PCR ITS-RFLP: A useful method for identifying filamentous fungi isolates on grapes.

Restriction digestion analysis of the ITS products was tested as an easy method to identify isolates of filamentous fungi on grapes. Endonucleases SduI, HinfI, MseI, HaeIII were used. Endonucleases BfmI, Cfr9I, Hpy188I, MaeII or PspGI were used as necessary to complete discrimination. The 43 species studied generated 42 different composite profiles. Only the species P. thomii and P. glabrum gave the same composite profile. 96.3% strains tested could be differentiated to the species level with only four enzymes. Hundred ninety nine strains of filamentous fungi were isolated from various vineyards in Burgundy and identified by this method. Penicillium (58.5%) was the genus the most frequently isolated and no strains of the genus Aspergillus was isolated. P. spinolusum was the most isolated species of Penicillium (22.70%). The species C. cladiosporioides, B. cinerea, E. nigrum, A. alternata, T. koningiopsis, P. diplodiella, C. herbarum, A. alternatum, T. cucumeris and F. oxysporum were also isolated. This technique is a rapid and reliable method appropriate for routine identification of filamentous fungi. This can be used to screen large numbers of isolates from various environments in a short time. This is the first exhaustive study of fungal diversity at species level in vineyard.

Assessment of survival of Listeria monocytogenes, Salmonella Infantis and Enterococcus faecalis artificially inoculated into experimental waste or compost.

AIMS: To evaluate survival of pathogenic strains, Listeria monocytogenes and Salmonella Infantis and a sanitation indicator Enterococcus faecalis in composts at different stages of the composting process and during storage. METHODS AND RESULTS: The studied pathogenic and indicator strains, originally isolated from compost, were inoculated into compost samples from the various stages of the composting process. During incubation, indigenous microflora diversity was monitored with DGGE analysis. After 90 days of incubation, strain survival was observed in compost sampled before the beginning of the cooling phase, and DGGE analysis demonstrated an increase of microbial diversity up to the cooling phase. However, inoculated strains were not detected in composts after 30, 60 or 90 days of incubation in compost sampled after the start of the cooling phase. Microbial diversity also became stable, and DGGE profiles reached a maximum number of bands at this stage. CONCLUSIONS: Strain survival was not observed in stabilized composts. The cooling phase seems to be the turning point for pathogen survival and at this stage the indigenous microflora appeared to play a significant role in suppression. SIGNIFICANCE AND IMPACT OF THE STUDY: The importance of indigenous microflora in the survival of pathogens in four different composts was demonstrated. Stabilized composts were recommended for spreading on land.

Dynamic molecular combing: stretching the whole human genome for high-resolution studies.

DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing. The precise measurement of hybridized DNA probes was achieved directly without requiring normalization. This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA. It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig, and of microdeletions in the tuberous sclerosis 2 gene on patients' DNA. The simplicity, reproducibility, and precision of this approach makes it a powerful tool for a variety of genomic studies.

[Spermiogenesis: histone acetylation triggers male genome reprogramming]. / Spermiogenèse: l'acétylation des histones déclenche la reprogrammation du génome mâle.

During their post-meiotic maturation, male germ cells undergo an extensive reorganization of their genome, during which histones become globally hyperacetylated, are then removed and progressively replaced by transition proteins and finally by protamines. The latter are known to tightly associate with DNA in the mature sperm cell. Although this is a highly conserved and fundamental biological process, which is a necessary prerequisite for the transmission of the male genome to the next generation, its molecular basis remains mostly unknown. We have identified several key factors involved in this process, and their detailed functional study has enabled us to propose the first model describing molecular mechanisms involved in post-meiotic male genome reprogramming. One of them, Bromodomain Testis Specific (BRDT), has been the focus of particular attention since it possesses the unique ability to specifically induce a dramatic compaction of acetylated chromatin. Interestingly, a mutation was found homozygous in infertile men which, according to our structural and functional studies, disrupts the function of the protein. A combination of molecular structural and genetic approaches has led to a comprehensive understanding of new major actors involved in the male genome reprogramming and transmission.

Influence of organic amendments on diuron leaching through an acidic and a calcareous vineyard soil using undisturbed lysimeters.

The influence of different organic amendments on diuron leaching was studied through undisturbed vineyard soil columns. Two composts (A and D), the second at two stages of maturity, and two soils (VR and Bj) were sampled. After 1 year, the amount of residues (diuron+metabolites) in the leachates of the VR soil (0.19-0.71%) was lower than in the Bj soil (4.27-8.23%), which could be explained by stronger diuron adsorption on VR. An increase in the amount of diuron leached through the amended soil columns, compared to the blank, was observed for the Bj soil only. This result may be explained by the formation of mobile complexes between diuron and water-extractable organic matter (WEOM) through the Bj soil, or by competition between diuron and WEOM for the adsorption sites in the soil. For both soils, the nature of the composts and their degree of maturity did not significantly influence diuron leaching.

