Mapping the vulnerability of groundwater to the contamination of four carbonate aquifers in Europe.

The vulnerability of four European aquifers with different hydrogeological and climatic characteristics was evaluated using the COP method. The results obtained were statistically analyzed by determination coefficients to measure which factor has greater importance in the vulnerability index. Furthermore, a new parameter has been designed to measure the vulnerability for the whole of the aquifer. The results demonstrate that COP is a useful method to assess the vulnerability of the test sites under consideration. The results obtained are coherent with the conceptual model of each pilot aquifer and the available hydrogeological information (hydrographs, isotopic data, tracer tests). Fissured carbonate aquifers (diffuse flow systems) are less vulnerable than karst aquifers (conduit flow systems) and the vulnerability index is more positively correlated with the O factor (unsaturated zone protection capacity) in the first case. The karst aquifers are more vulnerable than fissured aquifers and they show a higher correlation between the C factor (karst features) and the vulnerability index. Climatic variation (precipitation for example) influences the final vulnerability index of the aquifers according to the weight in the index and the spatial distribution.

Green coffee seed residue: A sustainable source of antioxidant compounds.

Oil extraction from green coffee seeds generates residual mass that is discarded by agribusiness and has not been previously studied. Bioactive secondary metabolites in coffee include antioxidant phenolic compounds, such as chlorogenic acids. Coffee seeds also contain caffeine, a pharmaceutically important methylxanthine. Here, we report the chemical profile, antioxidant activity, and cytotoxicity of hydroethanolic extracts of green Coffea arabica L. seed residue. The extracts of the green seeds and the residue have similar chemical profiles, containing the phenolic compounds chlorogenic acid and caffeine. Five monoacyl and three diacyl esters of trans-cinnamic acids and quinic acid were identified by ultra-performance liquid chromatography/electrospray ionization-quadruple time of flight mass spectrometry. The residue extract showed antioxidant potential in DPPH, ABTS, and pyranine assays and low cytotoxicity. Thus, coffee oil residue has great potential for use as a raw material in dietary supplements, cosmetic and pharmaceutical products, or as a source of bioactive compounds.

Factors controlling groundwater salinization and hydrogeochemical processes in coastal aquifers from southern Spain.

In detrital coastal aquifers, seawater and surface water may interact with groundwater in multiple ways. Understanding the interference of water fluxes in this type of environment is essential to effectively manage the groundwater resources in water-stressed regions, such as the Mediterranean coastal fringe. In this research, the characterization of the main hydrogeochemical processes and the interaction between surface water and groundwater in the Marbella-Estepona coastal aquifers (southern Spain) have been carried out by means of the combined use of different hydrogeochemical indicators along with isotope data. The results show that the diversity of source lithologies (peridotite, carbonate and/or metapelitic) substantially conditions the groundwater geochemistry. The analysis of ionic deltas made it possible a preliminary screening of the geochemical reactions that occur in the Marbella-Estepona aquifers, while the Discriminant Analysis allowed for a consistent classification of sampled groundwater types. The dissolution of calcite and dolomite determines the chemical composition of the groundwater from the eastern sector that are more conditioned by the rainwater infiltration. The dissolution of magnesium-bearing minerals (predominantly forming peridotite rocks) is observed in groundwater samples from the western and central sectors, whose chemical composition showed a greater influence of surface water. The spatial analysis of rCl-/Br- in groundwater has permitted to corroborate that saline intrusion is negligible, hardly affecting to its original water quality. The irregularly distributed recharge by precipitation (seasonal effect) and the atmospheric circulation of cloud fronts (coastal/continental effect) explains why most of groundwater sampled is isotopically impoverished with respect to the rainfall signature. The isotope approach also suggests the hydraulic relationship between surface water and groundwater in the study site. A deeper knowledge of spatial hydrogeochemical variations in coastal groundwater and the influence of water sources over them are crucial for a sustainable groundwater management and global change adaptation in equivalent Mediterranean water-stressed regions.

Spatial prediction of water quality variables along a main river channel, in presence of pollution hotspots.

In order to treat and evaluate the available data of water quality and fully exploit monitoring results (e.g. characterize regional patterns, optimize monitoring networks, infer conditions at unmonitored locations, etc.), it is crucial to develop improved and efficient methodologies. Accordingly, estimation of water quality along fluvial ecosystems is a frequent task in environment studies. In this work, a particular case of this problem is examined, namely, the estimation of water quality along a main stem of a large basin (where most anthropic activity takes place), from observational data measured along this river channel. We adapted topological kriging to this case, where each watershed contains all the watersheds of the upstream observed data ("nested support effect"). Data analysis was additionally extended by taking into account the upstream distance to the closest contamination hotspot as an external drift. We propose choosing the best estimation method by cross-validation. The methodological approach in spatial variability modeling may be used for optimizing the water quality monitoring of a given watercourse. The methodology presented is applied to 28 water quality variables measured along the Santiago River in Western Mexico.

