Assessing Phototoxicity in a Mammalian Cell Line: How Low Levels of Blue Light Affect Motility in PC3 Cells.

Phototoxicity is a significant constraint for live cell fluorescence microscopy. Excessive excitation light intensities change the homeostasis of the observed cells. Erroneous and misleading conclusions may be the problematic consequence of observing such light-induced pathophysiology. In this study, we assess the effect of blue light, as commonly used for GFP and YFP excitation, on a motile mammalian cell line. Tracking PC3 cells at different light doses and intensities, we show how motility can be used to reliably assess subtle positive and negative effects of illumination. We further show that the effects are a factor of intensity rather than light dose. Mitotic delay was not a sensitive indicator of phototoxicity. For early detection of the effect of blue light, we analysed the expression of genes involved in oxidative stress. This study addresses the need for relatively simple and sensitive methods to establish a dose-response curve for phototoxicity in mammalian cell line models. We conclude with a working model for phototoxicity and recommendations for its assessment.

Development of pGEMINI, a Plant Gateway Destination Vector Allowing the Simultaneous Integration of Two cDNA via a Single LR-Clonase Reaction.

Gateway technology has been used to facilitate the generation of a large number of constructs for the modification of plants for research purposes. However, many of the currently available vectors only allow the integration of a single cDNA of interest into an expression clone. The ability to over-express multiple genes in combination is essential for the study of plant development where several transcripts have a role to play in one or more metabolic processes. The tools to carry out such studies are limited, and in many cases rely on the incorporation of cDNA into expression systems via conventional cloning, which can be both time consuming and laborious. To our knowledge, this study reports on the first development of a vector allowing the simultaneous integration of two independent cDNAs via a single LR-clonase reaction. This vector "pGEMINI" represents a powerful molecular tool offering the ability to study the role of multi-cDNA constructs on plant development, and opens up the process of gene stacking and the study of gene combinations through transient or stable transformation procedures.

The olfactory sensory map in Drosophila.

The fruit fly (Drosophila melanogaster) exhibits robust odor-evoked behaviors in response to cues from diverse host plants and pheromonal cues from other flies. Understanding how the adult olfactory system supports the perception of these odorous chemicals and translates them into appropriate attraction or avoidance behaviors is an important goal in contemporary sensory neuroscience. Recent advances in genomics and molecular neurobiology have provided an unprecedented level of detail into how the adult Drosophila olfactory system is organized. Volatile odorants are sensed by two bilaterally symmetric olfactory sensory appendages, the third segment of the antenna and the maxillary palps, which respectively contain approximately 1200 and 120 olfactory sensory neurons (OSNs) each. These OSNs express a divergent family of seven transmembrane domain odorant receptors (ORs) with no homology to vertebrate ORs, which determine the odor specificity of a given OSN. Drosophila was the first animal for which all OR genes were cloned, their patterns of gene expression determined and axonal projections of most OSNs elucidated. In vivo electrophysiology has been used to decode the ligand response profiles of most of the ORs, providing insight into the initial logic of olfactory coding in the fly. This chapter will review the molecular biology, neuroanatomy and function of the peripheral olfactory system of Drosophila.