RNA-Puzzles: a CASP-like evaluation of RNA three-dimensional structure prediction

We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.     

High macro-collinearity between lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.) as revealed by comparative cytogenetic mapping

Common bean (P. vulgaris) and lima bean (P. lunatus) are the most important crop species from the genus Phaseolus. Both species have the same chromosome number (2n = 22) and previous cytogenetic mapping of BAC clones suggested conserved synteny. Nevertheless, karyotype differences were observed, suggesting structural rearrangements. In this study, comparative cytogenetic maps for chromosomes 3, 4 and 7 were built and the collinearity between the common bean and lima bean chromosomes was investigated. Thirty-two markers (30 BACs and 2 bacteriophages) from P. vulgaris were hybridized in situ on mitotic chromosomes from P. lunatus. Nine BACs revealed a repetitive DNA pattern with pericentromeric distribution and 23 markers showed unique signals. Nine of these markers were mapped on chromosome 3, eight on chromosome 4 and six on chromosome 7. The order and position of all analyzed BACs were similar between the two species, indicating a high level of macro-collinearity. Thus, although few inversions have probably altered centromere position in other chromosomes, the main karyotypic differences were associated with the repetitive DNA fraction.     

Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells

Background

Mucopolysaccharidosis type IIIA (MPS IIIA) is the most common of the mucopolysaccharidoses. The disease is caused by a deficiency of the lysosomal enzyme sulphamidase and results in the storage of the glycosaminoglycan (GAG), heparan sulphate. MPS IIIA is characterised by widespread storage and urinary excretion of heparan sulphate, and a progressive and eventually profound neurological course. Gene therapy is one of the few avenues of treatment that hold promise of a sustainable treatment for this disorder.

Methods

The murine sulphamidase gene cDNA was cloned into a lentiviral vector and high-titre virus produced. Human MPS IIIA fibroblast cultures were transduced with the sulphamidase vector and analysed using molecular, enzymatic and metabolic assays. High-titre virus was intravenously injected into six 5-week old MPS IIIA mice. Three of these mice were pre-treated with hyperosmotic mannitol. The weight of animals was monitored and GAG content in urine samples was analysed by polyacrylamide gel electrophoresis.

Results

Transduction of cultured MPS IIIA fibroblasts with the sulphamidase gene corrected both the enzymatic and metabolic defects. Sulphamidase secreted by gene-corrected cells was able to cross correct untransduced MPS IIIA cells. Urinary GAG was found to be greatly reduced in samples from mice receiving the vector compared to untreated MPS IIIA controls. In addition, the weight of treated mice became progressively normalised over the 6-months post-treatment.

Conclusion

Lentiviral vectors appear promising vehicles for the development of gene therapy for MPS IIIA.     

Long-term in vitro culture affects phenotypic plasticity of Neoregelia johannis plants

Plant Cell, Tissue and Organ Culture (PCTOC) - We carried out immunoassay of plant hormones [indoleacetic acid (IAA), cytokinins and abscisic acid (ABA)] in the extracts from explants...     

Trace elements in human milk: Correlation with blood levels, inter-element correlations and changes in concentration during the first month of lactation

Using inductively coupled plasma mass spectrometry (ICP-MS) based analytical procedures, the concentration of several trace elements (Mn, As, Pb, Co, Ni, Cu, Zn and Se) was determined in human milk samples collected from a group of healthy lactating Portuguese women (n=44), both on the 2nd day postpartum (i.e., colostrum; n=34) and at 1 month postpartum (i.e., mature milk; n=19). Blood samples (n=44), collected on the 2nd day after parturition, were also analyzed for the same trace elements. No major correlations were observed between the levels of the analyzed trace elements in blood and colostrum samples. All the studied elements, except for Co, Pb and Ni, showed a significant trend for a decrease in concentration in milk during the first month of lactation. This trend was more pronounced for Zn and Se, whose levels decreased to approximately 23% and 44% of their initial mean concentration, respectively. With the exception of Co (r=0.607) and Zn (r=0.487), no significant correlations were observed when comparing the levels of each trace element between samples of colostrum and mature milk. Several inter-element correlations were found within each type of milk sample. The most significant were: (i) Se vs Cu (r=0.828) and Se vs Co (r=0.605) in colostrum samples and (ii) Ni vs Pb (r=0.756), Ni vs Mn (r=0.743) and Se vs Co (r=0.714) in mature milk samples. An inverse correlation between Zn and Se was also found in both types of milk sample; however, it only reached statistical significance for mature milk (r=-0.624).     

