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The remarkable repetitive elements called CRISPRs (clustered regularly interspaced short palindromic repeats) consist of repeats interspaced with non-repetitive elements or 'spacers'. CRISPRs are present in both archaea and bacteria, in association with genes involved in DNA recombination and repair. In the Yersinia pestis genome, three such elements are found at three distinct loci, one of them being highly polymorphic. The authors have sequenced a total of 109 alleles of the three Y. pestis CRISPRs and they describe 29 new spacers, most being specific to one isolate. In nine strains of Yersinia pseudotuberculosis, 132 spacers were found, of which only three are common to Y. pestis isolates. In Y. pestis of the Orientalis biovar investigated in detail here, deletion of motifs is observed but it appears that addition of new motifs to a common ancestral element is the most frequent event. This takes place at the three different loci, although at a higher rate in one of the loci, and the addition of new motifs is polarized. Interestingly, the most recently acquired spacers were found to have a homologue at another locus in the genome, the majority of these inside an inactive prophage. This is believed to be the first time that the origin of the spacers in CRISPR elements has been explained. The CRISPR structure provides a new and robust identification tool.

BACKGROUND: In Archeae and Bacteria, the repeated elements called CRISPRs for "clustered regularly interspaced short palindromic repeats" are believed to participate in the defence against viruses. Short sequences called spacers are stored in-between repeated elements. In the current model, motifs comprising spacers and repeats may target an invading DNA and lead to its degradation through a proposed mechanism similar to RNA interference. Analysis of intra-species polymorphism shows that new motifs (one spacer and one repeated element) are added in a polarised fashion. Although their principal characteristics have been described, a lot remains to be discovered on the way CRISPRs are created and evolve. As new genome sequences become available it appears necessary to develop automated scanning tools to make available CRISPRs related information and to facilitate additional investigations. DESCRIPTION: We have produced a program, CRISPRFinder, which identifies CRISPRs and extracts the repeated and unique sequences. Using this software, a database is constructed which is automatically updated monthly from newly released genome sequences. Additional tools were created to allow the alignment of flanking sequences in search for similarities between different loci and to build dictionaries of unique sequences. To date, almost six hundred CRISPRs have been identified in 475 published genomes. Two Archeae out of thirty-seven and about half of Bacteria do not possess a CRISPR. Fine analysis of repeated sequences strongly supports the current view that new motifs are added at one end of the CRISPR adjacent to the putative promoter. CONCLUSION: It is hoped that availability of a public database, regularly updated and which can be queried on the web will help in further dissecting and understanding CRISPR structure and flanking sequences evolution. Subsequent analyses of the intra-species CRISPR polymorphism will be facilitated by CRISPRFinder and the dictionary creator. CRISPRdb is accessible at http://crispr.u-psud.fr/crispr.

Ground-state properties of exotic even-even nuclei with extreme neutron-to-proton ratios are described in the framework of self-consistent mean-field theory with pairing formulated in coordinate space. This theory properly accounts for the influence of the particle continuum, which is particularly important for weakly bound systems. The pairing properties of nuclei far from stability are studied with several interactions emphasizing different aspects, such as the range and density dependence of the effective interaction. Measurable consequences of spatially extended pairing fields are presented, and the sensitivity of the theoretical predictions to model details is discussed. \textcopyright{} 1996 The American Physical Society.

In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage lambda DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.

BACKGROUND: The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification. RESULTS: Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species). The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1) and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2). CONCLUSION: The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA) whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.

Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.

We present nonlinear self-consistent calculations of the charge density induced by isolated ${\mathrm{Li}}^{+}$, ${\mathrm{K}}^{+}$, ${\mathrm{Mg}}^{++}$, ${\mathrm{Al}}^{+++}$, and ${\mathrm{Ca}}^{++}$ ions when placed in an electron gas of the appropriate metallic density. By comparison with linear-response theory we show that in the first four metals nonlinear effects in the response of the conduction electrons to the ionic perturbations play an important role in determining the charge density and the interionic potential. However as in the case of Na studied in the previous paper these nonlinear effects can be simulated by using a suitably adjusted model potential. The calculated phonon dispersion curves for Li, K, and Al agree very well with experiment. Nonlinear effects are also very likely to be important in Ca but further work is necessary before conclusions can be drawn.

