Maternal lineages in native Canadian equine populations and their relationship to the Nordic and Mountain and Moorland pony breeds

A 378-bp section of the mitochondrial displacement loop was used to estimate genetic diversity in the native Canadian equine populations. The inclusion of 10 Mountain and Moorland, 3 Nordic pony breeds, 2 feral populations, and 5 horse breeds were also investigated as they may have influenced the development (or rejuvenation) of the native Canadian populations. A total of 281 samples were sequenced, which produced 75 haplotypes derived from 54 informative sites. On further investigation, 36 of these 75 haplotypes were found to be previously unreported. Overall, total diversity was lowest in the feral Sable Island population with a haplotype diversity (0.27 ± 0.12), nucleotide diversity (0.0007 ± 0.0004), and pairwise difference of 0.286 ± 0.317. This is not surprising due to the geographic isolation of this population. Haplotype diversity was highest (1.00 ± 0.13) in the New Forest population, pairwise difference was highest (8.061 ± 4.028) in the Icelandic breed, whereas nucleotide diversity was highest in the Exmoor breed (0.0209 ± 0.0025). Within the Canadian populations, haplotype diversity was highest in the Newfoundland pony (0.96 ± 0.08), whereas pairwise difference and nucleotide diversity was highest in the Canadian horse (7.090 ± 3.581 and 0.0188 ± 0.0042, respectively). Three different estimates of genetic distances were used to examine the phylogenetic relationships amongst these populations. All 3 estimates produced similar topologies. In general, the native Canadian populations were highly represented in the D clade, with particular emphasis in the D1 and D2 clades. This is an important factor when considering the phylogenetic conservation of these Canadian equine populations.     

Survival and immune response of drones of a Nosemosis tolerant honey bee strain towards N. ceranae infections

Honey bee colonies (Apis mellifera) have been selected for low level of Nosema in Denmark over decades and Nosema is now rarely found in bee colonies from these breeding lines. We compared the immune response of a selected and an unselected honey bee lineage, taking advantage of the haploid males to study its potential impact on the tolerance toward Nosema ceranae, a novel introduced microsporidian pathogen. After artificial infections of the N. ceranae spores, the lineage selected for Nosema tolerance showed a higher N. ceranae spore load, a lower mortality and an up-regulated immune response. The differences in the response of the innate immune system between the selected and unselected lineage were strongest at day six post infection. In particular genes of the Toll pathway were up-regulated in the selected strain, probably is the main immune pathway involved in N. ceranae infection response. After decades of selective breeding for Nosema tolerance in the Danish strain, it appears these bees are tolerant to N. ceranae infections.     

Human prorenin structure sheds light on a novel mechanism of its autoinhibition and on its non-proteolytic activation by the (pro)renin receptor

Antibodies and prorenin mutants have long been used to structurally characterize prorenin, the inactive proenzyme form of renin. They were designed on the basis of homology models built using other aspartyl protease proenzyme structures since no structure was available for prorenin. Here, we present the first X-ray structure of a prorenin. The current structure of prorenin reveals that, in this zymogene, the active site of renin is blocked by the N-terminal residues of the mature version of the renin molecule, which are, in turn, covered by an Ω-shaped prosegment. This prevents access of substrates to the active site. The departure of the prosegment on activation induces an important global conformational change in the mature renin molecule with respect to prorenin: similar to other related enzymes such as pepsin or gastricsin, the segment that constitutes the N-terminal β-strand in renin is displaced from the renin active site by about 180° straight into the position that corresponds to the N-terminal β-strand of the prorenin prosegment. This way, the renin active site will become completely exposed and capable of carrying out its catalytic functions. A unique inactivation mechanism is also revealed, which does not make use of a lysine against the catalytic aspartates, probably in order to facilitate pH-independent activation [e.g., by the (pro)renin receptor].     

Structural insights into antibody sequestering and neutralizing of Na+ channel α-type modulator from old world scorpion venom

The Old World scorpion Androctonus australis hector (Aah) produces one of the most lethal venoms for humans. Peptidic α-toxins AahI to AahIV are responsible for its potency, with AahII accounting for half of it. All four toxins are high affinity blockers of the fast inactivation phase of mammalian voltage-activated Na(+) channels. However, the high antigenic polymorphism of α-toxins prevents production of a polyvalent neutralizing antiserum, whereas the determinants dictating their trapping by neutralizing antibodies remain elusive. From an anti-AahII mAb, we generated an antigen binding fragment (Fab) with high affinity and selectivity for AahII and solved a 2.3 ?-resolution crystal structure of the complex. Sequestering of the C-terminal region of the bound toxin within a groove formed by the Fab combining loops is associated with a toxin orientation and main and side chain conformations that dictate the AahII antigenic specificity and efficient neutralization. From an anti-AahI mAb, we also preformed and crystallized a high affinity AahI-Fab complex. The 1.6 ?-resolution structure solved revealed a Fab molecule devoid of a bound AahI and with combining loops involved in packing interactions, denoting expulsion of the bound antigen upon crystal formation. Comparative analysis of the groove-like combining site of the toxin-bound anti-AahII Fab and planar combining surface of the unbound anti-AahI Fab along with complementary data from a flexible docking approach suggests occurrence of distinctive trapping orientations for the two toxins relative to their respective Fab. This study provides complementary templates for designing new molecules aimed at capturing Aah α-toxins and suitable for immunotherapy.     

