Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting.

A total of 189 Candida albicans isolates have been typed by multilocus enzyme electrophoresis. The results obtained confirm the clonal mode of reproduction of C. albicans. The C. albicans populations found in the oropharynx of human immunodeficiency virus (HIV)-infected patients, in the oropharynx of healthy carriers, or in association with invasive candidiasis could not be distinguished. No clone or group of clones could be associated with the appearance of clinical disorders or with a reduced in vitro susceptibility to the antifungal agent fluconazole. Multiple and sequential oral isolates from 24 HIV-infected patients were also typed by restriction enzyme analysis with the enzymes EcoRI and HinfI and by use of the Ca3 repetitive probe. The results obtained by the combination of all three typing methods show that all but one patient each carried a unique major C. albicans clone in their oropharynx. The 21 patients with sequential isolates had the same C. albicans clones in their throats during recurrent oropharyngeal candidiasis episodes, independently of clinical status or of changes of in vitro susceptibility to fluconazole. Finally, several isolates of the same C. albicans clone found simultaneously in the oropharynx of a patient may present different levels of susceptibility to fluconazole.

Identification of HIV integration sites in infected host genomic DNA.

The integration of the Human Immunodeficiency Virus (HIV) genetic information into the host genome is fundamental for its replication and long-term persistence in the host. Isolating and characterizing the integration sites can be useful for obtaining data such as identifying the specific genomic location of integration or understanding the forces dictating HIV integration site selection. The methods outlined in this article describe a highly efficient and precise technique for identifying HIV integration sites in the host genome on a small scale using molecular cloning techniques and standard sequencing or on a massive scale using 454 pyrosequencing.

DNA methylation profiles of human active and inactive X chromosomes.

X-chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell. To characterize epigenetic changes that accompany this process, we measured DNA methylation levels in 45,X patients carrying a single active X chromosome (X(a)), and in normal females, who carry one X(a) and one inactive X (X(i)). Methylated DNA was immunoprecipitated and hybridized to high-density oligonucleotide arrays covering the X chromosome, generating epigenetic profiles of active and inactive X chromosomes. We observed that XCI is accompanied by changes in DNA methylation specifically at CpG islands (CGIs). While the majority of CGIs show increased methylation levels on the X(i), XCI actually results in significant reductions in methylation at 7% of CGIs. Both intra- and inter-genic CGIs undergo epigenetic modification, with the biggest increase in methylation occurring at the promoters of genes silenced by XCI. In contrast, genes escaping XCI generally have low levels of promoter methylation, while genes that show inter-individual variation in silencing show intermediate increases in methylation. Thus, promoter methylation and susceptibility to XCI are correlated. We also observed a global correlation between CGI methylation and the evolutionary age of X-chromosome strata, and that genes escaping XCI show increased methylation within gene bodies. We used our epigenetic map to predict 26 novel genes escaping XCI, and searched for parent-of-origin-specific methylation differences, but found no evidence to support imprinting on the human X chromosome. Our study provides a detailed analysis of the epigenetic profile of active and inactive X chromosomes.