The effect of intracavernous injection of papaverine (ICIP) has been investigated in 45 men: 6 normally potent volunteers, 10 psychogenic, and 29 organic impotent males. The first two groups obtained erections within 10 minutes, lasting from 1 to 4 hours, after injection of 80 mg of papaverine. Those belonging to the organic group experienced delayed, weaker and shorter erections, related to various degrees of arterial and/or venous and neurologic lesions. Haemodynamic and radiologic studies done concomitantly, showed a strong vasodilation in the penile arteries (even when pathologic) and a diminished outflow from the cavernous bodies. Subsequently, a pilot study was performed in a series of 63 patients suffering from various degrees of angiogenic impotence, in order to study the therapeutic effect of repeated intracavernous injections of papaverine completed by intracavernous infusion of heparinized saline. Erections improved significantly in 66% of the patients with a mean follow up of one year.

BACKGROUND: Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies. RESULTS: This report presents a database (http://minisatellites.u-psud.fr) of tandem repeats from publicly available bacterial genomes which facilitates the identification and selection of tandem repeats. We illustrate the use of this database by the characterization of minisatellites from two important human pathogens, Yersinia pestis and Bacillus anthracis. In order to avoid simple sequence contingency loci which may be of limited value as epidemiological markers, and to provide genotyping tools amenable to ordinary agarose gel electrophoresis, only tandem repeats with repeat units at least 9 bp long were evaluated. Yersinia pestis contains 64 such minisatellites in which the unit is repeated at least 7 times. An additional collection of 12 loci with at least 6 units, and a high internal conservation were also evaluated. Forty-nine are polymorphic among five Yersinia strains (twenty-five among three Y. pestis strains). Bacillus anthracis contains 30 comparable structures in which the unit is repeated at least 10 times. Half of these tandem repeats show polymorphism among the strains tested. CONCLUSIONS: Analysis of the currently available bacterial genome sequences classifies Bacillus anthracis and Yersinia pestis as having an average (approximately 30 per Mb) density of tandem repeat arrays longer than 100 bp when compared to the other bacterial genomes analysed to date. In both cases, testing a fraction of these sequences for polymorphism was sufficient to quickly develop a set of more than fifteen informative markers, some of which show a very high degree of polymorphism. In one instance, the polymorphism information content index reaches 0.82 with allele length covering a wide size range (600-1950 bp), and nine alleles resolved in the small number of independent Bacillus anthracis strains typed here.

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Human paraoxonase 1 (PON-1) is a serum high-density lipoprotein-associated enzyme mainly secreted by the liver. It has endogenous and exogenous substrates and displays protective properties with respect to cardiovascular disease and organophosphate intoxication. In the HuH7 human hepatoma cell line, PON-1 activity and mRNA levels were increased by dietary polyphenolic compounds such as quercetin but also by toxic ligands of the aryl hydrocarbon receptor (AhR) such as 3-methylcholanthrene (3-MC). However, the 2,3,7,8-tetrachlorobenzo(p)dioxin (TCDD) was a poor inducer. Transient and stable transfection assays indicated that these compounds increased the PON-1 gene promoter activity in an AhR-dependent manner, since their effect was inhibited by 7-keto-cholesterol and AhR-directed short interfering RNA. Deletions and mutations studies showed that a xenobiotic responsive element (XRE)-like sequence within the PON-1 promoter mediated the effect of 3-MC and quercetin. In contrast with consensus XREs from the cytochrome P450 1A1 gene, the PON-1 XRE-like element mediated preferentially the effect of quercetin compared to the results seen with TCDD. Furthermore, AhR binding to this element was preferentially activated by quercetin. These observations provide a molecular mechanism for the regulation of the cardioprotective enzyme PON-1 by polyphenols. They suggest also that AhR ligands may differentially regulate gene expression depending on the DNA target sequence.

