RNA secondary structure, an important bioinformatics tool to enhance multiple sequence alignment: a case study (Sordariomycetes, Fungi)

In a case study of fungi of the class Sordariomycetes, we evaluated the effect of multiple sequence alignment (MSA) on the reliability of the phylogenetic trees, topology and confidence of major phylogenetic clades. We compared two main approaches for constructing MSA based on (1) the knowledge of the secondary (2D) structure of ribosomal RNA (rRNA) genes, and (2) automatic construction of MSA by four alignment programs characterized by different algorithms and evaluation methods, CLUSTAL, MAFFT, MUSCLE, and SAM. In the primary fungal sequences of the two functional rRNA genes, the nuclear small and large ribosomal subunits (18 S and 28 S), we identified four and six, respectively, highly variable regions, which correspond mainly to hairpin loops in the 2D structure. These loops are often positioned in expansion segments, which are missing or are not completely developed in the Archaeal and Eubacterial kingdoms. Proper sorting of these sites was a key for constructing an accurate MSA. We utilized DNA sequences from 28 S as an example for one-gene analysis. Five different MSAs were created and analyzed with maximum parsimony and maximum likelihood methods. The phylogenies inferred from the alignments improved with 2D structure with identified homologous segments, and those constructed using the MAFFT alignment program, with all highly variable regions included, provided the most reliable phylograms with higher bootstrap support for the majority of clades. We illustrate and provide examples demonstrating that re-evaluating ambiguous positions in the consensus sequences using 2D structure and covariance is a promising means in order to improve the quality and reliability of sequence alignments.     

Novel primate-specific genes, RMEL 1, 2 and 3, with highly restricted expression in melanoma, assessed by new data mining tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.     

Physiotherapy as an immunoactive therapy? A pilot study

OBJECTIVES: The aim of this study was to confirm the immunoregulatory and anti-inflammatory changes in the immunologic profile after two months of the facilitation physiotherapy in patients with multiple sclerosis; and to determine whether the changes in the immunologic profile correlate with the changes in dehydroepiandrosterone, the brain microstructure and clinical functions. Design & Setting: A group of 12 patients with multiple sclerosis was examined twice: at the beginning and 2 months later after the patients had undergone the facilitation therapy. Standardized tests evaluating chosen clinical functions (balance, righting, equilibrium and protective reactions, tremor, dysdiadochokinesis, dysmetry, fine hand function and walking), immune parameters (parameters of the humoral and cellular immunity), dehydroepiandrosterone and diffusion tensor imaging (the fractional anisotropy, mean diffusivity) were measured. The patients underwent the facilitation physiotherapy in two sessions lasting two hours each week for two months. Results: All clinical and diffusion tensor imaging parameters significantly improved following the therapy. Without the correction for multiple comparisons, there were significant changes in the IgG, IgG1 subclasses, in the numbers of Neutrophils and Lymphocytes, the T cells (CD3+) absolute number, the T cytotoxic subpopulation (CD3+CD8+) absolute number, B cells (CD19+) and the Natural killer cells. In addition, there was a significant correlation between the changes in the clinical functions and the changes in IgG1 (r=0.67), and between the changes in the mean diffusivity and the changes in CD3+CD8+ absolute (r=-0.61). The changes in the immune parameters and the mentioned correlations were not significant in view of the number of comparisons and thus necessitate further validation. No changes in the dehydroepiandrosterone concentration after the therapy were confirmed. Conclusion: The study suggests new possibilities of physiotherapy to influence the psycho-neuro-endocrine-immune response in patients with multiple sclerosis.     

Comparison of different kinds of probes used for analysis of variant telomeric sequences

In this work we aimed to compare and critically evaluate results obtained by different types of probes used for hybridisation to detect variant telomeric sequences with respect to their reliability and information value. Using slot-blot hybridisation we investigated three types of probes (oligonucleotides, cloned fragments and concatenated probes) under various conditions of hybridisation and washing. The concatenated probes exhibited the highest specificity although all three types are suitable for hybridisation of telomeric sequences under appropriate experimental conditions. We demonstrate how understanding generated from these data enables interpretation of hybridisation patterns of oligonucleotide probes to genomic DNAs.     

Cell surface properties as factors involved in Proteus vulgaris adhesion to stainless steel under starvation conditions

The purpose of these investigations was to evaluate the influence of limited nutrient availability in the culture medium on Proteus vulgaris biofilm formation on surfaces of stainless steel. The relationship between the P. vulgaris adhesion to the abiotic surfaces, the cellular ATP levels, cell surface hydrophobicity and changes in the profiles of extracellular proteins and lipopolysaccharides was examined. In all experimental variants the starvation conditions induced the bacterial cells to adhere to the surfaces of stainless steel. Higher ATP content and lower cell surface hydrophobicity of P. vulgaris cells was observed upon nutrient-limited conditions. Under starvation conditions a reduction in the levels of extracellular low molecular weight proteins was noticed. High molecular weight proteins formed the conditioning layer on stainless steel plates, making the bacteria adhesion process more favorable. The production of low molecular weight carbohydrates promoted more advanced stages of P. vulgaris biofilm formation process on the surfaces of stainless steel upon starvation.     

Sperm divergence in a passerine contact zone: Indication of reinforcement at the gametic level

Postcopulatory sexual selection may promote evolutionary diversification in sperm form, but the contribution of between‐species divergence in sperm morphology to the origin of reproductive isolation and speciation remains little understood. To assess the possible role of sperm diversification in reproductive isolation, we studied sperm morphology in two closely related bird species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia), that hybridize in a secondary contact zone spanning Central and Eastern Europe. We found: (1) striking divergence between the species in total sperm length, accompanied by a difference in the length of the mitochondrial sperm component; (2) greater divergence between species in sperm morphology in sympatry than in allopatry, with evidence for character displacement in sperm head length detected in L. megarhynchos; (3) interspecific hybrids showing sperm with a length intermediate between the parental species, but no evidence for decreased sperm quality (the proportion of abnormal spermatozoa in ejaculates). Our results demonstrate that divergence in sperm morphology between the two nightingale species does not result in intrinsic postzygotic isolation, but may contribute to postcopulatory prezygotic isolation. This isolation could be strengthened in sympatry by reinforcement.