Spider silk-bone sialoprotein fusion proteins for bone tissue engineering

The remarkable mechanical characteristics of the spider silk protein major ampullate spidroin protein suggest this polymer as a promising biomaterial to consider for the fabrication of scaffolds for bone regeneration. Herein, a new functionalized spider silk-bone sialoprotein fusion protein was designed, cloned, expressed, purified and the osteogenic activity studied. Bone sialoprotein (BSP) is a multidomain protein with the ability to induce cell attachment and differentiation and the deposition of calcium phosphates (CaP). Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) was used to assess the secondary structure of the fusion protein. In vitro mineralization studies demonstrated that this new fusion protein with BSP retained the ability to induce the deposition of CaP. Studies in vitro indicated that human mesenchymal stem cells had significant improvement towards osteogenic outcomes when cultivated in the presence of the new fusion protein vs. silk alone. The present work demonstrates the potential of this new fusion protein for future applications in bone regenerationPhD grant SFRH/BD/28603/2006; Chimera project, PTDC/EBB-EBI/109093/2008; NIH, P41 EB002520, EB003210 and DE017207.Foundation for Science and Technolog

Antispasmodic activity of aqueous extracts from Mentha x piperita native from Trás-os-Montes region (Portugal)

The wild mint Mentha x piperita (Mentha rotundifolia Huds.), has been used by the local people in a northern region of Portugal to prepare infusions for treating digestive pain and spasms, as an appetite stimulant and for treating headache and migraine. This suggests that these aqueous extracts have analgesic; antispasmodic and stomachic properties. In the present study the antioxidant potential of aqueous extracts of Mentha x piperita is investigated since natural antioxidants can scavenge the reactive oxygen species, ROS, and thus might attenuate inflammation pathways. The antispasmodic activity was also checked. The antioxidant potential of the extract was evaluated by the DPPH* method, by the quantification of the total phenolic compounds and by characterization of the main phenolic compounds. The antispasmodic effects were investigated by performing pharmacological assays using the distal ileum of guinea pigs. The aqueous extract exhibits antioxidant properties that may be due to its phenolic content. The main phenolic compounds were quinic, caffeic, rosmarinic and chlorogenic acids. The antispasmodic affects are observed after electrical stimulation of segments of distal ileum of guinea pigs and may be attributed, at least, to a alfa1 blockage

Mineralization of chitosan membrane using a double diffusion system for bone related applications

Chitosan membranes were subjected to a pre-treatment in a double diffusion system, with a calcium solution in one chamber and a phosphate solution in the other chamber. Both chambers were separated by the chitosan membrane and subject to three mineralization periods (5, 10 and 15 minutes). After this pre-treatment the bioactivity of the different calcium phosphate coatings formed was tested for different periods of immersion time, 7, 14 and 21 days at room temperature and 37ºC, in acellular simulated body fluid (1.0x). The results obtained demonstrated that the calcium phosphate coatings formed during the pre-treatment process are bioactive. It was found that the calcification is effective just in the side of the membrane exposed to the calcium solution chamber. This enabled to develop membranes with asymmetric osteoinductive properties that can be useful in different orthopedic applications

Correction: Biofunctionalized pectin hydrogels as 3D cellular microenvironments

Correction for 'Biofunctionalized pectin hydrogels as 3D cellular microenvironments' by Sara C. Neves et al., J. Mater. Chem. B, 2015, 3, 2096–2108

Exploring the saccharomyces cerevisiae volatile metabolome: indigenous versus commercial strains