Viral infections often induce potent CD8 T cell responses that play a key role in antiviral immunity. After viral clearance, the vast majority of the expanded CD8 T cells undergo apoptosis, leaving behind a stable number of memory cells. The relationship between the CD8 T cells that clear the acute viral infection and the long-lived CD8 memory pool remaining in the individual is not fully understood. To address this issue, we examined the T cell receptor (TCR) repertoire of virus-specific CD8 T cells in the mouse model of infection with lymphocytic choriomeningitis virus (LCMV) using three approaches: (a) in vivo quantitative TCR beta chain V segment and complementarity determining region 3 (CDR3) length repertoire analysis by spectratyping (immunoscope); (b) identification of LCMV-specific CD8 T cells with MHC class I tetramers containing viral peptide and costaining with TCR Vbeta-specific antibodies; and (c) functional TCR fingerprinting based on recognition of variant peptides. We compared the repertoire of CD8 T cells responding to acute primary and secondary LCMV infections, together with that of virus-specific memory T cells in immune mice. Our analysis showed that CD8 T cells from several Vbeta families participated in the anti-LCMV response directed to the dominant cytotoxic T lymphocyte (CTL) epitope (NP118-126). However, the bulk (approximately 70%) of this CTL response was due to three privileged T cell populations systematically expanding during LCMV infection. Approximately 30% of the response consisted of Vbeta10+ CD8 T cells with a beta chain CDR3 length of nine amino acids, and 40% consisted of Vbeta8.1+ (beta CDR3 = eight amino acids) and Vbeta8.2+ cells (beta CDR3 = six amino acids). Finally, we showed that the TCR repertoire of the primary antiviral CD8 T cell response was similar both structurally and functionally to that of the memory pool and the secondary CD8 T cell effectors. These results suggest a stochastic selection of memory cells from the pool of CD8 T cells activated during primary infection.

The density-functional theory (DFT) for a system of ions and electrons having overall charge neutrality is presented. For a given temperature and free-electron density, the theory self-consistently determines the various pair-distribution functions, bound states, and the effective ion charge $\overline{Z}=Z\ensuremath{-}{n}_{b}$, where ${n}_{b}$ is the mean number of bound electrons per ion. With the use of a sufficiently large correlation sphere to represent the "physically relevant" part of the plasma, a Kohn-Sham-type Schr\"odinger equation is solved for the electrons. The density-functional equations for the ions reduce to a Gibbs-Boltzmann equation containing an effective ion-correlation potential. The latter is obtained from the hypernetted-chain equation. The electron exchange-correlation potential is obtained from quantum many-body theory. The method is applied to an electron-proton plasma (EPP) for a number of physical situations. The machine-simulation (MS) results in the classical one-component-plasma limit, as well as the standard "proton-in-jellium" results, are correctly recovered in suitable limits. The MS results for the EPP plasma are limited by classical approximations, but are found to be in reasonable agreement with our DFT results which are probably more well founded. A shallow $1s$-like bound state appears with increase of temperature or decrease of density. The proton-pair distribution function in the EPP shows the onset of short-range order only when $\ensuremath{\Gamma}\ensuremath{\gtrsim}5$ or more. The electron-proton distribution function is found to be relatively insensitive to the details of the ion distribution for the static properties studied in this paper.

This paper studies the mechanisms underlying the apparent stability of the income distribution in Taiwan. An original decomposition method based on micro‐simulation techniques is proposed. Applied to the distribution of income in Taiwan since 1979, it permits isolating the respective impact of changes in: (a) the earning structure; (b) labor‐force participation behavior; and (c) the socio‐demographic structure of the population. The stability of the distribution in Taiwan appears as the result of various structural forces which happened to offset each other. The small drop observed in the inequality of individual earnings resulted from the combination of unequalizing changes in the wage structure and the effects of changes in female labor‐force participation as well as in the educational structure of the population. However, the same offsetting forces, together with changes in the composition of households, resulted in a small increase in the inequality of the distribution of equivalized household income.

In the following paper, two classes of infinite matrices are studied (matrices with exponentially or polynomially decreasing coefficients away from the diagonal). Properties of symbolic calculus type are shown: Inversibility on l^2 implies that the inverse has the same decay property. It is also shown that inversion is a “local” transformation of the matrix: A change in the coefficients is not felt far away from the area changed. Two applications concerning the properties of orthonormal bases of wavelets are given. Résumé Nous étudions dans cet article deux classes de matrices infinies (matrices dont les coefficients sont à décroissance exponentielle ou polynomiale en s’éloignant de la diagonale). Nous montrons qu’elles possèdent des propriétés du type « calcul symbolique » : L’inversibilité sur l^2 implique que les coefficients de la matrice inverse ont la même propriété de décroissance. De plus, l’inversion est une opération « locale » : Une perturbation des coefficients de A n’est pas ressentie sur son inverse loin de la zone de perturbation. Nous fournissons deux exemples d’applications concernant les propriétés des bases orthonormées d’ondelettes.