Identity crisis in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) shares many hallmarks with cancer. Cancer cells acquire their hallmarks by a pathological Darwinian evolution process built on the so-called cancer cell "identity crisis." Here we demonstrate that PAH shares the most striking features of the cancer identity crisis: the ectopic expression of normally silent tissue-specific genes.

Active maintenance of mHDA2/mHDAC6 histone-deacetylase in the cytoplasm.

The intracellular localization, and thereby the function, of a number of key regulator proteins tagged with a short leucine-rich motif (the nuclear export signal or NES) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus [1]. A common characteristic of these regulators is their transient action in the nucleus during either a specific phase of the cell cycle or in response to specific signals [1]. Here, we show that a particular member of the class II histone-deacetylases mHDA2/mHDAC6 [2] belongs to this family of cellular regulators that are present predominantly in the cytoplasm, but are also capable of shuttling between the nucleus and the cytoplasm. A very potent NES present at the amino terminus of mHDAC6 was found to play an essential role in this shuttling process. The sub-cellular localization of mHDAC6 appeared to be controlled by specific signals, since the arrest of cell proliferation was found to be associated with the translocation of a fraction of the protein into the nucleus. Data presented here suggest that mHDAC6 might be the first member of a functionally distinct class of deacetylases, responsible for activities not shared by other known histone deacetylases.

Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways.

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

Histone acetylation-mediated chromatin compaction during mouse spermatogenesis.

One of the most dramatic chromatin remodelling events takes place during mammalian spermatogenesis involving massive incorporation of somatic and testis-specific histone variants, as well as generalized histone modifications before their replacement by new DNA packaging proteins. Our data suggest that the induced histone acetylation occurring after meiosis may direct the first steps of genome compaction. Indeed, a double bromodomain-containing protein expressed in postmeiotic cells, Brdt, shows the extraordinary capacity to specifically condense acetylated chromatin in vivo and in vitro. In elongating spermatids, Brdt widely co-localizes with acetylated histones before accumulating in condensed chromatin domains. These domains preferentially maintain their acetylation status until late spermatogenesis. Based on these data, we propose that Brdt mediates a general histone acetylation-induced chromatin compaction and also maintains differential acetylation of specific regions, and is therefore involved in organizing the spermatozoon's genome.

[Epigenetics of the sperm cell]. / Epigénétique du spermatozoïde.

In addition to genetic information, the spermatozoon carries another type of information, named epigenetic, which is not associated with variations of the DNA sequence. In somatic cells, it is now generally admitted that epigenetic information is not only regulated by DNA methylation but also involves modifications of the genome structure, or epigenome. During male germ cell maturation, the epigenome is globally re-organized, since most histones, which are associated to DNA in somatic cells, are removed and replaced by sperm specific nuclear proteins, the protamines, responsible for the tight compaction of the sperm DNA. However, a small proportion of histones, and probably other proteins, are retained within the sperm nucleus, and the structure of the sperm genome is actually heterogeneous. This heterogeneity of the sperm epigenome could support an epigenetic information, transmitted to the embryo, which could be crucial for its development. Although it is nowadays possible to appreciate the global structure of the sperm genome, the precise constitution of the sperm epigenome remains unknown. In particular, very recent data suggest that specific regions of the genome could be associated with particular proteins and define specific structures. This structural partitioning of the sperm genome could convey important epigenetic information, crucial for the embryo development.

Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases.

Here we report a detailed analysis of waves of histone acetylation that occurs throughout spermatogenesis in mouse. Our data showed that spermatogonia and preleptotene spermatocytes contained acetylated core histones H2A, H2B and H4, whereas no acetylated histones were observed throughout meiosis in leptotene or pachytene spermatocytes. Histones remained unacetylated in most round spermatids. Acetylated forms of H2A and H2B, H3 and H4 reappeared in step 9 to 11 elongating spermatids, and disappeared later in condensing spermatids. The spatial distribution pattern of acetylated H4 within the spermatids nuclei, analyzed in 3D by immunofluorescence combined with confocal microscopy, showed a spatial sequence of events tightly associated with chromatin condensation. In order to gain an insight into mechanisms controlling histone hyperacetylation during spermiogenesis, we treated spermatogenic cells with a histone deacetylase inhibitor, trichostatin A (TSA), which showed a spectacular increase of histone acetylation in round spermatids. This observation suggests that deacetylases are responsible for maintaining a deacetylated state of histones in these cells. TSA treatment could not induce histone acetylation in condensing spermatids, suggesting that acetylated core histones are replaced by transition proteins without being previously deacetylated. Moreover, our data showed a dramatic decrease in histone deacetylases in condensing spermatids. Therefore, the regulation of histone deacetylase activity/concentration appears to play a major role in controling histone hyperacetylation and probably histone replacement during spermiogenesis.

