Generation and characterization of a recombinant vesicular stomatitis virus expressing the glycoprotein of Borna disease virus

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. BDV cell entry occurs via receptor-mediated endocytosis, a process initiated by the recognition of an as yet unidentified receptor at the cell surface by the BDV surface glycoprotein (G). The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of cellular receptors and detailed mechanisms involved in BDV cell entry. To overcome this problem, we generated and characterized a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSVDeltaG*/BDVG). Cells infected with rVSVDeltaG*/BDVG produced high titers (10(7) PFU/ml) of cell-free virus progeny, but this virus exhibited a highly attenuated phenotype both in cell culture and in vivo. Attenuation of rVSVDeltaG*/BDVG was associated with a delayed kinetics of viral RNA replication and altered genome/N mRNA ratios compared to results for rVSVDeltaG*/VSVG. Likewise, incorporation of BDV G into virions appeared to be restricted despite its high levels of expression and efficient processing in rVSVDeltaG*/BDVG-infected cells. Notably, rVSVDeltaG*/BDVG recreated the cell tropism and entry pathway of bona fide BDV. Our results indicate that rVSVDeltaG*/BDVG represents a unique tool for the investigation of BDV G-mediated cell entry, as well as the roles of BDV G in host immune responses and pathogenesis associated with BDV infection.     

Vasoactive intestinal peptide enhances growth and angiogenesis of human experimental prostate cancer in a xenograft model

We show that vasoactive intestinal peptide (VIP) exerts trophic and proangiogenic activities in experimental prostate cancer in vivo. Nude mice were subcutaneously injected with Matrigel impregnated with LNCaP prostate cancer cells. Cell treatment with 100 nM VIP for 1h before xenograft resulted in increased tumor growth after 8 and, more remarkably, 15 days of injection. The same occurred with the mRNA expression of the main angiogenic factor, vascular endothelial growth factor (VEGF), as shown by real-time RT-PCR quantification. The proangiogenic activity of VIP was further established by showing increases of hemoglobin levels, Masson trichromic staining, and immunohistochemical CD34 staining in tumors excised 15 days after subcutaneous injection of VIP-treated cells as compared to control conditions. All these parameters indicate that VIP increases vessel formation. This xenograft model is a useful tool to study in vivo the effects of VIP-related peptides in tumor growth and development of blood supply as well as their therapeutical potential in prostate cancer.     

Expression and one-step purification of recombinant Arabidopsis thaliana frataxin homolog (AtFH)

Frataxin, a nuclear-encoded mitochondrial protein, has been proposed to participate in Fe-S cluster assembly, mitochondrial energy metabolism, respiration, and iron homeostasis. However, its precise function remains elusive. Frataxin is highly conserved in living organisms with no major structural changes, in particular at the C-terminal protein domain, suggesting that it plays a key function in all organisms. Recently, a plant gene, AtFH, with significant homology to other members of the frataxin family has been described. To gain insight on the frataxin role in plants, the frataxin domain was expressed in Escherichia coli BL21-codonPlus (DE3)-RIL cells and purified using a Ni-chelating column. The purified protein, added to a mixture containing Fe(II) and H2O2, attenuates the Fenton reaction indicating that the recombinant plant frataxin is functional. The procedure described here produced high yield of 99% pure protein through only one chromatographic step, suitable for further structure-function studies.     

Phylogenetic reconstruction from non-genomic data

MOTIVATION: Recent results related to horizontal gene transfer suggest that phylogenetic reconstruction cannot be determined conclusively from sequence data, resulting in a shift from approaches based on polymorphism information in DNA or protein sequence to studies aimed at understanding the evolution of complete biological processes. The increasing amount of available information on metabolic pathways for several species makes it of greater relevance to understand the similarities and differences among such pathways. These similarities can then be used to infer phylogenetic trees not based exclusively in sequence data, therefore avoiding the previously mentioned problems. RESULTS: In this article, we present a method to assess the structural similarity of metabolic pathways for several organisms. Our algorithms work by using one of the three possible enzyme similarity measures (hierarchical, information content, gene ontology), and one of the two clustering methods (neighbor-joining, unweighted pair group method with arithmetic mean), to produce a phylogenetic tree both in Newick and graphic format. The web server implementing our algorithms is optimized to answer queries in linear time. AVAILABILITY: The software is available for free public use on a web server, at the address http://www.jaist.ac.jp/~clemente/cgi-bin/phylo.pl. It is available on demand in source code form for research use to educational institutions, non-profit research institutes, government research laboratories and individuals, for non-exclusive use, without the right of the licensee to further redistribute the source code.     

