High-yield production of a super-soluble miniature spidroin for biomimetic high-performance materials

The mechanical properties of artificial spider silks are approaching a stage where commercial applications become realistic. However, the yields of recombinant silk proteins that can be used to produce fibers with good mechanical properties are typically very low and many purification and spinning protocols still require the use of urea, hexafluoroisopropanol, and/or methanol. Thus, improved production and spinning methods with a minimal environmental impact are needed. We have previously developed a miniature spider silk protein that is characterized by high solubility in aqueous buffers and spinnability in biomimetic set-ups. In this study, we developed a production protocol that resulted in an expression level of >20 g target protein per liter in an Escherichia coli fedbatch culture, and subsequent purification under native conditions yielded 14.5 g/l. This corresponds to a nearly six-fold increase in expression levels, and a 10-fold increase in yield after purification compared to reports for recombinant spider silk proteins. Biomimetic spinning using only aqueous buffers resulted in fibers with a toughness modulus of 74 MJ/m(3), which is the highest reported for biomimetically as-spun artificial silk fibers. Thus, the process described herein represents a milestone for the economic production of biomimetic silk fibers for industrial applications

Impact of physio-chemical spinning conditions on the mechanical properties of biomimetic spider silk fibers

Artificial spider silk has emerged as a biobased fiber that could replace some petroleum-based materials that are on the market today. Recent progress made it possible to produce the recombinant spider silk protein NT2RepCT at levels that would make the commercialization of fibers spun from this protein economically feasible. However, for most applications, the mechanical properties of the artificial silk fibers need to be improved. This could potentially be achieved by redesigning the spidroin, and/or by changing spinning conditions. Here, we show that several spinning parameters have a significant impact on the fibers' mechanical properties by tensile testing more than 1000 fibers produced under 92 different conditions. The most important factors that contribute to increasing the tensile strength are fast reeling speeds and/or employing post-spin stretching. Stretching in combination with optimized spinning conditions results in fibers with a strength of >250 MPa, which is the highest reported value for fibers spun using natively folded recombinant spidroins that polymerize in response to shear forces and lowered pH.The mechanical properties of spider silk are known to be dependent on spinning conditions. Here, the tensile behavior of over 1000 biomimetic spider silk fibers spun under 92 different conditions are tested, resulting in a yield strength of more than 250 MPa

An Image-Analysis-Based Method for the Prediction of Recombinant Protein Fiber Tensile Strength

Silk fibers derived from the cocoon of silk moths and the wide range of silks produced by spiders exhibit an array of features, such as extraordinary tensile strength, elasticity, and adhesive properties. The functional features and mechanical properties can be derived from the structural composition and organization of the silk fibers. Artificial recombinant protein fibers based on engineered spider silk proteins have been successfully made previously and represent a promising way towards the large-scale production of fibers with predesigned features. However, for the production and use of protein fibers, there is a need for reliable objective quality control procedures that could be automated and that do not destroy the fibers in the process. Furthermore, there is still a lack of understanding the specifics of how the structural composition and organization relate to the ultimate function of silk-like fibers. In this study, we develop a new method for the categorization of protein fibers that enabled a highly accurate prediction of fiber tensile strength. Based on the use of a common light microscope equipped with polarizers together with image analysis for the precise determination of fiber morphology and optical properties, this represents an easy-to-use, objective non-destructive quality control process for protein fiber manufacturing and provides further insights into the link between the supramolecular organization and mechanical functionality of protein fibers

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially-driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a roadmap towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.JRC.I.5-Systems Toxicolog

Emodepside targets SLO-1 channels of Onchocerca ochengi and induces broad anthelmintic effects in a bovine model of onchocerciasis

Onchocerciasis (river blindness), caused by the filarial worm Onchocerca volvulus, is a neglected tropical disease mostly affecting sub-Saharan Africa and is responsible for >1.3 million years lived with disability. Current control relies almost entirely on ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. Here, we evaluated emodepside, a semi-synthetic cyclooctadepsipeptide registered for deworming applications in companion animals, for activity against adult filariae (i.e., as a macrofilaricide). We demonstrate the equivalence of emodepside activity on SLO-1 potassium channels in Onchocerca volvulus and Onchocerca ochengi, its sister species from cattle. Evaluation of emodepside in cattle as single or 7-day treatments at two doses (0.15 and 0.75 mg/kg) revealed rapid activity against microfilariae, prolonged suppression of female worm fecundity, and macrofilaricidal effects by 18 months post treatment. The drug was well tolerated, causing only transiently increased blood glucose. Female adult worms were mostly paralyzed; however, some retained metabolic activity even in the multiple high-dose group. These data support ongoing clinical development of emodepside to treat river blindness

