Functional characterisation and in silico modelling of MdPSY2 variants and MdPSY5 phytoene synthases from Malus domestica.

Carotenoids are plastid isoprenoid pigments that play critical roles in light harvesting, photoprotection, and phytohormone biosynthesis. They are also vitamin-A precursors and antioxidant molecules important for human nutrition. Apples (e.g. Malus x domestica Borkh), one of the most widely consumed fruits with high nutrient levels, have a very low carotenoid concentration in flesh, compared with other fruits and vegetables. This could be explained by a deficiency in carotenoid synthesis/accumulation and/or accelerated degradation. We analysed the contribution of M. domestica cv. 'Fuji' phytoene synthase (PSY) in the biosynthesis of carotenoids and determined that among four MdPSY genes present in the organism, MdPSY2 and MdPSY5 are highly expressed in leaves and during fruit ripening in line with an increment in carotenoid content in fruits. Furthermore, two representative polymorphic MdPSY2 variants were found, one with a Tyr358Phe substitution (MdPSY2_F) and the other that additionally has a six-amino-acid deletion in the signal peptide (MdPSY2_CG). MdPSY2, MdPSY5, MdPSY2_F and MdPSY2_CG are all localised in plastids. Interestingly, the polymorphic MdPSY2_F and MdPSY2_CG variants show lower enzymatic activity than the wild-type form in a heterologous complementation assay, which could be attributed to the Tyr358Phe substitution close to the active-site pocket, as was suggested by 3-D modelling analysis. The presence of polymorphic MdPSY2 variants with lower enzymatic activity could be partially responsible for the low carotenoid content in Fuji apple fruits.

Alternative Animal and Non-Animal Models for Drug Discovery and Development: Bonus or Burden?

Mammalian models have served as a basis for R&D over the past decades. Nevertheless, these models are expensive, laborious, may yield results that cannot always be translated into the human in vivo situation and, more recently, have reverberated great social and ethical dilemmas. Hence, the prospect of changes in the global scientific scenario and the Three Rs principle (Reduction, Replacement and Refinement) have encouraged the development of alternative methods to the use of mammals. Despite the efforts, suitable alternative tests are not available in all areas of biomedical research, as regulatory acceptance requires time, prior validation and robust financial and scientific investment. In this perspective, we aim to shed light on the concepts, challenges and perspectives for implementation of innovative alternative animal and non-animal methods in scientific research. The applicability and meaningfulness of invertebrate animal models, in silico analysis and reverse pharmacology are discussed, among other aspects of relevance in today's scenario. Overall, the use of alternative models, including Artemia salina (brine shrimp), Caenorhabditis elegans (roundworm), Danio rerio (zebra fish), Drosophila melanogaster (fruit fly), Galleria mellonella (greater waxmoth) and in silico modelling, increased 909% from 1990 to 2015, as compared to 154% of conventional mammals in the same period. Thus, technological and scientific advancements in the fields of toxicology and drug development seem to have diminished the need for mammalian models. Today, however, mammals still remain critically indispensable to provide - in most cases -reliable data subsidizing and validating translation into the clinical setting.