Improving model construction of profile HMMs for remote homology detection through structural alignment

Background  

Remote homology detection is a challenging problem in Bioinformatics. Arguably, profile Hidden Markov Models (pHMMs) are one of the most successful approaches in addressing this important problem. pHMM packages present a relatively small computational cost, and perform particularly well at recognizing remote homologies. This raises the question of whether structural alignments could impact the performance of pHMMs trained from proteins in the Twilight Zone, as structural alignments are often more accurate than sequence alignments at identifying motifs and functional residues. Next, we assess the impact of using structural alignments in pHMM performance.     

Seed bank dynamics of two obligate seeders, <Emphasis Type="Italic">Cistus monspeliensis</Emphasis> and <Emphasis Type="Italic">Rosmarinus officinalis</Emphasis>, in relation to time since fire

Many species in Mediterranean-type ecosystems regenerate after fire by seed germination from soil seed banks. Seed bank dynamics of two of those obligate seeders, Cistus monspeliensis and Rosmarinus officinalis, were investigated in relation to stand age since fire in southwestern Portugal. Soil seed density, annual seed input, annual seed losses through germination and seed persistence were compared between species at stands differing in age since fire (5, 10 and 35 years). Soil seed density and seed input increased over the first decade after fire and were lowest at 35-year-old stands for C. monspeliensis. In R. officinalis, few seeds were produced and found in the soil at early stages, and maximum seed input and soil seed density were attained at 35-year-old stands. Soil seed density was mostly driven by seed production in both species, which is largely dependent on plant traits and population dynamics related to fire. Overall, stand age since fire had a negligible effect on seed germination, seed persistence and viability. Ten to 39% of buried seeds were not recovered after 1 year, and viability of seeds recovered was 97–100% for C. monspeliensis and only 0–3% for R. officinalis. Variation in plant traits within the seeder syndrome was evidenced by this study. R. officinalis evidenced lower seed persistence, lower proportion of viable seed produced and lower density of viable soil seed than C. monspeliensis at any stage after fire. R. officinalis is expected to depend largely on previous year seed production for population replacement after fire.     

Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.     

Light and ultrastructural description of Meglitschia mylei n. sp. (myxozoa) from Myleus rubripinnis (Teleostei: Serrasalmidae) in the Amazon River system

Meglitschia mylei n. sp. found in the gall bladder of the teleostean fish Myleus rubripinnis (Serrasalmidae) from the middle Amazonian region of Brazil is described using light and transmission electron microscopy. The spores observed in the bile averaged 24.6±0.8 μm long, 8.7±0.4 μm wide and 5.1±0.3 μm thick and were strongly furcate and arcuate ∩-shaped composed of two symmetric equal-sized valves, up to ～70 nm thick. Each valve possessed one opposed tapering appendage, 20.1±0.7 μm long, oriented parallel towards the basal tip of the appendages and joined along a right suture line forming a thick strand. The strand goes around the central part of the spore, which in turn surrounds two equal and symmetric spherical polar capsules (PC), 2.1±0.3 μm in diameter, located at the same level. Each capsule contains a polar filament with five (rarely six) coils. The binucleate sporoplasm was irregular in shape, contained several sporoplasmosomes, ～175 nm in diameter and filled all the space of the two caudal appendages. Based on the arc shape of the spore with two tapering caudal appendages oriented to the basis of spores, on the number and position of the PC and of the polar filament coils and arrangements, and on the host specificity, we propose the name M. mylei n. sp. for this new myxozoan. Accordingly, this is the second described species of this genus.     

Evaluating models to assess the distribution of Buxus balearica in southern Spain

Question: Which is the best model to predict the habitat distribution of Buxus balearica Lam. in southern Spain? Location: Málaga and Granada, Spain, across an area of 38 180 km². Methods: Prediction models based on 17 environmental variables were tested. Six methods were compared: multivariate adaptive regression spline (MARS), maximum entropy approach to modelling species' distributions (Maxent), two generic algorithms based on environmental metrics dissimilarity (BIOCLIM and DOMAIN), Genetic Algorithm for Rule‐set Prediction (GARP), and supervised learning methods based on generalized linear classifiers (support vector machines, SVMs). To test the predictive power of the models we used the Kappa index. Results: Maxent most accurately predicted the habitat distribution of B. balearica, followed by MARS models. The other models tested yielded lower accuracy values. A comparison of the predictive power of the models revealed that climate variables made the highest contributions among the environmental variables studied. The variables that made the lowest contributions were the insolation models. To examine the sensitivity of the models to a reduction in the number of variables, a test showed that accuracy of over 0.90 was maintained by applying just three climatic variables (spring rainfall, mean temperature of the warmest month, and mean temperature of the coldest month). Maps derived from the algorithms of all models tested coincided well with the known distribution of the species. Conclusions: Model habitat prediction is a preliminary step towards highlighting areas of high habitat suitability of B. balearica. These data support the results of previous research, which show that MaxEnt is the best technique for modelling species distributions with small sample sizes.