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling

Artificial and natural silk materials have high mechanical property variability regardless of sample size

Silk fibres attract great interest in materials science for their biological and mechanical properties. Hitherto, the mechanical properties of the silk fibres have been explored mainly by tensile tests, which provide information on their strength, Young’s modulus, strain at break and toughness modulus. Several hypotheses have been based on these data, but the intrinsic and often overlooked variability of natural and artificial silk fibres makes it challenging to identify trends and correlations. In this work, we determined the mechanical properties of Bombyx mori cocoon and degummed silk, native spider silk, and artificial spider silk, and compared them with classical commercial carbon fibres using large sample sizes (from 10 to 100 fibres, in total 200 specimens per fibre type). The results confirm a substantial variability of the mechanical properties of silk fibres compared to commercial carbon fibres, as the relative standard deviation for strength and strain at break is 10–50%. Moreover, the variability does not decrease significantly when the number of tested fibres is increased, which was surprising considering the low variability frequently reported for silk fibres in the literature. Based on this, we prove that tensile testing of 10 fibres per type is representative of a silk fibre population. Finally, we show that the ideal shape of the stress–strain curve for spider silk, characterized by a pronounced exponential stiffening regime, occurs in only 25% of all tested spider silk fibres

Verticillium Wilt in Oilseed Rape—the Microbiome is Crucial for Disease Outbreaks as Well as for Efficient Suppression

Microbiome management is a promising way to suppress verticillium wilt, a severe disease in Brassica caused by Verticillium longisporum. In order to improve current biocontrol strategies, we compared bacterial Verticillium antagonists in different assays using a hierarchical selection and evaluation scheme, and we integrated outcomes of our previous studies. The result was strongly dependent on the assessment method chosen (in vitro, in vivo, in situ), on the growth conditions of the plants and their genotype. The most promising biocontrol candidate identified was a Brassica endophyte Serratia plymuthica F20. Positive results were confirmed in field trials and by microscopically visualizing the three-way interaction. Applying antagonists in seed treatment contributes to an exceptionally low ecological footprint, supporting efficient economic and ecological solutions to controlling verticillium wilt. Indigenous microbiome, especially soil and seed microbiome, has been identified as key to understanding disease outbreaks and suppression. We suggest that verticillium wilt is a microbiome-driven disease caused by a reduction in microbial diversity within seeds and in the soil surrounding them. We strongly recommend integrating microbiome data in the development of new biocontrol and breeding strategies and combining both strategies with the aim of designing healthy microbiomes, thus making plants more resilient toward soil-borne pathogens

Relationship of Cellular Topoisomerase Iiα Inhibition to Cytotoxicity and Published Genotoxicity of Fluoroquinolone Antibiotics in V79 Cells

Fluoroquinolone (FQ) antibiotics are bacteriocidal through inhibition of the bacterial gyrase and at sufficient concentrations in vitro, they can inhibit the homologous eukaryotic topoisomerase (TOPO) II enzyme. FQ exert a variety of genotoxic effects in mammalian systems through mechanisms not yet established, but which are postulated to involve inhibition of TOPO II enzymes. To assess the relationship of inhibition of cell nuclear TOPO II to cytotoxicity and reported genotoxicity, two FQ, clinafloxacin (CLFX) and lomefloxacin (LOFX), having available genotoxicity data showing substantial differences with CLFX being more potent than LOFX, were selected for study. The relative inhibitory activities of these FQ on nuclear TOPO IIα in cultured Chinese hamster lung fibroblasts (V79 cells) over dose ranges and at equimolar concentrations were assessed by measuring nuclear stabilized cleavage complexes of TOPO IIα-DNA. Cytotoxicity was measured by relative cell counts. Both FQ inhibited V79 cell nuclear TOPO IIα. The lowest-observed-adverse-effect levels for TOPO IIα inhibition were 55 μM for CLFX, and 516 μM for LOFX. The no-observed-adverse-effect-levels were 41 μM for CLFX, and 258 μM for LOFX. At equimolar concentrations (175 μM), CLFX was more potent than LOFX. Likewise, CLFX was more cytotoxic than LOFX. Thus, the two FQ, inhibited TOPO IIα in intact V79 cells, differed in their potencies and exhibited no-observed-adverse-effect levels. These findings are in concordance with published genotoxicity data and observed cytotoxicity