Differentiation of Isomeric Purine and Pyrimidine Mononucleotides by Fast Atom Bombardment Tandem Mass Spectrometry

Abstract

The use of positive ion fast atom bombardment mass-analysed ion kinetic energy (FAB/MIKE) spectroscopy to differentiate the 2′, 3′-and 5′-monophosphate isomers of adenosine, guanosine and cytidine is described.     

Longitudinal Study of Reproductive Performance of Female Cattle Produced by Somatic Cell Nuclear Transfer

The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT) is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1) superovulation followed by artificial insemination (AI) and embryo collection and (2) ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF). Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection (P = 0.77) or OPU-IVF (P = 0.97). These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics.     

Framing disputes and organizational legitimation: UK-based Sri Lankan Tamil diaspora groups' use of the ‘genocide’ frame since 2009

The ending of the war in Sri Lanka in 2009 led to significant changes in the political strategies pursued by Sri Lankan Tamil diaspora groups in the UK. One contentious feature of these groups' campaigns has been their use of the ‘genocide’ frame to describe the actions of the Sri Lankan state, which has been predominantly viewed either as a signal of these groups' strategic naivety or as a coded expression of a wider nationalist agenda. In this article I argue that its growing use in the post-war period is more complex and is best understood in relation to these organizations' strategies of legitimation. Deploying the genocide frame has served two key functions: to demonstrate groups' responsiveness to popular demands, and to challenge dominant international approaches to post-war Sri Lanka. Together these functions served to bolster groups' legitimacy in an environment characterized by political change and high levels of inter-organizational competition.     

EXTINCTION SPACE—A METHOD FOR THE QUANTIFICATION AND CLASSIFICATION OF CHANGES IN MORPHOSPACE ACROSS EXTINCTION BOUNDARIES

Three main modes of extinction are responsible for reductions in morphological disparity: (1) random (caused by a nonselective extinction event); (2) marginal (a symmetric, selective extinction event trimming the margin of morphospace); and (3) lateral (an asymmetric, selective extinction event eliminating one side of the morphospace). These three types of extinction event can be distinguished from one another by comparing changes in three measures of morphospace occupation: (1) the sum of range along the main axes; (2) the sum of variance; and (3) the position of the centroid. Computer simulations of various extinction events demonstrate that the pre‐extinction distribution of taxa (random or normal) in the morphospace has little influence on the quantification of disparity changes, whereas the modes of the extinction events play the major role. Together, the three disparity metrics define an “extinction‐space” in which different extinction events can be directly compared with one another. Application of this method to selected extinction events (Frasnian‐Famennian, Devonian‐Carboniferous, and Permian‐Triassic) of the Ammonoidea demonstrate the similarity of the Devonian events (selective extinctions) but the striking difference from the end‐Permian event (nonselective extinction). These events differ in their mode of extinction despite decreases in taxonomic diversity of similar magnitude.     

Expression of flotillins in the human placenta: potential implications for placental transcytosis

A proteomics survey of human placental syncytiotrophoblast (ST) apical plasma membranes revealed peptides corresponding to flotillin-1 (FLOT1) and flotillin-2 (FLOT2). The flotillins belong to a class of lipid microdomain-associated integral membrane proteins that have been implicated in clathrin- and caveolar-independent endocytosis. In the present study, we characterized the expression of the flotillin proteins within the human placenta. FLOT1 and FLOT2 were coexpressed in placental lysates and BeWo human trophoblast cells. Immunofluorescence microscopy of first-trimester and term placentas revealed that both proteins were more prominent in villous endothelial cells and cytotrophoblasts (CTs) than the ST. Correspondingly, forskolin-induced fusion in BeWo cells resulted in a decrease in FLOT1 and FLOT2, suggesting that flotillin protein expression is reduced following trophoblast syncytialization. The flotillin proteins co-localized with a marker of fluid-phase pinocytosis, and knockdown of FLOT1 and/or FLOT2 expression resulted in decreased endocytosis of cholera toxin B subunit. We conclude that FLOT1 and FLOT2 are abundantly coexpressed in term villous placental CTs and endothelial cells, and in comparison, expression of these proteins in the ST is reduced. These findings suggest that flotillin-dependent endocytosis is unlikely to be a major pathway in the ST, but may be important in the CT and endothelium.     