ON THE ROLE OF PERIOD-2 IN THE CIRCADIAN AND HOMEOSTATIC REGULATION OF SLEEP

Humans spend one third of their life sleeping, then we could raise the basic question: Why do we sleep? Despite the fact that we still don't fully understand its function, we made much progress in understanding at different levels how sleep is regulated. One model suggests that sleep is regulated by two processes: a homeostatic process that tracks the need for sleep and by a circadian rhythm that determines the preferred time-of-day sleep occurs. At the molecular level circadian rhythms are a property of interlocking transcriptional regula-tors referred to as clock genes. The heterodimeric transcription factors BMAL1::CLOCK/NPAS2 drive the transcription of many target genes including the clock genes Cryptochome1 (Cry1), Cry2, Period1 (Per1), and Per2. The encoded CRY/PER proteins are transcriptional inhibitors of BMAL1::CLOCK/NPAS2 thereby providing negative feedback to their own transcription. These genes seem, however, also involved in sleep homeostasis because the brain expression of clock genes, es-pecially that of Per2, increase as a function of time-spent-awake and because mice lacking clock genes display altered sleep homeostasis. The aim of first part of my doctoral work has been to advance our understanding the link that exists between sleep homeostasis and circadian rhythms investigating a possible mechanism by which sleep deprivation could alter clock gene expression by quantifying DNA-binding of the core-clock genes BMAL1, CLOCK and NPAS2 to their target chromatin loci including the E-box enhancers of the Per2 promoter. We made use of chromatin immunoprecipitation (ChIP) and quantitative poly-merase chain reaction (qPCR) to show that DNA-binding of CLOCK and BMAL1 to their target genes changes as a function of time-of-day in both liver and cerebral cortex. We then performed a 6h sleep deprivation (SD) and observed a significant decrease in DNA-binding of CLOCK and BMAL1 to Dbp. This is consistent with a decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was similarly decreased following SD. However, SD has been previously shown to in-crease Per2 expression in the cortex which seems paradoxical. Our results demonstrate that sleep-wake history can affect the molecular clock machinery directly at the level of the chromatin thereby altering the cortical expression of Dbp and Per2, and likely other targets. However, the precise dy-namic relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive. The second aim of my doctoral work has been to perform an in depth characterization of cir-cadian rhythmicity, sleep architecture, analyze the response to SD in full null-Per2 knock-out (Per2-/-) mice, and Per1-/- mice, as well as their double knock-out offspring (Per1,2-/-) and littermate wildtype (Wt) mice. The techniques used include locomotor activity recording by passive infrared (PIR) sen-sors, EEG/EMG surgery, recording, and analysis, and cerebral cortex extraction and quantification of mRNA levels by qPCR. Under standard LD12:12 conditions, we found that wakefulness onset, as well as the time courses of clock gene expression in the brain and corticosterone plasma levels were ad-vanced by about 2h in Per2-/- mice compared to Wt mice. When released under constant dark condi-tions almost all Per2-/- mice (97%) became arrhythmic immediately. From these observations, we conclude that while Per2-/- mice seem to be able to anticipate dark onset, this does not result from a self-sustained circadian clock. Our results suggest instead that the earlier onset of activity results from a labile, not-self sustained 22h rhythm linked to light onset suggesting the existence of a light-driven rhythm. Analyses of sleep under LD12:12 conditions revealed that in both Per2-/- and Per1,2-/- mice the same sleep phenotypes are observed compared to Wt mice: increased NREM sleep frag-mentation and inability to adequately compensate the loss of NREM sleep. That suggests a possible role of PER2 in sleep consolidation and recovery.

DNA mismatch repair gene MSH6 implicated in determining age at natural menopause.

The length of female reproductive lifespan is associated with multiple adverse outcomes, including breast cancer, cardiovascular disease and infertility. The biological processes that govern the timing of the beginning and end of reproductive life are not well understood. Genetic variants are known to contribute to ∼50% of the variation in both age at menarche and menopause, but to date the known genes explain <15% of the genetic component. We have used genome-wide association in a bivariate meta-analysis of both traits to identify genes involved in determining reproductive lifespan. We observed significant genetic correlation between the two traits using genome-wide complex trait analysis. However, we found no robust statistical evidence for individual variants with an effect on both traits. A novel association with age at menopause was detected for a variant rs1800932 in the mismatch repair gene MSH6 (P = 1.9 × 10(-9)), which was also associated with altered expression levels of MSH6 mRNA in multiple tissues. This study contributes to the growing evidence that DNA repair processes play a key role in ovarian ageing and could be an important therapeutic target for infertility.

Regional specificity of MRI contrast parameter changes in normal ageing revealed by voxel-based quantification (VBQ).

