Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.     

Glucose and ketone bodies production in hepatocytes isolated from fetuses at term--II. Effect of maternal adrenalectomy

Glucose and ketone bodies production has been studied in hepatocytes isolated from fetuses at term of fed and fasted adrenalectomized mothers. Maternal adrenalectomy diminishes the fetal liver weight. This effect is increased when the adrenalectomized pregnant rat is fasted for the last 2 days of gestation. Maternal adrenalectomy diminishes glucose production in hepatocytes isolated from fetuses at term. This diminution is markedly greater when the adrenalectomized pregnant rat is fasted for the last 48 hr of gestation. Maternal adrenalectomy diminishes ketone bodies production in hepatocytes isolated from fetuses at term.     

Rat striatal dopamine and tetrahydrobiopterin content following an intrastriatal injection of manganese chloride

Injection of manganese into the rat corpus striatum causes a rapid fall in the biopterin and dopamine (DA) content ipsilateral to the lesion. Two weeks after the lesion both biopterin and DA are partially recovered. Controls, injected with saline or magnesium, do not show alterations in their DA or cofactor levels. It is proposed that the fall in DA levels results from a rapid displacement of the amine from its storage sites by manganese followed by a decrease in the rate of DA synthesis causes by the drop in cofactor levels.     

Predator detection and avoidance by starlings under differing scenarios of predation risk

Practically all animals must find food while avoiding predators.An individual's perception of predation risk may depend on manyfactors, such as distance to refuge and group size, but it isunclear whether individuals respond to different factors ina similar manner. We tested whether flocks of foraging starlingsresponded in the same way to an increased perception of predationrisk by assessing three factors: (1) neighbor distances, (2)habitat obstruction, and (3) recent exposure to a predator.We found that in all three scenarios of increased risk, starlingsreduced their interscan intervals (food-searching bouts), whichincreased the frequency of their vigilance periods. We thenexamined how one of these factors, habitat obstruction, affectedescape speed by simulating an attack with a model predator.Starlings were slower to respond in visually obstructed habitats(long grass swards) and slower when they had their head downin obstructed habitats than when they had their head down inopen habitats. In addition, reaction times were quicker whenstarlings could employ their peripheral fields of vision. Ourresults demonstrate that different sources of increased riskcan generate similar behavioral responses within a species.The degree of visibility in the physical and social environmentaffects both the actual and perceived risk of predation.     

An Arabidopsis lipid map reveals differences between tissues and dynamic changes throughout development

Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web-accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi-bin/efpWeb.cgi .     

Allometric growth and adaptive value of exaggerated body parts in the larvae of Gratiana spadicea (Klug) (Coleoptera: Chrysomelidae)

1. Larvae of tortoise beetles present exaggerated body parts in association with an abdominal shield, which is made of faeces and exuviae that are deposited on the urogomphi throughout ontogeny. Growth trajectories and scaling relationships of these functional structures associated with the shield, if any, are unknown. 2. This study of Gratiana spadicea first tested, under field conditions, whether there is adaptive value associated with the shield regarding protection against predation and sunlight. Then, under laboratory conditions, the growth trajectory and allometric relationships among body parts were investigated, including scoli, individual and apparent furcae, and shield. The influence of food deprivation on the development of these structures was also determined. 3. Findings from previous studies were confirmed, suggesting that the adaptive value assigned to the shield is related to protection against predators. The present study demonstrated for the first time that the shield acts as a parasol in cassidines, decreasing the exposure of their larval body to sunlight. The scoli and apparent furca are exaggerated structures of G. spadicea, the development of which involves allometric growth and greater energetic investment (positive allometry) during ontogeny. There was proportionally less energetic investment for somatic construction of individual furca (negative allometry) due to the accumulation of the exuviae. 4. The possible consequences, in terms of developmental costs and survivorship benefits associated with the evolution of such exaggerated structures, are discussed.     

