Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.

A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

Genomewide association study using a high-density single nucleotide polymorphism array and case-control design identifies a novel essential hypertension susceptibility locus in the promoter region of endothelial NO synthase.

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37-1.73]; combined P=2.58 · 10(-13)). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25-1.44; P=1.032 · 10(-14)). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16-3.66) for systolic and 1.40 (95% CI: 0.25-2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.

Phylogeny and revision of the bee genus Rhinocorynura Schrottky (Hymenoptera, Apidae, Augochlorini), with comments on its female cephalic polymorphism

Phylogeny and revision of the bee genus Rhinocorynura Schrottky (Hymenoptera, Apidae, Augochlorini), with comments on its female cephalic polymorphism. A taxonomic revision and a phylogeny for the species of Rhinocorynura are provided. Six species are recognized: R. briseis, R. crotonis, R. inflaticeps and R. vernoniae stat. nov., the latter removed from synonymy with R. inflaticeps, in addition to two newly described species, R. brunnea sp. nov. and R. viridis sp. nov. Lectotypes for Halictus crotonis Ducke, 1906 and Halictus inflaticeps Ducke, 1906 are hereby designated. Another available name included in Rhinocorynura, Corynuropsis ashmeadi Schrottky, 1909, is removed from the genus and treated as species inquerenda in Augochlorini. Rhinocorynura is monophyletic in the phylogenetic analysis and the following relationships were found among its species: (R. crotonis (R. briseis ((R. brunnea sp. nov. + R. viridis sp. nov.) (R. inflaticeps + R. vernoniae)))). Biogeographic relationships within the genus and comparisons with related taxa are presented. Females of all species exhibit pronounced variation in body size, in two of them, R. inflaticeps and R. vernoniae, with structural modifications possibly linked to division of labor. Identification key and illustrations for the species are provided.

Developmental biology, polymorphism and ecological aspects of Stiretrus decemguttatus (Hemiptera, Pentatomidae), an important predator of cassidine beetles

Developmental biology, polymorphism and ecological aspects of Stiretrus decemguttatus (Hemiptera, Pentatomidae), an important predator of cassidine beetles. Stiretrus decemguttatus is an important predator of two species of cassidine beetles, Botanochara sedecimpustulata (Fabricius, 1781) and Zatrephina lineata (Fabricius, 1787) (Coleoptera, Cassidinae), on the Marajó Island, Brazil. It attacks individuals in all development stages, but preys preferentially on late-instar larvae. Its life cycle in the laboratory was 43.70 ± 1.09 days, with an egg incubation period of six days and duration from nymph and adult stages of 16.31 ± 0.11 and 22.10 ± 1.67 days, respectively. The duration of one generation (T) was 12.65 days and the intrinsic population growth rate (r) 0.25. These data reveal the adjustment of the life cycle of S. decemgutattus with those of the two preys, but suggest greater impact on Z. lineata. However, no preference over cassidine species was shown in the laboratory. Up to 17 different color patterns can be found in adults of S. decemguttatus, based on combinations of three basic sets of color markings. Some of them resemble the markings of chrysomelids associated with Ipomoea asarifolia (Convolvulaceae) and are possibly a mimetic ring. Three color patterns were identified in nymphs, none of which was associated with any specific adult color pattern.

Caste polymorphism of apterous line of the Neotropical termite Nasutitermes corniger (Motschulsky) (Isoptera, Termitidae)

Termite societies are structured by individuals that can be grouped into castes and instars. The development of these instars in most species occurs in irregular patterns and sometimes is distinguished subcastes in physical systems that originate polymorphic soldiers and workers. In this study, we characterized the morphological diversity of castes of apterous in Nasutitermes corniger. We collected four colonies of N. corniger, one every three months between May 2011 and February 2012. Individuals of the nest were separated into groups: larval stages, workers and presoldiers and soldiers. A morphometric analysis was performed on individuals from each group based on head width, metatibia, antenna, and thorax length. The data were submitted to discriminant analysis to confirm different morphological types inside these groups. The apterous line of N. corniger is composed of one first larval instar and two second larval instar. The workers caste has two lines of development with four instars in a larger line and three instars in a lower line. Two morphological types were identified in presoldiers and soldiers. The pattern of castes was similar to other species of the genus, in which bifurcation into two lines of workers, one smaller and one larger occurs after the first molt.

Genetic structure of populations of Pissodes castaneus (De Geer) (Coleoptera, Curculionidae) using amplified fragment length polymorphism

Genetic structure of populations of Pissodes castaneus (De Geer) (Coleoptera, Curculionidae) using amplified fragment length polymorphism. The objective of this study was to determine the genetic structure of populations of Pissodes castaneus from different areas and on different species of Pinus using the PCR-AFLP technique. Twenty samples were analyzed, representing 19 populations from Brazil and one from Florence, Italy, which is the region of origin of P. castaneus. The four combinations of primers generated a total of 367 fragments of DNA, and 100% of polymorphic loci, indicating high degree of molecular polymorphism. The dendrogram did not reveal trends for grouping the populations in relation to origin. The low genetic similarity (0.11 between the most distant groups) and genetic distances of 0.13 and 0.44 for 10 out of the 20 samples may indicate several founding events or multiple introductions of heterogeneous strains into Brazil. The allelic fixation index (Fst) was 0.3851, considered high, and the number of migrants (Nm) was 0.3991, indicating low gene flow among populations. The highest genetic distances were between the population from Irani, SC and Cambará do Sul, RS and Bituruna, PR, indicating an independent founding event or a particular allelic fixation in the former location. The high genetic diversity among populations points out that the populations are genetically heterogeneous with a diverse gene pool in the surveyed areas, what makes them to respond differently to control measures.