Normal ageing is associated with characteristic changes in brain microstructure. Although in vivo neuroimaging captures spatial and temporal patterns of age-related changes of anatomy at the macroscopic scale, our knowledge of the underlying (patho)physiological processes at cellular and molecular levels is still limited. The aim of this study is to explore brain tissue properties in normal ageing using quantitative magnetic resonance imaging (MRI) alongside conventional morphological assessment. Using a whole-brain approach in a cohort of 26 adults, aged 18-85years, we performed voxel-based morphometric (VBM) analysis and voxel-based quantification (VBQ) of diffusion tensor, magnetization transfer (MT), R1, and R2* relaxation parameters. We found age-related reductions in cortical and subcortical grey matter volume paralleled by changes in fractional anisotropy (FA), mean diffusivity (MD), MT and R2*. The latter were regionally specific depending on their differential sensitivity to microscopic tissue properties. VBQ of white matter revealed distinct anatomical patterns of age-related change in microstructure. Widespread and profound reduction in MT contrasted with local FA decreases paralleled by MD increases. R1 reductions and R2* increases were observed to a smaller extent in overlapping occipito-parietal white matter regions. We interpret our findings, based on current biophysical models, as a fingerprint of age-dependent brain atrophy and underlying microstructural changes in myelin, iron deposits and water. The VBQ approach we present allows for systematic unbiased exploration of the interaction between imaging parameters and extends current methods for detection of neurodegenerative processes in the brain. The demonstrated parameter-specific distribution patterns offer insights into age-related brain structure changes in vivo and provide essential baseline data for studying disease against a background of healthy ageing.

Respiratory chain dysfunction associated with multiple mitochondrial DNA deletions in antiretroviral therapy-related lipodystrophy.

Highly-active antiretroviral therapy (HAART) can induce a characteristic lipodystrophy syndrome characterized by peripheral fat wasting and central adiposity, usually associated with hyperlipidaemia and insulin resistance [1,2]. Indirect data have led some authors to propose that mitochondrial dysfunction could play a role in this syndrome [3,4].To date, as recently outlined by Kakuda et al. [5] in this journal, HIV-infected patients developing lipodystrophy have not been studied for mitochondrial changes or respiratory chain capacity...

Caracterização do dna ribossômico do isolado de Scleroderma UFSMSc1 de Eucalyptus grandis W. Hill ex-maiden

O ácido desoxiribunocléico ribossomal (rDNA) é utilizado como uma ferramenta importante para caracterizar o polimorfismo entre os fungos. Existem muitas cópias de rDNA as quais são arranjadas por espaços não codificados. Essas cópias são altamente conservadas entre espécies de fungos. O objetivo deste trabalho foi estudar a região do Espaço Interno Transcrito (ITS) e analisar as diferenças no polimorfismo da seqüência dessa região no fungo Scleroderma UFSMSc1 com seqüências dos isolados de Scleroderma e Pisolithus do banco de dados GenBank. O DNA do isolado de Scleroderma UFSMSc1 foi extraído por meio da solução de extração à base de CTAB. A partir do DNA, foram feitas reações de PCR com os oligonucleotídeos iniciadores universais ITS1 e ITS4, cujo produto amplificado foi purificado e seqüenciado. A região do ITS do fungo mostrou uma banda simples de aproximadamente 650 pares de base. Na análise da seqüência dessa região em comparação com algumas depositadas no GenBank, observou-se a formação de agrupamento com espécies de Scleroderma. Os resultados mostraram que essa técnica favorece a identificação de espécies de Scleroderma, visto que tais fungos são difíceis de ser identificados apenas por seus caracteres morfológicos.

Plant DNA barcodes and the influence of gene flow.

Success of species assignment using DNA barcodes has been shown to vary among plant lineages because of a wide range of different factors. In this study, we confirm the theoretical prediction that gene flow influences species assignment with simulations and a literature survey. We show that the genome experiencing the highest gene flow is, in the majority of the cases, the best suited for species delimitation. Our results clearly suggest that, for most angiosperm groups, plastid markers will not be the most appropriate for use as DNA barcodes. We therefore advocate shifting the focus from plastid to nuclear markers to achieve an overall higher success using DNA barcodes.

Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc.

The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. Here, we asked whether the Dpp signaling activity scales, i.e. expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, we found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. We also found that the target gene domains are not defined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone, an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth.

Simulations of action of DNA topoisomerases to investigate boundaries and shapes of spaces of knots.

