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Gomes N.C.M.,University of Aveiro | Cleary D.F.R.,University of Aveiro | Pinto F.N.,Federal University of Rio de Janeiro | Egas C.,Biocant Biotechnology Innovation Center | And 5 more authors.
PLoS ONE | Year: 2010

Background: Mangrove forests are of global ecological and economic importance, but are also one of the world's most threatened ecosystems. Here we present a case study examining the influence of the rhizosphere on the structural composition and diversity of mangrove bacterial communities and the implications for mangrove reforestation approaches using nursery-raised plants. Methodology/Principal Findings: A barcoded pyrosequencing approach was used to assess bacterial diversity in the rhizosphere of plants in a nursery setting, nursery-raised transplants and native (non-transplanted) plants in the same mangrove habitat. In addition to this, we also assessed bacterial composition in the bulk sediment in order to ascertain if the roots of mangrove plants affect sediment bacterial composition. We found that mangrove roots appear to influence bacterial abundance and composition in the rhizosphere. Due to the sheer abundance of roots in mangrove habitat, such an effect can have an important impact on the maintenance of bacterial guilds involved in nutrient cycling and other key ecosystem functions. Surprisingly, we also noted a marked impact of initial nursery conditions on the rhizosphere bacterial composition of replanted mangrove trees. This result is intriguing because mangroves are periodically inundated with seawater and represent a highly dynamic environment compared to the more controlled nursery environment. Conclusions/Significance: In as far as microbial diversity and composition influences plant growth and health, this study indicates that nursery conditions and early microbial colonization patterns of the replants are key factors that should be considered during reforestation projects. In addition to this, our results provide information on the role of the mangrove rhizosphere as a habitat for bacteria from estuarine sediments. © 2010 Gomes et al.

Faria T.Q.,University of Coimbra | Almeida Z.L.,University of Coimbra | Cruz P.F.,University of Coimbra | Jesus C.S.H.,University of Coimbra | And 2 more authors.
Physical Chemistry Chemical Physics | Year: 2015

The aggregation of proteins into insoluble amyloid fibrils is the hallmark of many, highly debilitating, human pathologies such as Alzheimer's or Parkinson's disease. Transthyretin (TTR) is a homotetrameric protein implicated in several amyloidoses like Senile Systemic Amyloidosis (SSA), Familial Amyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC), and the rare Central Nervous System selective Amyloidosis (CNSA). In this work, we have investigated the kinetics of TTR aggregation into amyloid fibrils produced by the addition of NaCl to acid-unfolded TTR monomers and we propose a mathematically simple kinetic mechanism to analyse the aggregation kinetics of TTR. We have conducted circular dichroism, intrinsic tryptophan fluorescence and thioflavin-T emission experiments to follow the conformational changes accompanying amyloid formation at different TTR concentrations. Kinetic traces were adjusted to a two-step model with the first step being second-order and the second being unimolecular. The molecular species present in the pathway of TTR oligomerization were characterized by size exclusion chromatography coupled to multi-angle light scattering and by transmission electron microscopy. The results show the transient accumulation of oligomers composed of 6 to 10 monomers in agreement with reports suggesting that these oligomers may be the causative agent of cell toxicity. The results obtained may prove to be useful in understanding the mode of action of different compounds in preventing fibril formation and, therefore, in designing new drugs against TTR amyloidosis. This journal is © the Owner Societies 2015.

Anjo S.I.,University of Coimbra | Santa C.,University of Coimbra | Manadas B.,University of Coimbra | Manadas B.,Biocant Biotechnology Innovation Center
Proteomics | Year: 2015

The quantification of large proteomes across multiple samples has become the major focus of proteomics. In addition to the advantages of in-gel digestion, the extensive time and sample handling required have precluded the use of this type of method for large quantitative screens. Therefore, an adaptation of the in-gel digestion method, termed short-GeLC, is proposed as a faster and more reproducible sample preparation method for quantitative approaches. The proposed methodology was compared with two well-established procedures for sample preparation, GeLC-MS and the classic liquid digestion followed by LC-MS, using a membrane protein-enriched sample. The results show that the short-GeLC approach substantially reduces the amount of sample handling and the overall time required for analysis compared with the gel-based methods without compromising the overall results at the protein identification level. Furthermore, the short-GeLC approach in combination with the SWATH acquisition method leads to the best quantitative results: more proteins were quantified, and the reproducibility was improved. Finally, this method performed well even on challenging samples enriched in membrane proteins. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Teixeira F.G.,University of Minho | Teixeira F.G.,PT Government Associate Laboratory | Panchalingam K.M.,University of Calgary | Anjo S.I.,University of CoimbraCoimbra | And 10 more authors.
Stem Cell Research and Therapy | Year: 2015

Abstract Introduction: The use of human umbilical cord Wharton Jelly-derived mesenchymal stem cells (hWJ-MSCs) has been considered a new potential source for future safe applications in regenerative medicine. Indeed, the application of hWJ-MSCs into different animal models of disease, including those from the central nervous system, has shown remarkable therapeutic benefits mostly associated with their secretome. Conventionally, hWJ-MSCs are cultured and characterized under normoxic conditions (21 % oxygen tension), although the oxygen levels within tissues are typically much lower (hypoxic) than these standard culture conditions. Therefore, oxygen tension represents an important environmental factor that may affect the performance of mesenchymal stem cells in vivo. However, the impact of hypoxic conditions on distinct mesenchymal stem cell characteristics, such as the secretome, still remains unclear. Methods: In the present study, we have examined the effects of normoxic (21 % O2) and hypoxic (5 % O2) conditions on the hWJ-MSC secretome. Subsequently, we address the impact of the distinct secretome in the neuronal cell survival and differentiation of human neural progenitor cells. Results: The present data indicate that the hWJ-MSC secretome collected from normoxic and hypoxic conditions displayed similar effects in supporting neuronal differentiation of human neural progenitor cells in vitro. However, proteomic analysis revealed that the use of hypoxic preconditioning led to the upregulation of several proteins within the hWJ-MSC secretome. Conclusions: Our results suggest that the optimization of parameters such as hypoxia may lead to the development of strategies that enhance the therapeutic effects of the secretome for future regenerative medicine studies and applications. © 2015 Teixeira et al.

Martins-Marques T.,University of Coimbra | Anjo S.I.,University of Coimbra | Pereira P.,University of Coimbra | Manadas B.,University of Coimbra | And 2 more authors.
Molecular and Cellular Proteomics | Year: 2015

The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of C×43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of C×43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, C×43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by C×43 is dictated by the nature of the proteins that interact with C×43. Therefore, the characterization of the C×43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of C×43. In the present report, we applied a quantitative SWATH-MS approach to characterize the C×43 interactome in rat hearts subjected to ischemia and ischemiareperfusion. Our results demonstrate that, in the heart, C×43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of C×43 with proteins involved in gene transcription strengthens the emerging concept that C×43 has a role in gene expression regulation. Importantly, our data shows that the interactome of C×43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of C×43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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