Electron microscopy of RecA-DNA complexes: Two different states, their functional significance and relation to the solved crystal structure

Seven different electron microscopy techniques habe been employed to study the RecA protein of E. coli. This review provides a summary of the conclusions that have been drawn from these studies, and attempts to relate these observations to models for the role of RecA protein in homologous recombination.

Genetic and environmental effects on the covariation betweencolour polymorphism and a life history trait

Variation in coloration with a strong underlying genetic basis is frequently found within animal populations but little is known about its function. Covariation between colour polymorphism and life-history traits can arise because morphs perform differently among environments or because they possess alternative alleles coding for key life-history traits. To test these two hypotheses, we studied a population of tawny owls Strix aluco, a bird displaying red, brown and grey morphs. We assessed the colour morph of breeding females, swapped eggs or hatchlings between pairs of nests, and examined how body condition in 3-week-old nestlings covaries with coloration of foster and genetic mothers. Redder foster and genetic mothers produced young in better condition. Because in two other years we observed that greyish females produced offspring in better condition than those of red females, the present study suggests that colour polymorphism signals genetic and phenotypic adaptations to cope with a fluctuating environment.

Crystal packing modifies ligand binding affinity: the case of aldose reductase.

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.

Quantitative analysis of crystal/grain sizes and their distributions in 2D and 3D

We review methods to estimate the average crystal (grain) size and the crystal (grain) size distribution in solid rocks. Average grain sizes often provide the base for stress estimates or rheological calculations requiring the quantification of grain sizes in a rock's microstructure. The primary data for grain size data are either 1D (i.e. line intercept methods), 2D (area analysis) or 3D (e.g., computed tomography, serial sectioning). These data have been used for different data treatments over the years, whereas several studies assume a certain probability function (e.g., logarithm, square root) to calculate statistical parameters as the mean, median, mode or the skewness of a crystal size distribution. The finally calculated average grain sizes have to be compatible between the different grain size estimation approaches in order to be properly applied, for example, in paleo-piezometers or grain size sensitive flow laws. Such compatibility is tested for different data treatments using one- and two-dimensional measurements. We propose an empirical conversion matrix for different datasets. These conversion factors provide the option to make different datasets compatible with each other, although the primary calculations were obtained in different ways. In order to present an average grain size, we propose to use the area-weighted and volume-weighted mean in the case of unimodal grain size distributions, respectively, for 2D and 3D measurements. The shape of the crystal size distribution is important for studies of nucleation and growth of minerals. The shape of the crystal size distribution of garnet populations is compared between different 2D and 3D measurements, which are serial sectioning and computed tomography. The comparison of different direct measured 3D data; stereological data and direct presented 20 data show the problems of the quality of the smallest grain sizes and the overestimation of small grain sizes in stereological tools, depending on the type of CSD. (C) 2011 Published by Elsevier Ltd.

Simultaneous identification of multiple mammalian species from mixed forensic samples based on mtDNA control region length polymorphism

Molecular species identification in mixed or contaminated biological material has always been problematic. We developed a simple and accurate method for mammal DNA identification in mixtures, based on interspecific mitochondrial DNA control region length polymorphism. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and amplification step, and is not based on a pre-defined panel of species. This protocol has been routinely employed by our laboratory for species identification in dozens of human and animal forensic caseworks. Six representative forensic caseworks involving the specific identification of mixed animal samples are reported in this paper, in order to demonstrate the applicability and usefulness of the method.

Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.

According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions.

Crystal structure of yeast peroxisomal multifunctional enzyme: structural basis for substrate specificity of (3R)-hydroxyacyl-CoA dehydrogenase units.

(3R)-hydroxyacyl-CoA dehydrogenase is part of multifunctional enzyme type 2 (MFE-2) of peroxisomal fatty acid beta-oxidation. The MFE-2 protein from yeasts contains in the same polypeptide chain two dehydrogenases (A and B), which possess difference in substrate specificity. The crystal structure of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase AB heterodimer, consisting of dehydrogenase A and B, determined at the resolution of 2.2A, shows overall similarity with the prototypic counterpart from rat, but also important differences that explain the substrate specificity differences observed. Docking studies suggest that dehydrogenase A binds the hydrophobic fatty acyl chain of a medium-chain-length ((3R)-OH-C10) substrate as bent into the binding pocket, whereas the short-chain substrates are dislocated by two mechanisms: (i) a short-chain-length 3-hydroxyacyl group ((3R)-OH-C4) does not reach the hydrophobic contacts needed for anchoring the substrate into the active site; and (ii) Leu44 in the loop above the NAD(+) cofactor attracts short-chain-length substrates away from the active site. Dehydrogenase B, which can use a (3R)-OH-C4 substrate, has a more shallow binding pocket and the substrate is correctly placed for catalysis. Based on the current structure, and together with the structure of the 2-enoyl-CoA hydratase 2 unit of yeast MFE-2 it becomes obvious that in yeast and mammalian MFE-2s, despite basically identical functional domains, the assembly of these domains into a mature, dimeric multifunctional enzyme is very different.