SERS as a tool for in vitro toxicology.

Measuring markers of stress such as pH and redox potential are important when studying toxicology in in vitro models because they are markers of oxidative stress, apoptosis and viability. While surface enhanced Raman spectroscopy is ideally suited to the measurement of redox potential and pH in live cells, the time-intensive nature and perceived difficulty in signal analysis and interpretation can be a barrier to its broad uptake by the biological community. In this paper we detail the development of signal processing and analysis algorithms that allow SERS spectra to be automatically processed so that the output of the processing is a pH or redox potential value. By automating signal processing we were able to carry out a comparative evaluation of the toxicology of silver and zinc oxide nanoparticles and correlate our findings with qPCR analysis. The combination of these two analytical techniques sheds light on the differences in toxicology between these two materials from the perspective of oxidative stress.

Identification of multiple integrin beta1 homologs in zebrafish (Danio rerio).

BACKGROUND: Integrins comprise a large family of alpha,beta heterodimeric, transmembrane cell adhesion receptors that mediate diverse essential biological functions. Higher vertebrates possess a single beta1 gene, and the beta1 subunit associates with a large number of alpha subunits to form the major class of extracellular matrix (ECM) receptors. Despite the fact that the zebrafish (Danio rerio) is a rapidly emerging model organism of choice for developmental biology and for models of human disease, little is currently known about beta1 integrin sequences and functions in this organism. RESULTS: Using RT-PCR, complete coding sequences of zebrafish beta1 paralogs were obtained from zebrafish embryos or adult tissues. The results show that zebrafish possess two beta1 paralogs (beta1-1 and beta1-2) that have a high degree of identity to other vertebrate beta1 subunits. In addition, a third, more divergent, beta1 paralog is present (beta1-3), which may have altered ligand-binding properties. Zebrafish also have other divergent beta1-like transcripts, which are C-terminally truncated forms lacking the transmembrane and cytoplasmic domains. Together with beta1-3 these truncated forms comprise a novel group of beta1 paralogs, all of which have a mutation in the ADMIDAS cation-binding site. Phylogenetic and genomic analyses indicate that the duplication that gave rise to beta1-1 and beta1-2 occurred after the divergence of the tetrapod and fish lineages, while a subsequent duplication of the ancestor of beta1-2 may have given rise to beta1-3 and an ancestral truncated paralog. A very recent tandem duplication of the truncated beta1 paralogs appears to have taken place. The different zebrafish beta1 paralogs have varied patterns of temporal expression during development. Beta1-1 and beta1-2 are ubiquitously expressed in adult tissues, whereas the other beta1 paralogs generally show more restricted patterns of expression. CONCLUSION: Zebrafish have a large set of integrin beta1 paralogs. beta1-1 and beta1-2 may share the roles of the solitary beta1 subunit found in other vertebrates, whereas beta1-3 and the truncated beta1 paralogs may have acquired novel functions.

Skin exposure to micro- and nano-particles can cause haemostasis in zebrafish larvae.

Low mass ambient exposure to airborne particles is associated with atherothrombotic events that may be a consequence of the combustion-derived nanoparticle content. There is concern also over the potential cardiovascular impact of manufactured nanoparticles. To better understand the mechanism by which toxic airborne particles can affect cardiovascular function we utilised zebrafish as a genetically tractable model. Using light and confocal fluorescence video-microscopy, we measured heart-rate and blood flow in the dorsal aorta and caudal artery of zebrafish larvae that had been exposed to a number of toxic and non-toxic microparticles and nanoparticles. Diesel exhaust particles (DEP), carboxy-charged Latex beads (carboxy-beads) and toxic alumina (Taimicron TM300), but not non-toxic alumina (Baikalox A125), were found to promote both skin and gut cell damage, increased leukocyte invasion into the epidermis, tail muscle ischaemia and haemostasis within the caudal artery of free swimming zebrafish larvae. The presence of sodium sulfite, a reducing agent, or warfarin, an anticoagulant, within the system water abrogated the effects of both toxic alumina and carboxy-beads but not DEP. Genetic manipulation of skin barrier function augmented skin damage and haemostasis, even for the non-toxic alumina. The toxic effects of carboxy-beads were still apparent after leukocyte numbers were depleted with anti-Pu.1 morpholino. We conclude that particle uptake across skin epithelium and gut mucosal barriers, or the presence of leukocytes, is not required for particle-induced haemostasis while a compromised skin barrier function accentuated tissue injury and haemostasis.