Browsing by Issue Date, starting with "2013-05"
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- Comparative Transcriptomic Analysis of the Burkholderia cepacia Tyrosine Kinase bceF Mutant Reveals a Role in Tolerance to Stress, Biofilm Formation, and VirulencePublication . Ferreira, A. S.; Silva, I. N.; Oliveira, V. H.; Becker, J. D.; Givskov, M.; Ryan, R. P.; Fernandes, F.; Moreira, L. M.The bacterial tyrosine-kinase (BY-kinase) family comprises the major group of bacterial enzymes endowed with tyrosine kinase activity. We previously showed that the BceF protein from Burkholderia cepacia IST408 belongs to this BY-kinase family and is involved in the biosynthesis of the exopolysaccharide cepacian. However, little is known about the extent of regulation of this protein kinase activity. In order to examine this regulation, we performed a comparative transcriptome profile between the bceF mutant and wild-type B. cepacia IST408. The analyses led to identification of 630 genes whose expression was significantly changed. Genes with decreased expression in the bceF mutant were related to stress response, motility, cell adhesion, and carbon and energy metabolism. Genes with increased expression were related to intracellular signaling and lipid metabolism. Mutation of bceF led to reduced survival under heat shock and UV light exposure, reduced swimming motility, and alteration in biofilm architecture when grown in vitro. Consistent with some of these phenotypes, the bceF mutant demonstrated elevated levels of cyclic-di-GMP. Furthermore, BceF contributed to the virulence of B. cepacia for larvae of the Greater wax moth, Galleria mellonella. Taken together, BceF appears to play a considerable role in many cellular processes, including biofilm formation and virulence. As homologues of BceF occur in a number of pathogenic and plant-associated Burkholderia strains, the modulation of bacterial behavior through tyrosine kinase activity is most likely a widely occurring phenomenon.
- Are There Really Twice as Many Bovid Species as We Thought?Publication . Heller, R.; Frandsen, P.; Lorenzen, E. D.; Siegismund, H. R.A major reappraisal of the taxonomy of ungulates (hoofed mammals) was presented in 2011 (Groves and Grubb 2011; G&G henceforth). The reappraisal presents a drastic revision of the taxonomic diversity of the group. It nearly doubles the number of bovid species—a group comprising cattle, bison, buffalo, goats, sheep, and antelopes—currently recognized (IUCN 2012), raising the number of species from 143 to 279. In our opinion, this represents taxonomic inflation; ecotypes or subspecies have been raised to the level of full species based not on new data, but solely on a change in the species concept used (Isaac et al. 2004). As we argue below, the excessive splitting of species is unconvincing in many cases. Furthermore, we warn that such taxonomic inflation in the bovids may impede management and conservation efforts (Isaac et al. 2004; Mace 2004; Frankham et al. 2012). The revised bovid species list of G&G was incorporated into the recently published “Handbook of the Mammals of the World—Volume 2” (Wilson and Mittermeier 2011; HMW henceforth) published in collaboration with the IUCN and Conservation International, 2 of the leading international authorities involved in the conservation of global biodiversity. Whereas G&G is a scientific revision intended for experts in the field, HMW is a multiauthored book series presenting the most up-to-date taxonomy of mammals for a broader readership. Collectively, these two volumes are likely to be highly influential and serve as a guideline for a wide-ranging audience including taxonomists, conservationists, ecologists, biodiversity managers, and policy makers. Any taxonomic revision that doubles the number of species within a family must anticipate critical evaluation, not least when it occurs in a prominent group such as the bovids. Here, we discuss the revision and its potential consequences, which we believe may be detrimental in many respects. The critiques we are raising are two-fold. First, we call into question the scientific grounds for the species splitting in G&G. Second, we criticize HMW for singularly adopting the bovid species list of G&G without subjecting it to critical evaluation. We discuss some of the practical downstream consequences of these actions.