In vivo nuclear magnetic resonance studies of glycolytic kinetics in Lactococcus lactis.

The metabolism of glucose by nongrowing cells of L. lactis strain MG5267 was studied under controlled conditions of pH, temperature, and gas atmosphere (anaerobic and aerobic) using a circulating system coupled to nuclear magnetic resonance (NMR) detection that allowed a noninvasive determination of intracellular pools of intermediate metabolites by 13C-NMR with a time resolution of 30 seconds. In addition, intracellular parameters, such as pH, NTP levels, and concentration of inorganic phosphate in the cytoplasm, could be monitored on-line by 31P-NMR with a time resolution of approx. 3 min. The time course for the concentrations of intracellular fructose 1,6-bisphosphate (FBP), 3-phosphoglycerate (3-PGA), and phosphoenolpyruvate (PEP), together with kinetic measurements of substrate consumption and endproducts formation, were used as a basis for the construction of a mechanistic model for glycolysis. In vivo measurements were complemented with determinations of phosphorylated metabolites in perchloric acid extracts. A top-down model was developed by simplifying the metabolism to the resolution allowed by the experimental data collected by in vivo NMR (grouped in seven metabolic steps). This simplified mechanistic model was adjusted to the metabolite concentrations determined by in vivo NMR. The results obtained led to the rationalization of the dynamics of glucose metabolism as being driven largely by ATP surplus. This excess causes accumulation of FBP due to NAD+ limitation, whose regeneration is dependent on downstream pyruvate reduction. The model was capable of predicting qualitative shifts in the metabolism of glucose when changing from anaerobic to aerobic conditions.     

Genetic variability of Triatoma rubrovaria (Reduviidae: Triatominae) from Brazil, Argentina and Uruguay as revealed by two different molecular markers

Randomly amplified polymorphic DNA (RAPD) and nuclear ribosomal DNA sequence analyses were used to assess the genetic population structure of the South American triatomine species Triatomo rubrovario throughout its geographical distribution. To investigate the genetic variability at both intraspecific and intrapopulational levels the RAPD profiles and the nucleotide sequences of the rDNA intergenic spacers, ITS-1 and ITS-2, were analysed and compared. The phenetic analysis based on RAPD profiles show three distinct clusters diverging by similarity coefficients ranging from 0.62 to 0.96. The ITS-1 and ITS-2 sequence variability detected may be considered very high, suggesting reproductive isolation between populations. A total of seven composite haplotypes (CH) were found, among which three are specific for Brazil, other three for Uruguay, and the last one common for the three countries studied. The population studied in Argentina does not represent an independent CH. Sequence analyses proved that the five populations studied are easily differentiable and that there is heterogeneity within each one. True mutations and indels are the responsible of sequence differences between haplotypes and populations, suggesting that divergence processes may presently go on within this species. The large intraspecific variability detected may underlie the known plasticity of T. rubrovaria, making it a potential intradomiciliary invader and consequently an appropriate vector for Chagas disease transmission. Therefore, this triatomine species must be continuously monitored throughout.     

Diversity of understory birds in old stands of native and Eucalyptus plantations

We compared the vegetation structure between old (>70 year) stands of planted diversified native forests and stands of Eucalyptus tereticornis embedded in a mosaic of Eucalyptus stands. We then tested for differences in the abundance, species richness, species composition, and ecological traits (forest dependence, sensitivity to forest fragmentation, and diet) of the understory bird assemblages inhabiting both kinds of stands. We expected differences in the structure of the bird assemblages because of the different origins and management strategies (contrary to native stands, Eucalyptus stands were selectively logged in the past). Three stands of each habitat (native and Eucalyptus) were sampled with mist nets during 11 months. Eucalyptus stands had a denser understory, whereas native plantations had a more developed vertical structure and a greater density of native trees. The abundance distribution of bird species was more homogeneous in Eucalyptus than in native stands. Eucalyptus had slightly higher species richness (36 species) than native stands (32 species). The composition of species and the occurrence of the diet, forest dependence, and sensitivity to forest fragmentation categories were similar between habitats. Some bird species (e.g. Turdus leucomelas), however, were more abundant in one habitat over the other. Old stands of Eucalyptus and planted native forest can harbor a diverse bird community similar in structure but not exactly equivalent for individual bird species. Planting native diversified forests and keeping set‐aside stands of the exotic tree should be viewed as complementary rather than alternative strategies for maintaining bird diversity within plantations.