Postnatal neural precursor cell regions in the rostral subventricular zone, hippocampal subgranular zone and cerebellum of the dog (Canis lupus familiaris)

Identification of neural stem and progenitor cells (NPCs) in vitro and in vivo is essential to the use of developmental and disease models of neurogenesis. The dog is a valuable large animal model for multiple neurodegenerative diseases and is more closely matched to humans than rodents with respect to brain organization and complexity. It is therefore important to determine whether immunohistochemical markers associated with NPCs in humans and rodents are also appropriate for the dog. The NPC markers CD15, CD133, nestin, GFAP and phosphacan (DSD-1) were evaluated in situ in the canine rostral telencephalon, hippocampal dentate gyrus, and cerebellum at different postnatal time-points. Positive staining results were interpreted in the context of region and cellular morphology. Our results showed that neurospheres and cells within the rostral subventricular zone (SVZ), dentate gyrus subgranular zone (SGZ), and white matter tracts of the cerebellum were immunopositive for CD15, nestin and GFAP. Neurospheres and the cerebellum were immunonegative for CD133, whereas CD133 staining was present in the postnatal rostral SVZ. Anti-phosphacan antibody staining delineated the neurogenic niches of the rostral lateral ventricle SVZ and the hippocampal SGZ. Positive staining for phosphacan was also noted in white matter tracts of the cerebellum and within the Purkinje layer. Our results showed that in the dog these markers were associated with regions shown to be neurogenic in rodents and primates.     

Vertical Transmission of Respiratory Syncytial Virus Modulates Pre- and Postnatal Innervation and Reactivity of Rat Airways

Background

Environmental exposure to respiratory syncytial virus (RSV) is a leading cause of respiratory infections in infants, but it remains unknown whether this infection is transmitted transplacentally from the lungs of infected mothers to the offspring. We sought to test the hypothesis that RSV travels from the respiratory tract during pregnancy, crosses the placenta to the fetus, persists in the lung tissues of the offspring, and modulates pre- and postnatal expression of growth factors, thereby predisposing to airway hyperreactivity.

Methodology

Pregnant rats were inoculated intratracheally at midterm using recombinant RSV expressing red fluorescent protein (RFP). Viral RNA was amplified by RT-PCR and confirmed by sequencing. RFP expression was analyzed by flow cytometry and viral culture. Developmental and pathophysiologic implications of prenatal infection were determined by analyzing the expression of genes encoding critical growth factors, particularly neurotrophic factors and receptors. We also measured the expression of key neurotransmitters and postnatal bronchial reactivity in vertically infected lungs, and assessed their dependence on neurotrophic signaling using selective biological or chemical inhibition.

Principal Findings

RSV genome was found in 30% of fetuses, as well as in the lungs of 40% of newborns and 25% of adults. RFP expression was also shown by flow cytometry and replicating virus was cultured from exposed fetuses. Nerve growth factor and its TrkA receptor were upregulated in RSV- infected fetal lungs and co-localized with increased cholinergic innervation. Acetylcholine expression and smooth muscle response to cholinergic stimulation increased in lungs exposed to RSV in utero and reinfected after birth, and blocking TrkA signaling inhibited both effects.

Conclusions/Significance

Our data show transplacental transmission of RSV from mother to offspring and persistence of vertically transmitted virus in lungs after birth. Exposure to RSV in utero is followed by dysregulation of neurotrophic pathways predisposing to postnatal airway hyperreactivity upon reinfection with the virus.     

It’s Time for Some “Site”-Seeing: Novel Tools to Monitor the Ubiquitin Landscape in Arabidopsis thaliana

Ubiquitination, the covalent binding of the small protein modifier ubiquitin to a target protein, is an important and frequently studied posttranslational protein modification. Multiple reports provide useful insights into the plant ubiquitinome, but mostly at the protein level without comprehensive site identification. Here, we implemented ubiquitin combined fractional diagonal chromatography (COFRADIC) for proteome-wide ubiquitination site mapping on Arabidopsis thaliana cell cultures. We identified 3009 sites on 1607 proteins, thereby greatly increasing the number of known ubiquitination sites in this model plant. Finally, The Ubiquitination Site tool (http://bioinformatics.psb.ugent.be/webtools/ubiquitin_viewer/) gives access to the obtained ubiquitination sites, not only to consult the ubiquitination status of a given protein, but also to conduct intricate experiments aiming to study the roles of specific ubiquitination events. Together with the antibodies recognizing the ubiquitin remnant motif, ubiquitin COFRADIC represents a powerful tool to resolve the ubiquitination maps of numerous cellular processes in plants.