Minisatellites have been found in association with important features of human genome biology such as gene regulation, chromosomal fragile sites, and imprinting. Our knowledge of minisatellite biology has greatly increased in the past 10 years owing to the identification and careful analysis of human hypermutable minisatellites, experimental models in yeast, and recent in vitro studies of minisatellite recombination properties. In parallel, minisatellites have been put forward as potential biomarkers for the monitoring of genotoxic agents such as ionizing radiation. We summarize and discuss recent observations on minisatellites. In addition we take advantage of recent whole chromosome sequence data releases to provide a unifying view which may facilitate the annotation of tandem repeat sequences.

We have calculated the exchange and correlation corrections to the free energy (${F}_{\mathrm{xc}}$) and the chemical potential (${\ensuremath{\mu}}_{\mathrm{xc}}$) of an electron system at arbitrary degeneracies and temperatures. These are needed for density-functional, average-atom-type calculations of properties of plasmas and liquid metals. The problem of inverting the parametric relation between ${\ensuremath{\mu}}_{\mathrm{xc}}$ and the density correction ${n}_{\mathrm{xc}}$ is considered. We present easy-to-use analytic fits to ${F}_{\mathrm{xc}}$ and ${\ensuremath{\mu}}_{\mathrm{xc}}$ as functions of the temperature and density. We also consider the effect of a lineraly responding ion background on the electron chemical potential as well as the ion chemical potential, as these are needed in calculations of ionelectron systems at arbitrary degeneracies and densities.

A laser-induced breakdown spectroscopy technique for analyzing biological matter for the detection of biological hazards is investigated. Eight species were considered in our experiment: six bacteria and two pollens in pellet form. The experimental setup is described, then a cumulative intensity ratio is proposed as a quantitative criterion because of its linearity and reproducibility. Time-resolved laser-induced breakdown spectroscopy (TRELIBS) exhibits a good ability to differentiate among all these species, whatever the culture medium, the species or the strain. Thus we expect that TRELIBS will be a good candidate for a sensor of hazards either on surfaces or in ambient air.

BACKGROUND: Currently available reference methods for the molecular epidemiology of the Mycobacterium tuberculosis complex either lack sensitivity or are still too tedious and slow for routine application. Recently, tandem repeat typing has emerged as a potential alternative. This report contributes to the development of tandem repeat typing for M. tuberculosis by summarising the existing data, developing additional markers, and setting up a freely accessible, fast, and easy to use, internet-based service for strain identification. RESULTS: A collection of 21 VNTRs incorporating 13 previously described loci and 8 newly evaluated markers was used to genotype 90 strains from the M. tuberculosis complex (M. tuberculosis (64 strains), M. bovis (9 strains including 4 BCG representatives), M. africanum (17 strains)). Eighty-four different genotypes are defined. Clustering analysis shows that the M. africanum strains fall into three main groups, one of which is closer to the M. tuberculosis strains, and an other one is closer to the M. bovis strains. The resulting data has been made freely accessible over the internet http://bacterial-genotyping.igmors.u-psud.fr/bnserver to allow direct strain identification queries. CONCLUSIONS: Tandem-repeat typing is a PCR-based assay which may prove to be a powerful complement to the existing epidemiological tools for the M. tuberculosis complex. The number of markers to type depends on the identification precision which is required, so that identification can be achieved quickly at low cost in terms of consumables, technical expertise and equipment.

Abstract. Within the framework of the European Employment Strategy, the European Union has defined a set of indicators to monitor employment quality — the so‐called Laeken indicators. This article discusses and implements these indicators. From a theoretical perspective, it shows that the concept of work quality encompasses several dimensions, which are likely to be related to national institutions, particularly industrial relations and welfare systems. It then proceeds with a comparative analysis of quality in work across the 27 Member States, which confirms the existence of several models in Europe and suggests that the Laeken indicators should be supplemented by additional measures.