Development of a strategy to functionalize a dextrin-based hydrogel for animal cell cultures using a starch-binding module fused to RGD sequence

Several approaches can be used to functionalize biomaterials, such as hydrogels, for biomedical applications. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence, present in several proteins from the extra-cellular matrix (ECM), is a ligand for integrin-mediated cell adhesion; this sequence was recognized as a major functional group responsible for cellular adhesion. In this work a bi-functional recombinant protein, containing a starch binding module (SBM) and RGD sequence was used to functionalize a dextrin-based hydrogel. The SBM, which belongs to an α-amylase from Bacillus sp. TS-23, has starch (and dextrin, depolymerized starch) affinity, acting as a binding molecule to adsorb the RGD sequence to the hydrogel surface. Results The recombinant proteins SBM and RGD-SBM were cloned, expressed, purified and tested in in vitro assays. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with recombinant proteins were performed using mouse embryo fibroblasts 3T3. A polystyrene cell culture plate was used as control. The results showed that the RGD-SBM recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell spreading on the hydrogel surface was observed only in the presence of the RGD-SBM. Conclusion The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel. Many proteins in nature that hold a RGD sequence are not cell adhesive, probably due to the conformation/accessibility of the peptide. We therefore emphasise the successful expression of a bi-functional protein with potential for different applications.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil)Fundação para a Ciência e a Tecnologia (FCT

Production of recombinant carbohydrate-binding modules fused to RGD : functional studies using bacterial cellulose

The attachment of cells to biomedical materials can be improved by using adhesion molecules, present in the extracellular matrix substances, such as fibronectin, vitronectin, or laminin. In many cases, Arg-Gly-Asp (RGD) was found to be the major functional amino acid sequence responsible for cellular adhesion. In the present study, a method for producing chimerics proteins, RGDCBM, with functions similar to fibronectin, which contains a cellulose-binding module (CBM), was developed. The CBM used was from the cellulosoma of the bacteria Clostridium thermocellum. The genes encoding these CBM-containing chimeric proteins were cloned, and the protein expressed and purified. Bacterial cellulose (BC) secreted by Gluconacetobacter xylinus was produced. Polystyrene surfaces and bacterial cellulose sheets where ‘‘coated’’ with these RGD-containing proteins, and then used in adhesion/biocompatibility tests, using a mouse embryo fibroblasts culture. The results showed that the proteins containing the RGD or GRGDY sequence were able to improve the adhesion of the fibroblast on the polystyrene plate, furthermore proteins containing the RGD sequence were more effective than the proteins containing the GRGDY sequence. Preliminary adhesion studies of fibroblast cultures on cellulose sheets, functionalized with the recombinant proteins, showed positive effects on the adhesion and proliferation of the cells. The results demonstrated that the proteins containing the RGD sequence were able to increase significantly the adhesion of fibroblast to BC surfaces when compared with the controls (cellulose treated with the CBM or buffer). The results also demonstrated that the protein containing one RGD sequence have a stronger effect than the protein containing two RGDs

Modelización de productos forestales no maderables en Europa: revisión

Non-wood forest products (NWFP) like cork, edible mushrooms, pine nuts, acorns, resins, medicinal plants, and floral greens, among others, provide important recreational and commercial activities in the rural forested areas of the world, being in certain regions more profitable than traditional timber harvesting. Despite the importance of non-wood forest products and services, forest management and planning methods and models in Europe have been traditionally wood oriented, leading to a lack for tools helping forest management based on optimizing these products. In the present work we’ll show and discuss the main factors and challenges limiting the development of classical empirical models for NWFP, and we will review the existing models for the main NWFP in Europe: cork, pine nuts, berries, mushrooms and resins.Los productos forestales no maderables (PFNM), como el corcho, los hongos coemstibles, piñones, bellotas, resinas y plantas medicinales u ornamentales, entre otros, son fuente tanto de servicios recreativos como de actividad económica en una parte importante de los bosques del mundo. En muchas de estas zonas, el aprovechamiento de estos productos supone un ingreso superior al obtenido en el aprovechamiento maderable tradicional. Sin embargo, y pese a la importancia de estos productos y servicios no maderables, la gestión forestal, las herramientas de apoyo y los modelos forestales existentes en Europa han sido normalmente desarrollados desde una perspectiva centrada en la producción de madera. Esto se ha traducido en la escasez de herramientas de apoyo a la decisión de la gestión basadas en la optimización de la producción no maderable. En el presente trabajo se exponen y discuten los principales factores que limitan el desarrollo de los modelos para productos no maderables, y se revisan los modelos existentes para los principales productos no maderables en Europa: corcho, piñones, frutos del bosque, hongos y resina

Bacterial communities associated with Monochamus galloprovincialis, the insect-vector of pine wilt disease