- SNP typing reveals similarity in Mycobacterium tuberculosis genetic diversity between Portugal and Northeast BrazilPublication . Marques, Isabel; Soares, Patricia; Nebenzahl-Guimaraes, Hanna; Costa, Joao; Miranda, Anabela; Duarte, Raquel; Alves, Adriana; Macedo, Rita; Duarte, Tonya A.; Barbosa, Theolis; Oliveira, Martha; Nery, Joilda S.; Boechat, Neio; Pereira, Susan M.; Barreto, Mauricio L.; Pereira-Leal, Jose; Gomes, Maria Gabriela Miranda; Penha-Goncalves, CarlosHuman tuberculosis is an infectious disease caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). Although spoligotyping and MIRU-VNTR are standard methodologies in MTBC genetic epidemiology, recent studies suggest that Single Nucleotide Polymorphisms (SNP) are advantageous in phylogenetics and strain group/lineages identification. In this work we use a set of 79 SNPs to characterize 1987 MTBC isolates from Portugal and 141 from Northeast Brazil. All Brazilian samples were further characterized using spolygotyping. Phylogenetic analysis against a reference set revealed that about 95% of the isolates in both populations are singly attributed to bacterial lineage 4. Within this lineage, the most frequent strain groups in both Portugal and Brazil are LAM, followed by Haarlem and X. Contrary to these groups, strain group T showed a very different prevalence between Portugal (10%) and Brazil (1.5%). Spoligotype identification shows about 10% of mis-matches compared to the use of SNPs and a little more than 1% of strains unidentifiability. The mis-matches are observed in the most represented groups of our sample set (i.e., LAM and Haarlem) in almost the same proportion. Besides being more accurate in identifying strain groups/lineages, SNP-typing can also provide phylogenetic relationships between strain groups/lineages and, thus, indicate cases showing phylogenetic incongruence. Overall, the use of SNP-typing revealed striking similarities between MTBC populations from Portugal and Brazil.
- The Confounding Effect of Population Structure on Bayesian Skyline Plot Inferences of Demographic HistoryPublication . Heller, Rasmus; Chikhi, Lounes; Siegismund, Hans RedlefMany coalescent-based methods aiming to infer the demographic history of populations assume a single, isolated and panmictic population (i.e. a Wright-Fisher model). While this assumption may be reasonable under many conditions, several recent studies have shown that the results can be misleading when it is violated. Among the most widely applied demographic inference methods are Bayesian skyline plots (BSPs), which are used across a range of biological fields. Violations of the panmixia assumption are to be expected in many biological systems, but the consequences for skyline plot inferences have so far not been addressed and quantified. We simulated DNA sequence data under a variety of scenarios involving structured populations with variable levels of gene flow and analysed them using BSPs as implemented in the software package BEAST. Results revealed that BSPs can show false signals of population decline under biologically plausible combinations of population structure and sampling strategy, suggesting that the interpretation of several previous studies may need to be re-evaluated. We found that a balanced sampling strategy whereby samples are distributed on several populations provides the best scheme for inferring demographic change over a typical time scale. Analyses of data from a structured African buffalo population demonstrate how BSP results can be strengthened by simulations. We recommend that sample selection should be carefully considered in relation to population structure previous to BSP analyses, and that alternative scenarios should be evaluated when interpreting signals of population size change.
