either ibotenate or S-bromowillardiine. Two classical antioxidants, N-acetylcysteine and catalase-PEG, provided the same neuroprotective effect as PRDX5. For in vitro experiments, neocortical neurons were exposed to 300 microM NMDA alone, NMDA plus recombinant PRDX5, or NMDA, recombinant PRDX5 and dithiothreitol, a classical electron donor for peroxiredoxins. Recombinant PRDX5 plus dithiothreitol displayed a synergistic neuroprotective effect on NMDA-induced neuronal death. These findings indicate that reactive oxygen species production participates in the formation of NMDA receptor-mediated brain lesions in newborn mice and that antioxidant compounds, such as PRDX5, provide some neuroprotection in these models.     

Impairing the microRNA biogenesis pathway induces proteome modifications characterized by size bias and enrichment in antioxidant proteins

Perturbation of individual microRNAs, or of the microRNA pathway, plays a role in carcinogenesis. In certain cancer cells, inhibition of the microRNA biogenesis pathway leads to a growth arrest state (CoGAM for Colony Growth Arrest induced by Microprocessor inhibition), which can be rescued by re-expression of individual microRNAs such as miR-20a. We now report that inhibition of the microRNA biogenesis pathway induced proteome changes characterized by a size bias in differentially expressed proteins, with induction of small proteins and inhibition of large ones. This size bias was observed in cells undergoing CoGAM, as well as in CoGAM-resistant cells, and in CoGAM-sensitive cells rescued by miR-20a. In this case, GO analysis of induced proteins identified by mass spectrometry revealed a significant enrichment in proteins involved in resistance to oxidative stress. In addition, H(2) O(2) treatment of Saccharomyces cerevisiae or mammalian cells led to similarly size-biased proteome modifications. Our results point to size bias as a relevant readout of proteome modifications, in particular in conditions of stress such as inhibition of the microRNA biogenesis pathway or oxidative stress. They also suggest research avenues to study the role of the microRNA pathway in proteostasis.     

Cancer Cells Induced to Express Mesenchymal Phenotype Release Exosome-like Extracellular Vesicles Carrying Tissue Factor

Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant interactions with their surroundings, including the vasculature. Whether the release/uptake of extracellular vesicles (EVs) plays a role during these processes has not been studied. EVs are heterogeneous membrane structures that originate either at the surface (microparticles), or within (exosomes) activated or transformed cells, and are involved in intercellular trafficking of bioactive molecules. Here, we show that epithelial cancer cells (A431, DLD-1) adopt mesenchymal features (epithelial-to-mesenchymal transition-like state) upon activation of epidermal growth factor receptor (EGFR) coupled with blockade of E-cadherin. This treatment leads to a coordinated loss of EGFR and tissue factor (TF) from the plasma membrane and coincides with a surge in emission of small, exosome-like EVs containing both receptors. TF (but not EGFR) is selectively up-regulated in EVs produced by mesenchymal-like cancer cells and can be transferred to cultured endothelial cells rendering them highly procoagulant. We postulate that epithelial-to-mesenchymal transition-like changes may alter cancer cell interactions with the vascular systems through altered vesiculation and TF shedding.     

Axial speed of sound is related to tendon's nonlinear elasticity

Axial speed of sound (SOS) measurements have been successfully applied to noninvasively evaluate tendon load, while preliminary studies showed that this technique also has a potential clinical interest in the follow up of tendon injuries. The ultrasound propagation theory predicts that the SOS is determined by the effective stiffness, mass density and Poisson's ratio of the propagating medium. Tendon stiffness characterizes the tissue's mechanical quality, but it is often measured in quasi-static condition and for entire tendon segments, so it might not be the same as the effective stiffness which determines the SOS. The objectives of the present study were to investigate the relationship between axial SOS and tendon's nonlinear elasticity, measured in standard laboratory conditions, and to evaluate if tendon's mass density and cross-sectional area (CSA) affect the SOS level. Axial SOS was measured during in vitro cycling of 9 equine superficial digital tendons. Each tendon's stiffness was characterized with a tangent modulus (the continuous derivative of the true stress/true strain curve) and an elastic modulus (the slope of this curve's linear region). Tendon's SOS was found to linearly vary with the square root of the tangent modulus during loading; tendon's SOS level was found correlated to the elastic modulus's square root and inversely correlated to the tendon's CSA, but it was not affected by tendon's mass density. These results confirm that tendon's tangent and elastic moduli, measured in laboratory conditions, are related to axial SOS and they represent one of its primary determinants.