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.     

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 .     

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.     

The Identification of Congeners and Aliens by Drosophila Larvae

We investigated the role of Drosophila larva olfactory system in identification of congeners and aliens. We discuss the importance of these activities in larva navigation across substrates, and the implications for allocation of space and food among species of similar ecologies. Wild type larvae of cosmopolitan D. melanogaster and endemic D. pavani, which cohabit the same breeding sites, used species-specific volatiles to identify conspecifics and aliens moving toward larvae of their species. D. gaucha larvae, a sibling species of D. pavani that is ecologically isolated from D. melanogaster, did not respond to melanogaster odor cues. Similar to D. pavani larvae, the navigation of pavani female x gaucha male hybrids was influenced by conspecific and alien odors, whereas gaucha female x pavani male hybrid larvae exhibited behavior similar to the D. gaucha parent. The two sibling species exhibited substantial evolutionary divergence in processing the odor inputs necessary to identify conspecifics. Orco (Or83b) mutant larvae of D. melanogaster, which exhibit a loss of sense of smell, did not distinguish conspecific from alien larvae, instead moving across the substrate. Syn ^97CS and rut larvae of D. melanogaster, which are unable to learn but can smell, moved across the substrate as well. The Orco (Or83b), Syn ^97CS and rut loci are necessary to orient navigation by D. melanogaster larvae. Individuals of the Trana strain of D. melanogaster did not respond to conspecific and alien larval volatiles and therefore navigated randomly across the substrate. By contrast, larvae of the Til-Til strain used larval volatiles to orient their movement. Natural populations of D. melanogaster may exhibit differences in identification of conspecific and alien larvae. Larval locomotion was not affected by the volatiles.     

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g^-1) ] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g^-1, while the threshold for the ST was greater than 0.1.10³ CFU^-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g^-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.     

Expression and subcellular localization of a 35-kDa carbonic anhydrase IV in a human pancreatic ductal cell line (Capan-1).

The high intraluminal concentrations of HCO(3)(-) in the human pancreatic ducts have suggested the existence of a membrane protein supplying the Cl(-)/HCO(3)(-) exchanger. Membrane-bound carbonic anhydrase IV (CA IV) is one of the potential candidates for this protein. The difficulties in isolating human pancreatic ducts have led the authors to study the molecular mechanisms of HCO(3)(-) secretion in cancerous cell lines. In this work, we have characterized the CA IV expressed in Capan-1 cells. A 35-kDa CA IV was detected in cell homogenates and purified plasma membranes. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase-C indicated that this CA IV was not anchored by a glycosylphosphatidylinositol (GPI). In contrast, its detection on purified plasma membranes by an antibody specifically directed against the carboxyl terminus of human immature GPI-anchored CA IV indicated that it was anchored by a C-terminal hydrophobic segment. Immunoelectron microscopy and double-labeling immunofluorescence revealed that this CA IV was present on apical plasma membranes, and in the rough endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate compartment, the Golgi complex, and secretory granules, suggesting its transport via the classical biosynthesis/secretory pathway. The expression in Capan-1 cells of a 35-kDa CA IV anchored in the apical plasma membrane through a hydrophobic segment, as is the case in the healthy human pancreas, should make the study of its role in pancreatic HCO(3)(-) secretion easier.