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.     

Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate

Arabidopsis thaliana PHO1 is primarily expressed in the root vascular cylinder and is involved in the transfer of inorganic phosphate (Pi) from roots to shoots. To analyze the role of PHO1 in transport of Pi, we have generated transgenic plants expressing PHO1 in ectopic A. thaliana tissues using an estradiol-inducible promoter. Leaves treated with estradiol showed strong PHO1 expression, leading to detectable accumulation of PHO1 protein. Estradiol-mediated induction of PHO1 in leaves from soil-grown plants, in leaves and roots of plants grown in liquid culture, or in leaf mesophyll protoplasts, was all accompanied by the specific release of Pi to the extracellular medium as early as 2-3 h after addition of estradiol. Net Pi export triggered by PHO1 induction was enhanced by high extracellular Pi and weakly inhibited by the proton-ionophore carbonyl cyanide m-chlorophenylhydrazone. Expression of a PHO1-GFP construct complementing the pho1 mutant revealed GFP expression in punctate structures in the pericycle cells but no fluorescence at the plasma membrane. When expressed in onion epidermal cells or in tobacco mesophyll cells, PHO1-GFP was associated with similar punctate structures that co-localized with the Golgi/trans-Golgi network and uncharacterized vesicles. However, PHO1-GFP could be partially relocated to the plasma membrane in leaves infiltrated with a high-phosphate solution. Together, these results show that PHO1 can trigger Pi export in ectopic plant cells, strongly indicating that PHO1 is itself a Pi exporter. Interestingly, PHO1-mediated Pi export was associated with its localization to the Golgi and trans-Golgi networks, revealing a role for these organelles in Pi transport.     

Sequelae after AF ablation: Efficacy and Safety go Hand in Hand

Although nowadays performed on a routine basis, catheter ablation of atrial fibrillation is associated with the potential for major complications. Improving the safety remains therefore an important challenge. This article summarizes the different types of complications associated with AF ablation grouping them into clinically overt major complications, subclinical injury and permanent injury. Furthermore, it describes the potential predictors for complications and highlights the dynamic interplay between efficacy and safety.     

Semisynthetic neoboutomellerone derivatives as ubiquitin-proteasome pathway inhibitors

The interesting pharmacological properties of neoboutomellerones 1 and 2 were the basis for the assembly of a small library of analogues consisting of natural products isolated from the plant Neoboutonia melleri and of semisynthetic derivatives. As the two enone systems (C23-C24a and C1-C3) and the two hydroxyls groups (C22 and C26) of neoboutomellerones are required for activity, modifications were focused on these functional groups. Biological evaluation by using a cellular assay for proteasome activity provided clues regarding the mechanism of action of these natural products and synthetic derivatives. Certain neoboutomellerone derivatives inhibited the proliferation of human WM-266-4 melanoma tumor cells at submicromolar concentration and warrant evaluation as anticancer agents.     

The emerging importance of the SPX domain-containing proteins in phosphate homeostasis

Plant growth and development are strongly influenced by the availability of nutrients in the soil solution. Among them, phosphorus (P) is one of the most essential and most limiting macro-elements for plants. In the environment, plants are often confronted with P starvation as a result of extremely low concentrations of soluble inorganic phosphate (Pi) in the soil. To cope with these conditions, plants have developed a wide spectrum of mechanisms aimed at increasing P use efficiency. At the molecular level, recent studies have shown that several proteins carrying the SPX domain are essential for maintaining Pi homeostasis in plants. The SPX domain is found in numerous eukaryotic proteins, including several proteins from the yeast PHO regulon, involved in maintaining Pi homeostasis. In plants, proteins harboring the SPX domain are classified into four families based on the presence of additional domains in their structure, namely the SPX, SPX-EXS, SPX-MFS and SPX-RING families. In this review, we highlight the recent findings regarding the key roles of the proteins containing the SPX domain in phosphate signaling, as well as providing further research directions in order to improve our knowledge on P nutrition in plants, thus enabling the generation of plants with better P use efficiency.