- A Novel Quantitative Fluorescent Reporter Assay for RAG Targets and RAG ActivityPublication . Trancoso, Inês; Bonnet, Marie; Gardner, Rui; Carneiro, Jorge; Barreto, Vasco M.; Demengeot, Jocelyne; Sarmento, Leonor M.Recombination-Activating Genes (RAG) 1 and 2 form the site specific recombinase that mediates V(D)J recombination, a process of DNA editing required for lymphocyte development and responsible for their diverse repertoire of antigen receptors. Mistargeted RAG activity associates with genome alteration and is responsible for various lymphoid tumors. Moreover several non-lymphoid tumors express RAG ectopically. A practical and powerful tool to perform quantitative assessment of RAG activity and to score putative RAG-Recognition signal sequences (RSS) is required in the fields of immunology, oncology, gene therapy, and development. Here we report the detailed characterization of a novel fluorescence-based reporter of RAG activity, named GFPi, a tool that allows measuring recombination efficiency (RE) by simple flow cytometry analysis. GFPi can be produced both as a plasmid for transient transfection experiments in cell lines or as a retrovirus for stable integration in the genome, thus supporting ex vivo and in vivo studies. The GFPi assay faithfully quantified endogenous and ectopic RAG activity as tested in genetically modified fibroblasts, tumor derived cell lines, developing pre-B cells, and hematopoietic cells. The GFPi assay also successfully ranked the RE of various RSS pairs, including bona fide RSS associated with V(D)J segments, artificial consensus sequences modified or not at specific nucleotides known to affect their efficiencies, or cryptic RSS involved in RAG-dependent activation of oncogenes. Our work validates the GFPi reporter as a practical quantitative tool for the study of RAG activity and RSS efficiencies. It should turn useful for the study of RAG-mediated V(D)J and aberrant rearrangements, lineage commitment, and vertebrate evolution.
- Expression Profile of microRNAs Regulating Proliferation and Differentiation in Mouse Adult Cardiac Stem CellsPublication . Brás-Rosário, Luis; Matsuda, Alex; Pinheiro, Ana Isabel; Gardner, Rui; Lopes, Telma; Amaral, Andreia; Gama-Carvalho, MargaridaThe identification of cardiac cells with stem cell properties changed the paradigm of the heart as a post mitotic organ. These cells proliferate and differentiate into cardiomyocytes, endothelial and vascular smooth muscle cells, providing for cardiac cell homeostasis and regeneration. microRNAs are master switches controlling proliferation and differentiation, in particular regulating stem cell biology and cardiac development. Modulation of microRNAs -regulated gene expression networks holds the potential to control cell fate and proliferation, with predictable biotechnologic and therapeutic applications. To obtain insights into the regulatory networks active in cardiac stem cells, we characterized the expression profile of 95 microRNAs with reported functions in stem cell and tissue differentiation in mouse cardiac stem cells, and compared it to that of mouse embryonic heart and mesenchymal stem cells. The most highly expressed microRNAs identified in cardiac stem cells are known to target key genes involved in the control of cell proliferation and adhesion, vascular function and cardiomyocyte differentiation. We report a subset of differentially expressed microRNAs that are proposed to act as regulators of differentiation and proliferation of adult cardiac stem cells, providing novel insights into active gene expression networks regulating their biological properties.
- Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root NodulesPublication . Limpens, Erik; Moling, Sjef; Hooiveld, Guido; Pereira, Patrícia A.; Bisseling, Ton; Becker, Jörg D.; Küster, HelgeLegumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital "in situ". This digital "in situ" offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.
- Filling the gaps of dinosaur eggshell phylogeny: Late Jurassic Theropod clutch with embryos from PortugalPublication . Araújo, Ricardo; Castanhinha, Rui; Martins, Rui M. S.; Mateus, Octávio; Hendrickx, Christophe; Beckmann, F.; Schell, N.; Alves, L. C.The non-avian saurischians that have associated eggshells and embryos are represented only by the sauropodomorph Massospondylus and Coelurosauria (derived theropods), thus missing the basal theropod representatives. We report a dinosaur clutch containing several crushed eggs and embryonic material ascribed to the megalosaurid theropod Torvosaurus. It represents the first associated eggshells and embryos of megalosauroids, thus filling an important phylogenetic gap between two distantly related groups of saurischians. These fossils represent the only unequivocal basal theropod embryos found to date. The assemblage was found in early Tithonian fluvial overbank deposits of the Lourinhã Formation in West Portugal. The morphological, microstructural and chemical characterization results of the eggshell fragments indicate very mild diagenesis. Furthermore, these fossils allow unambiguous association of basal theropod osteology with a specific and unique new eggshell morphology.