Influence of silk–silica fusion protein design on silica condensation in vitro and cellular calcification

Biomaterial design via genetic engineering can be utilized for the rational functionalization of proteins to promote biomaterials integration and tissue regeneration. Spider silk has been extensively studied for its biocompatibility, biodegradability and extraordinary material properties. As a protein-based biomaterial, recombinant DNA derived derivatives of spider silks have been modified with biomineralization domains which lead to silica deposition and potentially accelerated bone regeneration. However, the influence of the location of the R5 (SSKKSGSYSGSKGSKRRIL) silicifying domain fused with the spider silk protein sequence on biosilicification process remains to be determined. Here we designed two silk-R5 fusion proteins that differed in the location of the R5 peptide, C- vs. N-terminus, where the spider silk domain consisted of a 15mer repeat of a 33 amino acid consensus sequence of the major ampullate dragline Spidroin 1 from Nephila clavipes (SGRGGLGG QG AGAAAAAGGA GQGGYGGLGSQGT). The chemical, physical and silica deposition properties of these recombinant proteins were assessed and compared to a silk 15mer control without the R5 present. The location of the R5 peptide did not have significant effect on wettability and surface energies, while the C - terminal location of the R5 promoted more controlled silica precipitation, suggesting differences in protein folding and possibly different access to charged amino acids that drive the silicification process. Further, cell compatibility in vitro, as well as the ability to promote human bone marrow derived mesenchymal stem cells (hMSC) differentiation were demonstrated for both variants of the fusion proteins

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

Quantifying the efficiency of hydroxyapatite mineralising peptides

We present a non-destructive analytical calibration tool to allow quantitative assessment of individual calcium phosphates such as hydroxyapatite (HAP) from mixtures including brushite. Many experimental approaches are used to evaluate the mineralising capabilities of biomolecules including peptides. However, it is difficult to quantitatively compare the efficacy of peptides in the promotion of mineralisation when inseparable mixtures of different minerals are produced. To address this challenge, a series of hydroxyapatite and brushite mixtures were produced as a percent/weight (0–100%) from pure components and multiple (N=10) XRD patterns were collected for each mixture. A linear relationship between the ratio of selected peak heights and the molar ratio was found. Using this method, the mineralising capabilities of three known hydroxyapatite binding peptides, CaP(S) STLPIPHEFSRE, CaP(V) VTKHLNQISQSY and CaP(H) SVSVGMKPSPRP, was compared. All three directed mineralisation towards hydroxyapatite in a peptide concentration dependent manner. CaP(V) was most effective at inducing hydroxyapatite formation at higher reagent levels (Ca2+ = 200mM), as also seen with peptide-silk chimeric materials, whereas CaP(S) was most effective when lower concentrations of calcium (20mM) and phosphate were used. The approach can be extended to investigate HAP mineralisation in the presence of any number of mineralisation promoters or inhibitors

Role of microbes in plant protection using intersection of nanotechnology and biology

Published online: 15 Aug 2018Plant pathogens are one of the dominating components which restrain crop productivity. Preliminary step headed for managing plant disease is to accurately recognize the pathogen under lab, glasshouse, and field conditions. Modern approach, such as culture-based, antibody-based rapid methods and quantitative polymerase chain reaction (Q-PCR), entrusts on multiple assays to precisely identify the specific plant pathogens which are further time-consuming and lack high sensitivity. Nanobiotechnology ameliorates crop productivity through transmission of genes to target sites for breeding of varieties resistant to different plant pathogens with focus on improving sensitivity. Intersection of nanotechnology and biology also improves specificity and agility of pathogen detection which further facilitates crop disease management. Bio-fabrication of nanoparticles like silver (Ag) and copper (Cu) is used as novel antimicrobials for the management of pathogenic microorganisms that inhibits fungal hyphae and conidial germination in agricultural crops. Biological agents reduce metal which leads to capping of nanoparticles through the secretion of various enzymes. A modern class of protein nanocompartments called as encapsulins that encapsulate cargo proteins are found in bacteria and archaea. Nanobiotechnology also reduces detection times of crop pathogens and cost by the development of biosensors and phage proteins. In this chapter we emphasize on microbial semblance in nanobiotechnology applications that precede to integrated disease management of agricultural crops including precise diagnostic layout of plant diseases and modification of crop environments to adversely affect crop pathogens

Biogenesis of medium-chain-length polyhydroxyalkanoates

26 p.-3 fig.-2 tab.Medium-chain-length polyhydroxyalkanoates (mcl-PHA) are biotechnologically useful natural products found in many bacteria. This biopolymer functions as a carbon and energy storage reservoir in cells but has physical and mechanical properties that make it a promising bioplastic with applications ranging from adhesives to medical implants. Therefore, there is much interest in understanding the biology of mcl-PHA synthesis and metabolism. Increased knowledge of PHA biology serves as a foundation for the bioengineering of PHA and its eventual use as a biologically derived product. This chapter covers the state of knowledge on mcl-PHA, including its synthesis and its central role in cellular metabolism. Moreover, this chapter discusses methods for bioengineering mcl-PHA production in bacteria as well as synthetic biology methods for its study and production in the natural mcl-PHA producer, Pseudomonas putida.Research on polymer biotechnology in the laboratory of M. Auxiliadora Prieto is supported by funding from the European Union’s Horizon 2020 research and innovation program under grant agreements number 633962 and 679050. We also acknowledge support from the Community of Madrid (P2013/MIT2807) and the Spanish Ministry of Economy (BIO201344878R, BIO2014-61515-EXP).Peer reviewedPostprin