Determination of Opiates in Whole Blood and Vitreous Humor: A Study of the Matrix Effect and an Experimental Design to Optimize Conditions for the Enzymatic Hydrolysis of Glucuronides

Faculty of Medicine University of Sao Paulo

Radiation of the Tnt1 retrotransposon superfamily in three Solanaceae genera

Abstract Background Tnt1 was the first active plant retrotransposon identified in tobacco after nitrate reductase gene disruption. The Tnt1 superfamily comprises elements from Nicotiana (Tnt1 and Tto1) and Lycopersicon (Retrolyc1 and Tlc1) species. The study presented here was conducted to characterise Tnt1-related sequences in 20 wild species of Solanum and five cultivars of Solanum tuberosum. Results Tnt1-related sequences were amplified from total genomic DNA using a PCR-based approach. Purified fragments were cloned and sequenced, and clustering analysis revealed three groups that differ in their U3 region. Using a network approach with a total of 453 non-redundant sequences isolated from Solanum (197), Nicotiana (140) and Lycopersicon (116) species, it is demonstrated that the Tnt1 superfamily can be treated as a population to resolve previous phylogenetic multifurcations. The resulting RNAseH network revealed that sequences group according to the Solanaceae genus, supporting a strong association with the host genome, whereas tracing the U3 region sequence association characterises the modular evolutionary pattern within the Tnt1 superfamily. Within each genus, and irrespective of species, nearly 20% of Tnt1 sequences analysed are identical, indicative of being part of an active copy. The network approach enabled the identification of putative "master" sequences and provided evidence that within a genus these master sequences are associated with distinct U3 regions. Conclusion The results presented here support the hypothesis that the Tnt1 superfamily was present early in the evolution of Solanaceae. The evidence also suggests that the RNAseH region of Tnt1 became fixed at the host genus level whereas, within each genus, propagation was ensured by the diversification of the U3 region. Different selection pressures seemed to have acted on the U3 and RNAseH modules of ancestral Tnt1 elements, probably due to the distinct functions of these regions in the retrotransposon life cycle, resulting in both co evolution and adaptation of the element population with its host.

Morphometric analysis of the placenta in the New World mouse Necromys lasiurus (Rodentia, Cricetidae): a comparison of placental development in cricetids and murids

Background: Stereology is an established method to extrapolate three-dimensional quantities from two-dimensional images. It was applied to placentation in the mouse, but not yet for other rodents. Herein, we provide the first study on quantitative placental development in a sigmodontine rodent species with relatively similar gestational time. Placental structure was also compared to the mouse, in order to evaluate similarities and differences in developmental patterns at the end of gestation. Methods: Fetal and placental tissues of Necromys lasiurus were collected and weighed at 3 different stages of gestation (early, mid and late gestation) for placental stereology. The total and relative volumes of placenta and of its main layers were investigated. Volume fractions of labyrinth components were quantified by the One Stop method in 31 placentae collected from different individuals, using the Mercator® software. Data generated at the end of gestation from N. lasiurus placentae were compared to those of Mus musculus domesticus obtained at the same stage. Results: A significant increase in the total absolute volumes of the placenta and its main layers occurred from early to mid-gestation, followed by a reduction near term, with the labyrinth layer becoming the most prominent area. Moreover, at the end of gestation, the total volume of the mouse placenta was significantly increased compared to that of N. lasiurus although the proportions of the labyrinth layer and junctional zones were similar. Analysis of the volume fractions of the components in the labyrinth indicated a significant increase in fetal vessels and sinusoidal giant cells, a decrease in labyrinthine trophoblast whereas the proportion of maternal blood space remained stable in the course of gestation. On the other hand, in the mouse, volume fractions of fetal vessels and sinusoidal giant cells decreased whereas the volume fraction of labyrinthine trophoblast increased compared to N. lasiurus placenta. Conclusions: Placental development differed between N. lasiurus and M. musculus domesticus. In particular, the low placental efficiency in N. lasiurus seemed to induce morphological optimization of fetomaternal exchanges. In conclusion, despite similar structural aspects of placentation in these species, the quantitative dynamics showed important differences.