Integrated radiation hybrid and yeast artificial chromosome map of chromosome 9p.

A panel of 93 radiation-reduced hybrids have been screened using PCR amplification and oligonucleotide primers for sequence-tagged sites (STSs) specific for 114 single-copy loci mapping to the short arm of chromosome 9. An x-ray dose of 6,000 rads gave an average retention frequency of approximately 23%. We have constructed a framework map containing 31 markers ordered by analyzing coretention patterns, with support for the order greater than 1,000:1. In addition, we have placed the remaining markers which could not be mapped to a single interval with this support to a range of intervals on the framework map. The STS oligonucleotide primers used in the construction of the radiation hybrid (RH) map have been used to isolate and order yeast artificial chromosomes (YACs) assigned to 9p identified from the CEPH megaYAC library. Eighty-nine STS markers have screened positive with at least one YAC. A total of 88 individual YACs (with an average size of 0.9 MB) have been placed on the map in a series of contigs and in some cases mapped cytogenetically by fluorescence in situ hybridization. Additionally, the YAC information has been used in conjunction with the RH framework placements to generate an integrated map containing 65 loci including 51 uniquely positioned markers, with an average resolution of 0.79 Mb.

Inoculation of an atrazine-degrading strain, Chelatobacter heintzii Cit1, in four different soils: effects of different inoculum densities.

The possibility to improve atrazine degradation in soils by bioaugmentation was studied. The atrazine-mineralizing strain, Chelatobacter heintzii Cit1, was inoculated in four sterile and four non-sterile soils, at varying inoculum densities. Two soils, which had shown enhanced atrazine mineralization, were used to determine which inoculum density was capable of restoring their original mineralizing capacity after sterilization. The two other soils, with intermediate and low capacity to mineralize atrazine, were used in order to demonstrate that atrazine mineralization in such soils could be improved by inoculation. Mineralization kinetics were fitted using the Gompertz model. In the case of soils adapted to atrazine mineralization, inoculation of C. heintzii did not accelerate the rate of atrazine mineralization, which was essentially performed by the indigenous microflora. However, with soils that did not mineralize atrazine, the introduction of 10(4) cfug(-1) resulted in a 3-fold increase of atrazine mineralization capacity.

Identification of exons in a region of human chromosome 6q known to contain tumour suppressor genes.

An important objective in human genomic mapping is the isolation of expressed sequences from defined chromosomal regions. The 6q27 region of the long arm of chromosome 6 is an important target in this endeavour because a number of tumour suppressor genes have been mapped to this region. We have used exon trapping and amplification in conjunction with a chromosome specific genomic library to generate ESTs in 6q27. Nine novel expressed sequences have been identified and these provide additional markers and potential candidate genes for the tumour suppressor genes known to be in 6q27.

[Organizing the sperm nucleus]. / Organisation nucléaire du spermatozoïde.

Thanks to the success of new assisted reproductive technology, including sperm microinjection (i.c.s.i.), men with severe spermatogenesis impairments can now become biological fathers. Whether the germinal cell used for i.c.s.i. is conveying appropriate genetic and epigenetic information is an important concern. However, to date, there is a huge lack of data on which information is epigenetically conveyed to the offspring and how. The basic support for epigenetic marks is the nucleus structure. During spermatogenesis, a major re-organization of the male germ cells nucleus structure occurs, which includes a global condensation associated with a removal of most core somatic histones and their replacement by sperm-specific nuclear proteins. The available data on the molecular mechanisms involved in this process and how it could relate to the setting of male-specific epigenetic information is reviewed and discussed in light of our current knowledge about nuclear structure and functions.

A specific CBP/p300-dependent gene expression programme drives the metabolic remodelling in late stages of spermatogenesis.

Histone hyperacetylation is thought to drive the replacement of histones by transition proteins that occur in elongating spermatids (ElS) after a general shut down of transcription. The molecular machineries underlying this histone hyperacetylation remain still undefined. Here, we focused our attention on the role of Cbp and p300 in histone hyperacetylation and in the preceding late-gene transcriptional activity in ElS. A strategy was designed to partially deplete Cbp and p300 in ElS. These cells progressed normally through spermiogenesis and showed normal histone hyperacetylation and removal. However, a genome-wide transcriptomic analysis, performed in the round spermatids (RS) and ElS, revealed the existence of a gene regulatory circuit encompassing genes presenting high expression levels in pre-meiotic cells, undergoing a repressed state in spermatocytes and early post-meiotic cells, but becoming reactivated in ElS, just prior to the global shutdown of transcription. Interestingly, this group of genes was over-represented within the genes affected by Cbp/p300 knock down and were all involved in metabolic remodelling. This study revealed the occurrence of a tightly regulated Cbp/p300-dependent gene expression programme that drives a specific metabolic state both in progenitor spermatogenic cells and in late transcriptionally active spermatids and confirmed a special link between Cpb/p300 and cell metabolism programming previously shown in somatic cells.