Winemaking is a highly industrialized process and a number of commercial Saccharomyces cerevisiae strains are used around the world, neglecting the diversity of native yeast strains that are responsible for the production of wines peculiar flavours. The aim of this study was to in-depth establish the S. cerevisiae volatile metabolome and to assess inter-strains variability. To fulfill this objective, two indigenous strains (BT2652 and BT2453 isolated from spontaneous fermentation of grapes collected in Bairrada Appellation, Portugal) and two commercial strains (CSc1 and CSc2) S. cerevisiae were analysed using a methodology based on advanced multidimensional gas chromatography (HS-SPME/GC×GC-ToFMS) tandem with multivariate analysis. A total of 257 volatile metabolites were identified, distributed over the chemical families of acetals, acids, alcohols, aldehydes, ketones, terpenic compounds, esters, ethers, furan-type compounds, hydrocarbons, pyrans, pyrazines and S-compounds. Some of these families are related with metabolic pathways of amino acid, carbohydrate and fatty acid metabolism as well as mono and sesquiterpenic biosynthesis. Principal Component Analysis (PCA) was used with a dataset comprising all variables (257 volatile components), and a distinction was observed between commercial and indigenous strains, which suggests inter-strains variability. In a second step, a subset containing esters and terpenic compounds (C10 and C15), metabolites of particular relevance to wine aroma, was also analysed using PCA. The terpenic and ester profiles express the strains variability and their potential contribution to the wine aromas, specially the BT2453, which produced the higher terpenic content. This research contributes to understand the metabolic diversity of indigenous wine microflora versus commercial strains and achieved knowledge that may be further exploited to produce wines with peculiar aroma properties

Unrevealing the interactive effects of climate change and oil contamination on lab-simulated estuarine benthic communities

There is growing concern that modifications to the global environment such as ocean acidification and increased ultraviolet radiation may interact with anthropogenic pollutants to adversely affect the future marine environment. Despite this, little is known about the nature of the potential risks posed by such interactions. Here, we performed a multifactorial microcosm experiment to assess the impact of ocean acidification, ultraviolet radiation B (UV-B) and oil hydrocarbon contamination on sediment chemistry, the microbial community (composition and function) and biochemical marker response of selected indicator species. We found that increased ocean acidification and oil contamination in the absence of UV-B will significantly alter bacterial composition by, among other changes, greatly reducing the relative abundance of Desulfobacterales, known to be important oil hydrocarbon degraders. Along with changes in bacterial composition, we identified concomitant shifts in the composition of aromatic hydrocarbons in the sediment and an increase in oxidative stress effects on our indicator species. Interestingly, our study identifies UV-B as a critical component in the interaction between these factors, since its presence alleviates harmful effects caused by the combination of reduced pH and oil pollution. The model system used here shows that the interactive effect of reduced pH and oil contamination can adversely affect the structure and functioning of sediment benthic communities, with the potential to exacerbate the toxicity of oil hydrocarbons in marine ecosystems

Antimicrobial functionalized genetically engineered spider silk

Genetically engineered fusion proteins offer potential as multifunctional biomaterials for medical use. Fusion or chimeric proteins can be formed using recombinant DNA technology by combining nucleotide sequences encoding different peptides or proteins that are otherwise not found together in nature. In the present study, three new fusion proteins were designed, cloned and expressed and assessed for function, by combining the consensus sequence of dragline spider silk with three different antimicrobial peptides. The human antimicrobial peptides human neutrophil defensin 2 (HNP-2), human neutrophil defensins 4 (HNP-4) and hepcidin were fused to spider silk through bioengineering. The spider silk domain maintained its self-assembly features, a key aspect of these new polymeric protein biomaterials, allowing the formation of b-sheets to lock in structures via physical interactions without the need for chemical crosslinking. These new functional silk proteins were assessed for antimicrobial activity against Gram e Escherichia coli and Gram þ Staphylococcus aureus and microbicidal activity was demonstrated. Dynamic light scattering was used to assess protein aggregation to clarify the antimicrobial patterns observed. Attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and circular dichroism (CD) were used to assess the secondary structure of the new recombinant proteins. In vitro cell studies with a human osteosarcoma cell line (SaOs-2) demonstrated the compatibility of these new proteins with mammalian cells.Fundação para a Ciência e a Tecnologia (FCT) - Bolsa de doutoramento (SFRH/BD/28603/2006); Chimera project (PTDC/EBB-EBI/109093/2008); NIH and Tissue Engineering Resource Center EB003210, P41 EB002520, DE017207