Intracellular pathways involved in bone regeneration triggered by recombinant silk-silica chimeras

Biomineralization at the organic-inorganic interface is critical to many biology material functions in vitro and in vivo. Recombinant silk-silica fusion peptides are organic-inorganic hybrid material systems that can be effectively used to study and control biologically-mediated mineralization due to the genetic basis of sequence control. However, to date, the mechanisms by which these functionalized silk-silica proteins trigger the differentiation of human mesenchymal stem cells (hMSCs) to osteoblasts remain unknown. To address this challenge, we analyzed silk-silica surfaces for silica-hMSC receptor binding and activation, and the intracellular pathways involved in the induction of osteogenesis on these bioengineered biomaterials. The induction of gene expression of αVβ3 integrin, all three Mitogen-activated Protein Kinsases (MAPKs) as well as c-Jun, Runt-related Transcription Factor 2 (Runx2) and osteoblast marker genes was demonstrated upon growth of the hMSCs on the silk-silica materials. This induction of key markers of osteogenesis correlated with the content of silica on the materials. Moreover, computational simulations were performed for silk/silica-integrin binding which showed activation of αVβ3 integrin in contact with silica. This integrated computational and experimental approach provides insight into interactions that regulate osteogenesis towards more efficient biomaterial designs

The effect of agitation speed, enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer’s spent grain

Brewer’s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 ºC to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2 3 full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer’s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo), Brazil. Novozymes ( FAPESP )Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Grapevine rootstocks shape underground bacterial microbiome and networking but not potential functionality

BackgroundThe plant compartments of Vitis vinifera, including the rhizosphere, rhizoplane, root endosphere, phyllosphere and carposphere, provide unique niches that drive specific bacterial microbiome associations. The majority of phyllosphere endophytes originate from the soil and migrate up to the aerial compartments through the root endosphere. Thus, the soil and root endosphere partially define the aerial endosphere in the leaves and berries, contributing to the terroir of the fruit. However, V. vinifera cultivars are invariably grafted onto the rootstocks of other Vitis species and hybrids. It has been hypothesized that the plant species determines the microbiome of the root endosphere and, as a consequence, the aerial endosphere. In this work, we test the first part of this hypothesis. We investigate whether different rootstocks influence the bacteria selected from the surrounding soil, affecting the bacterial diversity and potential functionality of the rhizosphere and root endosphere.MethodsBacterial microbiomes from both the root tissues and the rhizosphere of Barbera cultivars, both ungrafted and grafted on four different rootstocks, cultivated in the same soil from the same vineyard, were characterized by 16S rRNA high-throughput sequencing. To assess the influence of the root genotype on the bacterial communities’ recruitment in the root system, (i) the phylogenetic diversity coupled with the predicted functional profiles and (ii) the co-occurrence bacterial networks were determined. Cultivation-dependent approaches were used to reveal the plant-growth promoting (PGP) potential associated with the grafted and ungrafted root systems.ResultsRichness, diversity and bacterial community networking in the root compartments were significantly influenced by the rootstocks. Complementary to a shared bacterial microbiome, different subsets of soil bacteria, including those endowed with PGP traits, were selected by the root system compartments of different rootstocks. The interaction between the root compartments and the rootstock exerted a unique selective pressure that enhanced niche differentiation, but rootstock-specific bacterial communities were still recruited with conserved PGP traits.ConclusionWhile the rootstock significantly influences the taxonomy, structure and network properties of the bacterial community in grapevine roots, a homeostatic effect on the distribution of the predicted and potential functional PGP traits was found

Assessment strategy for bacterial lignin depolymerization : Kraft lignin and synthetic lignin bioconversion with Pseudomonas putida