A comparison of ferric-chelate reductase and chlorophyll and growth ratios as indices of selection of quince,pear and olive genotypes under iron deficiency stress

Quince (Cydonia oblonga Mill.), pear (Pyrus communis L.) and olive (Olea europaea L.) genotypes were evaluated for their tolerance to iron deficiency stress by growing young plants in three types of aerated nutrient solutions: (1) with iron, (2) without iron or (3) low in iron and with 10 mM bicarbonate. Plants were obtained either from rooted softwood cuttings or from germination of seeds. The degree of tolerance was evaluated with several indices: (1) the chlorophyll content, (2) the root Fe³⁺ reducing capacity and (3) the whole plant relative growth. Fifteen hours before Fe³⁺ reducing capacity determination, iron was applied to the roots of plants with iron-stress, since this method resulted in increasing the reductase activity. All quince and pear genotypes increased the root Fe³⁺ reducing capacity when grown in the treatments for iron-stress, in relation to control plants of the same genotypes. In olive cultivars, the Fe³⁺ reducing capacity was lower in the iron-stress treatments than in the control one. Studying the relationship between relative growth and chlorophyll content for each genotype under iron-stress, in relation to both indices in control plants, a classification of species and genotypes was established. According to that, most olive cultivars and some pear rootstocks and cultivars appear more iron-efficient than quince rootstocks. Our study shows that in some woody species, determining root Fe³⁺ reducing capacity is not the best method to establish tolerance to iron deficiency stress.     

Body Size,Growth and Life Span: Implications for the Polewards Range Shift of Octopus tetricus in South-Eastern Australia

Understanding the response of any species to climate change can be challenging. However, in short-lived species the faster turnover of generations may facilitate the examination of responses associated with longer-term environmental change. Octopus tetricus, a commercially important species, has undergone a recent polewards range shift in the coastal waters of south-eastern Australia, thought to be associated with the southerly extension of the warm East Australian Current. At the cooler temperatures of a polewards distribution limit, growth of a species could be slower, potentially leading to a bigger body size and resulting in a slower population turnover, affecting population viability at the extreme of the distribution. Growth rates, body size, and life span of O. tetricus were examined at the leading edge of a polewards range shift in Tasmanian waters (40°S and 147°E) throughout 2011. Octopus tetricus had a relatively small body size and short lifespan of approximately 11 months that, despite cooler temperatures, would allow a high rate of population turnover and may facilitate the population increase necessary for successful establishment in the new extended area of the range. Temperature, food availability and gender appear to influence growth rate. Individuals that hatched during cooler and more productive conditions, but grew during warming conditions, exhibited faster growth rates and reached smaller body sizes than individuals that hatched into warmer waters but grew during cooling conditions. This study suggests that fast growth, small body size and associated rapid population turnover may facilitate the range shift of O. tetricus into Tasmanian waters.     

Reduced Global Genetic Differentiation of Exploited Marine Fish Species

Knowledge on genetic structure is key to understand species connectivity patterns and to define the spatiotemporal scales over which conservation management plans should be designed and implemented. The distribution of genetic diversity (within and among populations) greatly influences species ability to cope and adapt to environmental changes, ultimately determining their long-term resilience to ecological disturbances. Yet, the drivers shaping connectivity and structure in marine fish populations remain elusive, as are the effects of fishing activities on genetic subdivision. To investigate these questions, we conducted a meta-analysis and compiled genetic differentiation data (F_ST/Φ_ST estimates) for more than 170 fish species from over 200 published studies globally distributed. We modeled the effects of multiple life-history traits, distance metrics, and methodological factors on observed population differentiation indices and specifically tested whether any signal arising from different exposure to fishing exploitation could be detected. Although the myriad of variables shaping genetic structure makes it challenging to isolate the influence of single drivers, results showed a significant correlation between commercial importance and genetic structure, with widespread lower population differentiation in commercially exploited species. Moreover, models indicate that variables commonly used as proxy for connectivity, such as larval pelagic duration, might be insufficient, and suggest that deep-sea species may disperse further. Overall, these results contribute to the growing body of knowledge on marine genetic connectivity and suggest a potential effect of commercial fisheries on the homogenization of genetic diversity, highlighting the need for additional research focused on dispersal ecology to ensure long-term sustainability of exploited marine species.