Multicolor PRINS and multicolor PNA.

Both PRimed IN Situ (PRINS) and Peptide Nucleic Acid (PNA) technologies have emerged as research techniques, but they have quickly evolved to applications in biological diagnosis assays. The two procedures now constitute efficient alternatives to the conventional fluorescence in situ hybridization (FISH) procedure for in situ chromosome identification and aneuploidy detection. They present several advantages (specificity, speed, discriminating ability) that make them very attractive for a number of cytogenetic purposes. Multicolor PRINS and PNA protocols have been described for the specific identification of human chromosomes. Various applications have already been developed in human genetics and new adaptations are ongoing.

Design of novel starch-based Pickering emulsions as platforms for skin photoprotection.

Green coffee oil and modified starch were recently found to have an enhanced protection effect against UV radiation. Therefore, this work aimed to develop an innovative sunscreen formulation based on Pickering emulsions concept, i.e., surfactant-free emulsions stabilized by physical UV filters associated natural oils as a key strategy for prevention against UV-induced skin damage. The Pickering emulsions of different compositions were characterized in terms of pH, mechanical, physical and microbiological stability by a thorough pharmaceutical control. In addition, the sun protection factor (SPF) as well as the in vitro and in vivo biological properties of the final formulations, including Episkin®, HRIPT and sunscreen water resistance. Formulation studies demonstrated the addition of physical UV filters was beneficial, leading to the inclusion of ZnO and TiO2 to ensure a high SPF against UVA and UVB, respectively. Although starch particles presented no intrinsic photoprotection properties, they proved to be a SPF promoter by a synergistic effect. Green coffee oil was the selected natural oil due to the highest SPF, when compared to other natural oils tested. Besides the excellent sunscreen activity confirmed by in vitro and in vivo results, the final formulations proved to be also suitable for topical use according to the rheological assessment and stability throughout the study period (3months). In conclusion, the combination of three multifunctional solid particles and green coffee oil, contributed to achieve a stable and effective innovative sunscreen with a wide range of UV radiation protection.

[The cytogenetics of human oocytes: 40 years of progress]. / Cytogénétique des ovocytes humains: 40 ans de progrès.

Chromosomal abnormalities account for the majority of pre- and post- implantation embryo wastage in humans. Most of these abnormalities result from maternal meiotic errors, which preferentially occur during the first meiotic division. Consequently, the cytogenetic analysis of human oocytes has then been considered as a highly valuable source of data for the investigation of both the occurrence and the origin of chromosomal abnormalities in human. During the last 4 decades, the cytogenetic analysis of human oocytes has never stopped progressing, according to the advents of new technologies. Both karyotyping and molecular cytogenetic studies have been reported to date, providing a large body of data on the incidence and the distribution of chromosomal abnormalities in human female gametes. However, these studies display a great variability in results, which may be essentially attributable to the limitations of these techniques when applied to human oocytes. The most relevant analysis have led to the estimate that 15-20% of human oocytes present chromosome abnormalities, and they have emphasized the implication of both whole chromosome non-disjunction and chromatid separation in the occurrence of aneuploidy in human oocytes. The effect of advanced maternal age on the incidence of aneuploidy in human oocytes has also been clearly evidenced by recent reports based on large sample of oocytes or polar bodies.

Vulnerability mapping and protection zoning of karst springs. Validation by multitracer tests.

Protection zoning of karst springs and wells used for water supply is a key aspect in many countries, calling for specific methodologies adapted to the particular characteristics of karst media. This work presents a new approach, in view of the present state of the art and based on experiences with contamination vulnerability mapping at the pilot site of the Villanueva del Rosario karst system (southern Spain). Source (intrinsic) vulnerability maps were prepared and compared using three European procedures for karst aquifers. The vulnerability maps were then tested using dye tracers. The COP+K method and Slovene Approach appear to provide reliable results in terms of intrinsic vulnerability mapping. Nevertheless, all the methods have a margin of error. The COP+K map is adopted as the baseline to delineate the protection zones, through the conversion from vulnerability classes to degrees of protection.

Rapid in situ detection of chromosome 21 by PRINS technique.

The "PRimed IN Situ labeling" (PRINS) method is an interesting alternative to in situ hybridization for chromosomal detection. In this procedure, chromosome labeling is performed by in situ annealing of specific oligonucleotide primers, followed by primer elongation by a Taq polymerase in the presence of labeled nucleotides. Using this process, we have developed a simple and semi-automatic method for rapid in situ detection of human chromosome 21. The reaction was performed on a programmable temperature cycler, with a chromosome 21 specific oligonucleotide primer. Different samples of normal and trisomic lymphocytes and amniotic fluid cells were used for testing the method. Specific labeling of chromosome 21 was obtained in both metaphases and interphase nuclei in a 1 hour reaction. The use of oligonucleotide primer for in situ labeling overcomes the need for complex preparations of specific DNA probes. The present results demonstrate that PRINS may be a simple and reliable technique for rapidly detecting aneuploidies.