In order to better understand bacterial depolymerization of lignin, a new analytical approach was proposed using Pseudomonas putida KT2440 as delignifying bacterium and Escherichia coli as non-delignifying control. Two different types of lignins, technical Kraft lignin and synthetic dehydrogenopolymer (DHP), were submitted to a bioconversion kinetic study over 7 days. The concomitant analysis of the supernatant acid-precipitable lignin fraction and water-soluble extractives by high-performance size-exclusion chromatography (HPSEC) and gas chromatography – mass spectrometry (GC–MS) highlighted the specific action of P. putida towards these substrates, with the transitory formation of phenolic metabolites (dihydroferulic acid for Kraft lignins and dimers for DHP) and the prevention of Kraft lignin self-assemblying. In both cases lignin apparent depolymerization followed by repolymerization was observed. The analysis of the bacterial pellets indicated the time-increasing content of lignins associated to bacterial cells, which could account for the apparent structural changes observed with E. coli in the supernatant

Biosynthesis of selenium nanoparticles by Azoarcus sp. CIB

10 p.-6 fig.Background: Different bacteria have been reported so far that link selenite resistance to the production of metallic selenium nanoparticles (SeNPs). Although SeNPs have many biotechnological applications in diverse areas, the molecular mechanisms involved in their microbial genesis are not fully understood. The Azoarcus genus is a physiologically versatile group of beta-proteobacteria of great environmental relevance. Azoarcus sp. CIB is a facultative anaerobe that combines the ability to degrade under aerobic and/or anaerobic conditions a wide range of aromatic compounds, including some toxic hydrocarbons such as toluene and m-xylene, with an endophytic life style in the root of rice. We unravel here an additional physiological feature of the strain CIB that is related to its resistance to selenium oxyanions and the formation of SeNPs.Results: This work is the first report of a member of the Azoarcus genus that is able to anaerobically grow in the presence of selenite. Electron microscopy preparations and X-ray spectroscopy analyses demonstrate the reduction of selenite to spherical electron-dense SeNPs whose average size was 123 ± 35 nm of diameter. Our data suggest that the main molecular mechanism of selenite resistance resides on an energy-dependent selenite exporter. Azoarcus cells trigger the synthesis of SeNPs when they reach the stationary-phase of growth, and either the exhaustion of electron donor or acceptor, both of which lead to starvation conditions, produce the reduction of selenite to red elemental selenium. Azoarcus becomes a promising biocatalyst, either as whole cells or cellular extracts, for the anaerobic and/or aerobic green synthesis of SeNPs.Conclusions: Azoarcus turns out to be a new eco-friendly system to reduce selenite and produce spherical SeNPs.Moreover, this is the first report of a rice endophyte able to produce SeNPs. Since Azoarcus is also able to degrade both aerobically and anaerobically toxic aromatic compounds of great environmental concern, it becomes a suitable candidate for a more sustainable agricultural practice and for bioremediation strategies.This work was supported by Ministry of Economy and Competitiveness of Spain Grants BIO2012-39501 and MAT2014-59222R, Fundación RamónAreces XVII CN, and by European Union FP7 Grant 311815 (Synpol Project). H.Fernández-Llamosas was the recipient of a Research Personnel Formation (FPI) fellowship from the Ministry of Economy and Competitiveness of Spain.Peer reviewe

Deconstruction of the hemicellulose fraction from lignocellulosic materials into simple sugars

Hemicelluloses hold a great promise for the production of added-value compounds in the biorefinery framework. Specifically, the xylan-rich hemicelluloses from hardwoods and agro-industrial residues present themselves as effective feedstock choices for the biotechnological production of xylitol. This paper reviews the various hemicellulose structures present in such materials and critically evaluates the available processing options to produce xylose-rich fermentable hydrolysates. Currently, acid-based processes still present the best trade-off between operation easiness and xylose yield and recovery. Nevertheless, concerns regarding the impact of the fractionation processes on the overall upgradability of all biomass fractions (namely, cellulose and specially lignin) may turn the route to other strategies. Specifically, the combined/sequential use of processes targeting hemicellulose dissolution and hydrolysis might hold great promise for the economical production of pentoses