[Epigenetic perturbations and cancer: innovative therapeutic strategies against cancer]. / Perturbations épigénétiques et cancer : nouvelles stratégies anticancéreuses.

A complex system of molecular milestones ensures labelling of the genome, driving its organization and functions. These milestones correspond to particular marks associated to active and repressed genes, as well as to non-coding regions or those containing repetitive sequences. Most of these marks are chemical modifications of DNA, corresponding to cytosine methylation, or various posttranslational modifications of histones, the proteins which package the genome. These chemical modifications of DNA or histones are reversible and are catalysed and removed by enzymatic activities associated with factors ensuring critical cellular functions. Indeed, these enzymes are directly connected with signalling pathways, sensing extra- and intracellular environments. Altogether these mechanisms globally control the expression status of genes in each cell, meaning that certain genes are kept active, while most of the genome remains silent. Subtle metabolic changes or intra and extracellular modifications, by altering the marking associated to genes, can have long-term consequences on their expression status. Genes coding for essential regulators of cellular proliferation and differentiation could be among these genes, such as tumor suppressor genes for instance. Hence the knowledge of all these so-called "epigenetic" mechanisms will shed new light on the environmental impact on the control of gene expression and associated diseases, including malignant transformation. The understanding of these mechanisms will also pave the way for innovative therapeutic strategies to fight cancer. This review is aiming to give an overview to the reader of the relevance of epigenetic mechanisms for the understanding and treatment of cancer.

Functional characterization of ATAD2 as a new cancer/testis factor and a predictor of poor prognosis in breast and lung cancers.

Cancer cells frequently express genes normally active in male germ cells. ATAD2 is one of them encoding a conserved factor harbouring an AAA type ATPase domain and a bromodomain. We show here that ATAD2 is highly expressed in testis as well as in many cancers of different origins and that its high expression is a strong predictor of rapid mortality in lung and breast cancers. These observations suggest that ATAD2 acts on upstream and basic cellular processes to enhance oncogenesis in a variety of unrelated cell types. Accordingly, our functional studies show that ATAD2 controls chromatin dynamics, genome transcriptional activities and apoptotic cell response. We could also highlight some of the important intrinsic properties of its two regulatory domains, including a functional cross-talk between the AAA ATPase domain and the bromodomain. Altogether, these data indicate that ATAD2 overexpression in somatic cells, by acting on basic properties of chromatin, may contribute to malignant transformation.

Diabetes management in OLDES project.

EU project OLDES (Older People's e-services at home) develops easy to use and low cost ICT platform in order to offer a better quality of life to elderly people directly in their homes through innovative systems of tele-accompany, tele-assistance and tele-medicine. The elderly are able to access the services and send relevant medical data from their home by being connected to the central server via a low cost PC which is based on Negroponte paradigm. The OLDES platform interface uses television screens controlled through a remote control customized for the elderly. The feasibility of OLDES project is evaluated by the pilot study concentrating on compensation of diabetic patients. Compensation of diabetes is achieved by monitoring glucose glycemia level, blood pressure and weight. Moreover, the patient feeds into OLDES system daily consumption of food using interactive food scales and obtains advice if necessary.

Epigenetic reprogramming of the male genome during gametogenesis and in the zygote.

During post-meiotic maturation, male germ cells undergo a formidable reorganization and condensation of their genome. During this phase most histones are globally acetylated and then replaced by sperm-specific basic proteins, named protamines, which compact the genome into a very specific structure within the sperm nucleus. Several studies suggest that this sperm-specific genome packaging structure conveys an important epigenetic message to the embryo. This paper reviews what is known about this fundamental, yet poorly understood, process, which involves not only global changes of the structure of the haploid genome, but also localized specific modifications of particular genomic regions, including pericentric heterochromatin and sex chromosomes. After fertilization, the male genome undergoes a drastic decondensation, and rapidly incorporates new histones. However, it remains different from the maternal genome, bearing specific epigenetic marks, especially in the pericentric heterochromatin region. The functional implications of male post-meiotic and post-fertilization genome reprogramming are not well known, but there is experimental evidence to show that it affects early embryonic development.