Bacterial communities associated with the Bursaphelenchus xylophilus (pine wood nematode, PWN) are suggested to play a role in pine wilt disease (PWD) development. However, it’s not clear where the PWN acquires these communities. In this sense, it is possible that bacterial communities colonizing Monochamus spp. may affect the bacterial communities associated with the PWN. In this work, we present the characterization of bacterial communities of the Portuguese insect vector Monochamus galloprovincialis using culture independent methods, and investigate the common bacterial communities between the insect-vector and the pathogenic agent, PWN. Monochamus galloprovincialis is mainly composed by γ-proteobacteria, Firmicutes and Bacteroidetes, sharing common bacterial genera with B. xylophilus (i.e. Serratia, Bacillus, and Pseudomonas). These results can bring new insights into the role of the insect vector in the PWN-bacteria interaction

Evidence for the fixation of gene duplications by positive selection in \u3ci\u3eDrosophila\u3c/i\u3e

Gene duplications play a key role in the emergence of novel traits and in adaptation. But despite their centrality to evolutionary processes, it is still largely unknown how new gene duplicates are initially fixed within populations and later maintained in genomes. Long-standing debates on the evolution of gene duplications could be settled by determining the relative importance of genetic drift vs. positive selection in the fixation of new gene duplicates. Using the Drosophila Global Diversity Lines (GDL), we have combined genome-wide SNP polymorphism data with a novel set of copy number variant calls and gene expression profiles to characterize the polymorphic phase of new genes. We found that approximately half of the roughly 500 new complete gene duplications segregating in the GDL lead to significant increases in the expression levels of the duplicated genes and that these duplications are more likely to be found at lower frequencies, suggesting a negative impact on fitness. However, we also found that six of the nine gene duplications that are fixed or close to fixation in at least one of the five populations in our study show signs of being under positive selection, and that these duplications are likely beneficial because of dosage effects, with a possible role for additional mutations in two duplications. Our work suggests that in Drosophila, theoretical models that posit that gene duplications are immediately beneficial and fixed by positive selection are most relevant to explain the long-term evolution of gene duplications in this species. Supplemental materials attached below

Groundwater quality comparison between rural farms and riparian wells in the western Amazon, Brazil

Groundwater quality of a riparian forest is compared to wells in surrounding rural areas at Urupá River basin. Groundwater types were calcium bicarbonated at left margin and sodium chloride at right, whereas riparian wells exhibited a combination of both (sodium bicarbonate). Groundwater was mostly solute-depleted with concentrations within permissible limits for human consumption, except for nitrate. Isotopic composition suggests that inorganic carbon in Urupá River is mostly supplied by runoff instead of riparian groundwater. Hence, large pasture areas in addition to narrow riparian forest width in this watershed may have an important contribution in the chemical composition of this river.FAPESPCNPq - CT-HIDROCNPq - Milêni

Impact of Euro-Atlantic blocking patterns in Iberia precipitation using a novel high resolution dataset

In this work we reassess the impacts of blocking patterns on precipitation regimes in the Iberian Peninsula, distinguishing between north Atlantic and European blocking. For this we take full advantage of the recently developed high-resolution datasets for the Iberian countries. Precipitation anomalies during blocking events obtained with this dataset allow a much finer regional characterization of the impacts in both average and extreme daily precipitation, particularly when compared to widely used low-resolution reanalysis datasets. Blocked patterns induce a negative-positive dipole of precipitation anomalies from northwest to southeast Iberia. Increases are widespread during Atlantic blocks and pronounced in southern and eastern areas of Iberia, while during European blocks they are more spatially restricted, with increases above 50 % in coastal Mediterranean areas, which represents a considerable fraction of the annual precipitation. Blocking impacts in precipitation are nearly opposite to those found during strong zonal flow situations, but there are also some asymmetries in the precipitation responses. A significant increase in cyclones and cut-off lows frequency southwards of blocking structures is related to precipitation excesses over southern and eastern areas, where dynamical factors and local processes play a crucial role. On the contrary, precipitation deficits in northwest Iberia during blocking episodes are better explained by a reduction in north Atlantic frontal activity and simultaneous decreases in large-scale moisture advection towards northern Iberia. We show that these anomalies during blocking result from changes in precipitation amount rather than from increases in rainy days, pointing to more extreme rainfall regimes, particularly in southeastern Iberia. Finally, an Extreme Value Analysis was performed, fitting Generalized Pareto Distributions to precipitation extremes. Results show that the different extreme precipitation regimes of northwest and Mediterranean regions are partially determined by opposite anomalies of the zonal flow. Thus, heavy precipitation events in Mediterranean areas are usually short-lived and frequently associated with blocking conditions, while in northwest Iberia the total accumulations during rainfall episodes are more important for triggering extreme events and they are mainly related to strong westerly flows.Peer reviewe