The configuration space available to randomly cyclized polymers is divided into subspaces accessible to individual knot types. A phantom chain utilized in numerical simulations of polymers can explore all subspaces, whereas a real closed chain forming a figure-of-eight knot, for example, is confined to a subspace corresponding to this knot type only. One can conceptually compare the assembly of configuration spaces of various knot types to a complex foam where individual cells delimit the configuration space available to a given knot type. Neighboring cells in the foam harbor knots that can be converted into each other by just one intersegmental passage. Such a segment-segment passage occurring at the level of knotted configurations corresponds to a passage through the interface between neighboring cells in the foamy knot space. Using a DNA topoisomerase-inspired simulation approach we characterize here the effective interface area between neighboring knot spaces as well as the surface-to-volume ratio of individual knot spaces. These results provide a reference system required for better understanding mechanisms of action of various DNA topoisomerases.

A common ancestor DNA motif for invertebrate and vertebrate hormone response elements.

The ecdysone-responsive DNA sequence of the Drosophila hsp27 gene promoter contains four direct and inverted repeats reminiscent of those that compose the vertebrate palindromic estrogen response element (ERE) and the thyroid hormone/retinoic acid response element (TRE/RRE). Interestingly, a 3 bp substitution in the wild-type Hsp27 ecdysone response element (EcdRE) increases both its similarity with the vertebrate ERE and TRE/RRE and its capacity to confer ecdysone responsiveness to a heterologous promoter. Remarkably, increasing the spacing between the inverted repeats of this strong EcdRE by two nucleotides converts it into an ERE. Inversely, decreasing the spacing between the two inverted repeats of the vertebrate consensus palindromic ERE, from three to one nucleotide, converts it into a functional EcdRE. Thus, the only difference between an invertebrate EcdRE and a vertebrate palindromic ERE or TRE/RRE is in the spacing between the conserved inverted repeated motifs forming these palindromic HREs. The finding that the sequence motif 5'-GGTCA-3' present in the vertebrate ERE and TRE/RRE is also a functionally important characteristic of an invertebrate HRE, suggests that a common ancestor regulatory DNA sequence gave rise to all HREs known so far. We discuss the possibility that this progenitor motif is the GGTCA sequence.

Mitochondrial DNA variation along an altitudinal gradient in the greater white-toothed shrew, Crocidura russula.

The distribution of mitochondrial control region-sequence polymorphism was investigated in 15 populations of Crocidura russula along an altitudinal gradient in western Switzerland. High-altitude populations are smaller, sparser and appear to undergo frequent bottlenecks. Accordingly, they showed a loss of rare haplotypes, but unexpectedly, were less differentiated than lowland populations. Furthermore, the major haplotypes segregated significantly with altitude. The results were inconsistent with a simple model of drift and dispersal. They suggested instead a role for historical patterns of colonization, or, alternatively, present-day selective forces acting on one of the mitochondrial genes involved in metabolic pathways.

Quantification of human adenoviruses in European recreational waters

The presence of human adenoviruses in recreational water might cause disease in the population upon exposure. Human adenoviruses detected by PCR could also serve as indicators of the virological water quality. In order to assess the applicability of human adenoviruses to the evaluation of the faecal contamination in European bathing waters, a real-time quantitative PCR assay was developed for the quantification of human adenoviruses in 132 samples collected from 24 different recreational marine and freshwater sites in nine European countries.Selected samples presenting positive nested-PCR results for human adenoviruses were analyzed using quantitative PCR and 80 samples from a total of 132 produced quantitative results with mean values of 3.2x102 10 per 100 ml of water, human adenovirus 41 being the most prevalent serotype. Human adenoviruses were quantified in samples from all 15 surveillance laboratories. Statistical analysis showed no homogeneous linear relation between humanadenoviruses and E. coli, intestinal enterococci or somatic coliphages concentrations in the tested samples when considering all the data together. Significant correlations between human adenoviruses and at least one of the other indicators were observed only when data from individual Laboratories were considered. The quantification of human adenoviruses may provide complementary information in relation to the use of bacterial standards in the control of water quality in bathing water.