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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.


PubMed | Applied Photophysics Ltd., University of Coimbra, IBMC Institute for Molecular and Cell Biology, Biocant Biotechnology Innovation Center and 2 more.
Type: Journal Article | Journal: The Biochemical journal | Year: 2016

Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood. Recently, the structure of laforin with bound maltohexaose was determined and despite the importance of such breakthrough, some molecular interaction details remained missing. We herein report a thorough biophysical characterization of laforin-carbohydrate interaction using soluble glycans. We demonstrated an increased preference of laforin for the interaction with glycans with higher order of polymerization and confirmed the importance of tryptophan residues for glycan interaction. Moreover, and in line with what has been described for other CBMs and lectins, our results confirmed that laforin-glycan interactions occur with a favourable enthalpic contribution counter-balanced by an unfavourable entropic contribution. The analysis of laforin-glycan interaction through the glycan side by saturation transfer difference (STD)-NMR has shown that the CBM-binding site can accommodate between 5 and 6 sugar units, which is in line with the recently obtained crystal structure of laforin. Overall, the work in the present study complements the structural characterization of laforin and sheds light on the molecular mechanism of laforin-glycan interaction, which is a pivotal requisite to understand the physiological and pathological roles of laforin.


Jensen K.H.,Harvard University | Jensen K.H.,Technical University of Denmark | Valente A.X.C.N.,Biocant Biotechnology Innovation Center | Valente A.X.C.N.,University of Coimbra | And 2 more authors.
Physics of Fluids | Year: 2014

We examine the fluid mechanics of viscous flow through filters consisting of perforated thin plates. We classify the effects that contribute to the hydraulic resistance of the filter. Classical analyses assume a single pore size and account only for filter thickness. We extend these results to obtain an analytical formula for the pressure drop across the microfilter versus the flow rate that accounts for the non-uniform distribution of pore sizes, the hydrodynamic interactions between the pores given their layout pattern, and wall slip. Further, we discuss inertial effects and their order of scaling. © 2014 AIP Publishing LLC.


PubMed | Sharif University of Technology, Iran Polymer And Petrochemical Institute, Biocant Biotechnology Innovation Center, Portuguese Institute of Blood and Transplantation and University of Coimbra
Type: Journal Article | Journal: ACS applied materials & interfaces | Year: 2016

Superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with antimicrobial agents are promising infection-targeted therapeutic platforms when coupled with external magnetic stimuli. These antimicrobial nanoparticles (NPs) may offer advantages in fighting intracellular pathogens as well as biomaterial-associated infections. This requires the development of NPs with high antimicrobial activity without interfering with the biology of mammalian cells. Here, we report the preparation of biocompatible antimicrobial SPION@gold core-shell NPs based on covalent immobilization of the antimicrobial peptide (AMP) cecropin melittin (CM) (the conjugate is named AMP-NP). The minimal inhibitory concentration (MIC) of the AMP-NP for Escherichia coli was 0.4 g/mL, 10-times lower than the MIC of soluble CM. The antimicrobial activity of CM depends on the length of the spacer between the CM and the NP. AMP-NPs are taken up by endothelial (between 60 and 170 pg of NPs per cell) and macrophage (between 18 and 36 pg of NPs per cell) cells and accumulate preferentially in endolysosomes. These NPs have no significant cytotoxic and pro-inflammatory activities for concentrations up to 200 g/mL (at least 100 times higher than the MIC of soluble CM). Our results in membrane models suggest that the selectivity of AMP-NPs for bacteria and not eukaryotic membranes is due to their membrane compositions. The AMP-NPs developed here open new opportunities for infection-site targeting.


Cleary D.F.R.,University of Aveiro | Becking L.E.,Naturalis Biodiversity Center | Becking L.E.,Wageningen University | de Voogd N.J.,Naturalis Biodiversity Center | And 4 more authors.
FEMS Microbiology Ecology | Year: 2013

Marine lakes are unique ecosystems that contain isolated populations of marine organisms. Isolated from the surrounding marine habitat, many lakes house numerous endemic species. In this study, microbial communities of sponges inhabiting these lakes were investigated for the first time using barcoded pyrosequencing of 16S rRNA gene amplicons. Our main goals were to compare the bacterial richness and composition of two sponge species (Suberites diversicolor and Cinachyrella australiensis) inhabiting both marine lakes and adjacent open coastal systems. Host species and habitat explained almost 59% of the variation in bacterial composition. There was a significant difference in composition between both host species. Within S. diversicolor, there was little discernible difference between bacterial communities inside and outside lakes. The bacterial community of this species was, furthermore, dominated (63% of all sequences) by three very closely related alphaproteobacterial taxa identified as belonging to the recently described order Kiloniellales. Cinachyrella australiensis, in contrast, hosted markedly different bacterial communities inside and outside lakes with very few shared abundant taxa. Cinachyrella australiensis in open habitat only shared 9.4% of OTUs with C. australiensis in lake habitat. Bacteria were thus both highly species specific and, in the case of C. australiensis, habitat specific. © 2013 Federation of European Microbiological Societies.


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.


Bhise N.S.,Harvard University | Bhise N.S.,Harvard-MIT Division of Health Sciences and Technology | Ribas J.,Harvard University | Ribas J.,Harvard-MIT Division of Health Sciences and Technology | And 16 more authors.
Journal of Controlled Release | Year: 2014

Novel microfluidic tools allow new ways to manufacture and test drug delivery systems. Organ-on-a-chip systems - microscale recapitulations of complex organ functions - promise to improve the drug development pipeline. This review highlights the importance of integrating microfluidic networks with 3D tissue engineered models to create organ-on-a-chip platforms, able to meet the demand of creating robust preclinical screening models. Specific examples are cited to demonstrate the use of these systems for studying the performance of drug delivery vectors and thereby reduce the discrepancies between their performance at preclinical and clinical trials. We also highlight the future directions that need to be pursued by the research community for these proof-of-concept studies to achieve the goal of accelerating clinical translation of drug delivery nanoparticles. © 2014 Elsevier B.V.


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.


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.


PubMed | Biocant Biotechnology Innovation Center
Type: Journal Article | Journal: Marine drugs | Year: 2012

Deep-sea environments are largely unexplored habitats where a surprising number of species may be found in large communities, thriving regardless of the darkness, extreme cold, and high pressure. Their unique geochemical features result in reducing environments rich in methane and sulfides, sustaining complex chemosynthetic ecosystems that represent one of the most surprising findings in oceans in the last 40 years. The deep-sea Lucky Strike hydrothermal vent field, located in the Mid Atlantic Ridge, is home to large vent mussel communities where Bathymodiolus azoricus represents the dominant faunal biomass, owing its survival to symbiotic associations with methylotrophic or methanotrophic and thiotrophic bacteria. The recent transcriptome sequencing and analysis of gill tissues from B. azoricus revealed a number of genes of bacterial origin, hereby analyzed to provide a functional insight into the gill microbial community. The transcripts supported a metabolically active microbiome and a variety of mechanisms and pathways, evidencing also the sulfur and methane metabolisms. Taxonomic affiliation of transcripts and 16S rRNA community profiling revealed a microbial community dominated by thiotrophic and methanotrophic endosymbionts of B. azoricus and the presence of a Sulfurovum-like epsilonbacterium.

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