Chemistry of locked nucleic acids (LNA): Design, synthesis, and bio-physical properties

Preparation of LNA nucleosides requires a number of synthetic steps but very efficient procedures have been developed, as have protocols for synthesis of LNA oligonucleotides on automated DNA synthesizers. In all cases, LNA oligonucleotides have exhibited good aqueous solubility as would be expected from their close structural resemblance to the natural nucleic acids. The universality of LNA mediated high-affinity and specific hybridization has been demonstrated extensively with a large number of duplex forming LNA-oligonucleotides. Most importantly, most of the members of the LNA molecular family have been shown to exert their substantial affinity increase (i) in combination with standard DNA, RNA and contemporary analogues and (ii) whether inserted as single nucleosides in an oligonucleotide or as blocks of contiguous nucleotides, an important point. The works on TFO's is expanding the usefulness of LNA to double strand recognition and it has been demonstrated that LNA it is a promising structure for further base modifications in the pursuit of global sequence specific recognition of DNA.     

Complex patterns of geographic variation in heat tolerance and Hsp70 expression levels in the common frog Rana temporaria

1.

We tested for geographical variation in heat tolerance and Hsp70 expression levels of Rana temporaria tadpoles along a 1500 km long latitudinal gradient in Sweden.     

Nuclease resistant methylphosphonate-DNA/LNA chimeric oligonucleotides

Synthesis of chimeric 9-mer oligonucleotides containing methylphosphonate-linkages and locked nucleic acid (LNA) monomers, their binding affinity towards complementary DNA and RNA, and their 3′-exonucleolytic stability are described. The obtained methylphosphonate-DNA/LNA chimeric oligonucleotides display similarly high RNA affinity and RNA selectivity as a corresponding 9-mer DNA/LNA chimeric oligonucleotide, but much higher resistance towards 3′-exonucleolytic degradation.     

Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension

Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x_L and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x_L over-expressing subline (hVMbcl-x_L) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-x_L cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 ± 0.8% to 17.2 ± 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-x_L-over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-x_L cell cultures compared with control. We conclude that Bcl-x_L and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells.     

Species richness and composition assessment of spiders in a Mediterranean scrubland

Intensive fieldwork has been undertaken in Portugal in order to develop a standardized and optimized sampling protocol for Mediterranean spiders. The present study had the objectives of testing the use of semi-quantitative sampling for obtaining an exhaustive species richness assessment of spiders and testing the effects of day, time of day, collector and sampling method on the collected species richness and composition of a Mediterranean scrubland. The collecting summed 224 samples corresponding to one person-hour of effective fieldwork each. In total, 115 species were captured, of which 110 were recorded inside a delimited one-hectare plot, corresponding to more than 70% of the about 160 estimated species. Although no estimator reached the asymptote, the Michaelis-Menten curve behaviour indicates that the estimated richness should be accurate. Most different sampling approaches (day, time of day, collector and sampling method) were found to influence richness, abundance or composition of the samples to some extent, although sampling method had the strongest influence whereas “collector” showed no effect at all. The results support the idea that the only variables that need to be controlled in similar protocols are the sampling methods and the time of day when each method is executed. We conclude that populations in structurally simple habitats present narrower peaks of adult abundance, which implies higher percentages of juveniles in samples. Finally, results also indicate that habitats with a relatively simple structure like scrublands may require as much sampling effort, in order to reach similar proportions of captured species in relation to the estimated richness, as habitats that are much more complex.     

On reliable discovery of molecular signatures

Background  

Molecular signatures are sets of genes, proteins, genetic variants or other variables that can be used as markers for a particular phenotype. Reliable signature discovery methods could yield valuable insight into cell biology and mechanisms of human disease. However, it is currently not clear how to control error rates such as the false discovery rate (FDR) in signature discovery. Moreover, signatures for cancer gene expression have been shown to be unstable, that is, difficult to replicate in independent studies, casting doubts on their reliability.