ITS-NANO - Prioritising nanosafety research to develop a stakeholder driven intelligent testing strategy

Background To assess the risk of all nanomaterials (NMs) on a case-by-case basis is challenging in terms of financial, ethical and time resources. Instead a more intelligent approach to knowledge gain and risk assessment is required. Methods A framework of future research priorities was developed from the accorded opinion of experts covering all major stake holder groups (government, industry, academia, funders and NGOs). It recognises and stresses the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling approaches as key components of the current and future risk assessment of NMs. Results The framework for future research has been developed from the opinions of over 80 stakeholders, that describes the research priorities for effective development of an intelligent testing strategy (ITS) to allow risk evaluation of NMs. In this context, an ITS is a process that allows the risks of NMs to be assessed accurately, effectively and efficiently, thereby reducing the need to test NMs on a case-by-case basis. For each of the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling, key-priority research areas are described via a series of stepping stones, or hexagon diagrams structured into a time perspective. Importantly, this framework is flexible, allowing individual stakeholders to identify where their own activities and expertise are positioned within the prioritisation pathway and furthermore to identify how they can effectively contribute and structure their work accordingly. In other words, the prioritisation hexagon diagrams provide a tool that individual stakeholders can adapt to meet their own particular needs and to deliver an ITS for NMs risk assessment. Such an approach would, over time, reduce the need for testing by increasing the reliability and sophistication of in silico approaches. The manuscript includes an appraisal of how this framework relates to the current risk assessment approaches and how future risk assessment could adapt to accommodate these new approaches. A full report is available in electronic format (pdf) at http://www.nano.hw.ac.uk/research-projects/itsnano.html webcite. Conclusion ITS-NANO has delivered a detailed, stakeholder driven and flexible research prioritisation (or strategy) tool, which identifies specific research needs, suggests connections between areas, and frames this in a time-perspective

Adhesivejoiningtechnologiesactivatedbyelectro-magnetic external trims

Joining is a key and fundamental aspect of vehicle design and manufacturing process.The development of efficient, simple, inexpensive and reversible adhesive bonding technologies offers low cost, improved life cycle and recycling solutions for a vehicle. This innovative technology is based on magneto sensitive nanoparticles dispersed into the adhesive. Different typologies of susceptors, embedded in thermoplastic hot-melt adhesives, have been tested to investigate the thermal behavior of these innovative materials when exposed to electromagnetic fields. These innovative technologies are intended to optimize the bonding process offering new opportunities connected with costruction, improved resistance to applied loads, easy and rapid dismantling and smart recycling. Experimental procedures to optimize the electromagnetic process have been performed. Results about both shear strength and sliding temperature of the modified adhesives are presented

Double immunohistochemistry on human osteosarcomas.

<p>Co-immunostainings of osteosarcoma tissue array either with the anti-P2X7R-ec plus anti-Ki67 antibodies (A–D), or with the two primary anti-P2X7R antibodies (E–H) were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">Materials and Methods</a>. (A,C): representative P2X7RB positive osteosarcoma showing a higher number of Ki67 positive nuclei (stained in blue) respect to a representative P2X7RA+B tumour (B,D). (E–H): representative P2X7RA+B positive tumours demonstrating the presence of a mix of cells some (stained in brown) positive for P2X7RB only, some others (stained in blue-brown) positive for both P2X7RA and B. Two different magnifications, 20x (A,B,E,F) and 40x (C,D,G,H) are shown.</p

P2X7RA and B increase ATP release and NFATc1 activity in Te85 transfected cells.

<p>(A): Extracellular ATP was measured in the culture supernatants with ENLITEN rLuciferase/Luciferin reagents as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">materials and methods</a>. Te85 clones were plated at 5x10⁵ cells per well in 96 well plates and, following adhesion, incubated for 24 hours in the absence (untreated) or presence of either 100 µM BzATP, 100 µM A740003 or 4 U/ml Apyrase. ATP release was expressed as fold increase over Te85 wt reference sample. Data are means ± SE; N = 3. (B) Nuclear fractions of the different clones were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">materials and methods</a>. Activated NFATc1 was measured by ELISA in absence (untreated) or in presence of either 50 µM BzATP or 10 µM cyclosporin (CSA). CSA vehicle (DMSO) was used as control. Results are compared to nuclear NFATc1 levels in Jurkat cells stimulated with PHA supplied by manufacturer. In graph means ± SE of absorbance are shown, N = 9. Colour coding: green Te85 wt, red Te85 P2RX7A, cyan Te85 P2RX7B, purple Te85 P2RX7A+B.</p

Schematic summary of obtained data and hypothetical deductions.

<p>Schematic summary of obtained data and hypothetical deductions.</p

P2X7RA and B expression reduces RANK-L while differently modulates OPG messenger and mineralization.

<p>RANK-L and OPG mRNA levels were evaluated by real-time PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">Materials and Methods</a>. Messenger expression was normalized on G3PDH internal control and displayed as fold increase over Te85 wt reference sample. (A): RANK-L mRNA expression (B): OPG mRNA expression. Data are shown as mean ± SE, N = 12, ***p<0.001 versus Te85 wt. (C): Mineralization by Te85 clones was assessed over a 21 days period as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107224#s2" target="_blank">Materials and Methods</a>. (A): Quantification of the amount of alizarin red S staining of each clone. Data are shown as means ± SE (N = 6) analysed by Kruskal-Wallace test with Dunn's multiple comparison post-test. ***<i>P</i><0.001 versus Te85 wt and Te85-P2X7RA; *<i>P</i><0.05 vs Te85 wt; ^$$<i>P</i><0.01 versus Te85 P2X7RA. Colour coding: green Te85 wt, red Te85 P2X7RA, cyan Te85 P2X7RB, purple Te85 P2X7RA+B.</p

Characterization of P2X7R isoforms A and B expressed in Te85 transfected clones.

<p>(A): RT-PCR showing selective expression of P2X7RA or P2X7RB in Te85 clones. Housekeeping control gene was G3PDH, osteoblast specific gene was collagen I. (B): P2X7R surface expression determined by flow cytometry with anti-P2X7R-ec. Graph shows the Mean Fluorescence Intensity (MFI) after subtraction of secondary antibody values. Data are shown as means ± SE, N = 3. ***<i>P</i><0.001 versus Te85 wt; ^###<i>P</i><0.001 versus Te85 P2RX7B; §<i>P</i><0.05 versus Te85 P2RX7A. (C): Representative traces showing intracellular calcium increment following stimulation with 500 µM BzATP. (D): Calcium variations evoked by P2X7R activation showed as means ± SE, N = 10. ***p<0.001 versus Te85 wt. (C): Representative traces showing ethidium bromide uptake following stimulation with 500 µM BzATP. (D): Percentages of ethidium permeabilization on digitonin control showed as means ± SE, N = 10. ***<i>p</i><0.001 versus Te85 wt. Colour coding: green Te85 wt, red Te85 P2X7RA, cyan Te85 P2X7RB, purple Te85 P2X7RA+B.</p