Aneuploidy detection in human sperm nuclei using PRINS technique.

Rapid and specific identification of chromosomes can be attained in situ using the PRimed IN Situ (PRINS) labelling technique. We have adapted this technique to mature human sperm in combination with a protocol for simultaneous decondensation and denaturation of sperm nuclei. This strategy allowed us to obtain double labelling of human spermatozoa in a < 2-hr reaction. In the present study, we report the estimates of disomy for chromosomes 3, 7, 10, 11, and 17 on 64,642 spermatozoa from 2 normal males. The incidences of disomy ranged from 0.28-0.34%. There were no significant interindividual or interchromosomal differences in disomy rates.

Interphasic analysis of aneuploidy in cancer cell lines using primed in situ labeling.

The primed in situ (PRINS) labeling technique has been adapted to chromosomal screening of interphasic tumoral cells. A panel of ten chromosome-specific alpha-satellite DNA primers was used to evaluate numerical chromosome abnormalities in two colon cancer cell lines (Caco-2 and HT-29) and in three of their subpopulations (PF11, TC7, and HT29-MTX). In each cell line, the copy number distribution for different chromosomes showed different patterns. The observation of significant variations in the chromosome constitutions between subpopulations derived from the same original tumor suggests the common occurrence of chromosome copy number heterogeneity in tumoral cell lines. This study demonstrates that the PRINS procedure offers a simple and reliable method for in situ chromosomal screening, which could be efficiently used for karyotypic analysis of tumoral cells.

Preimplantation embryo chromosome analysis by primed in situ labeling method.

OBJECTIVE: To present the use of primed in situ labeling method in preimplantation diagnosis. DESIGN: Double- and triple-primed in situ labeling were performed on 10 morphologically abnormal preimplantation embryos, using combinations of specific primers for chromosomes 9, 13, 16, 18, 21, X, and Y. SETTING: Embryos were obtained from patients at the Montpellier University Hospital. PATIENT(S): Seven women undergoing IVF at the Montpellier University Hospital. INTERVENTION(S): Isolated interphase nuclei from poor quality preimplantation embryos were prepared for primed in situ labeling technique. MAIN OUTCOME MEASURE(S): Numerical abnormalities assessed by primed in situ labeling analysis. RESULT(S): Using directly fluorescent-labeled nucleotides, the labeling reaction for three chromosomes did not exceed 2.30 hours. Only three analyzed embryos appeared to be chromosomally normal. Mosaicism, aneupoidy, and haploidy were observed in the seven other embryos. CONCLUSION(S): The primed in situ labeling method offers a simple and reliable screening tool for gender determination and aneuploidy detection. The use of this technique may contribute to significantly improve the procedure of preimplantation diagnosis.

[Application of the PRINS technique for chromosome examination in fetal cells present in maternal blood]. / Application de la technique PRINS à l'examen chromosomique des cellules foetales présentes dans le sang maternal.

Both detection and chromosomal analysis of fetal cells present in the maternal circulation can be performed using Histopaque double density gradient centrifugation followed by primed in situ (PRINS) labeling technique. This approach has been tested on blood samples from 15 pregnant women and 6 control donors with primers specific for chromosomes 9, X and Y. The cell separation technique allows recovery of both mononuclear cells and polynuclear cells with a 97% efficiency. PRINS labeling was successful in 100% cells from control blood samples. Among patient samples, 2 "false-negative" results were observed. These preliminary results suggest that the present protocol might be efficient for non-invasive prenatal chromosome analysis.

Characterisation of dissolved organic matter in karst spring waters using intrinsic fluorescence: relationship with infiltration processes.

From analysis of spectrophotometric properties of dissolved organic matter (OM) and the hydrochemical responses of some karst springs under different hydrologic conditions, an assessment of the origin and transfer pathway of OM present in karst spring waters, from soil and epikarst toward the spring, has been conducted for three karst aquifers in southern Spain: Alta Cadena, Sierra de Enmedio and Los Tajos. Intrinsic fluorescence (excitation-emission matrices or EEMs), together with major water chemistry (electrical conductivity, temperature, alkalinity, Cl⁻, Mg⁺²) and P(CO2) along with natural hydrochemical tracers (TOC and NO3⁻, have been monitored in 19 springs which drain the three karst aquifers examined in this study. The spring water EEM spectra indicate that fulvic acid-like substances, produced in the soil as a consequence of the decomposition of OM, are the dominant fluorophores, although some of the OM appears to originate from in situ microbiological activity but could be indicative of contamination present in recharge waters from livestock. During each recharge event, TOC and NO3⁻ concentrations increased and variations in fluorescence intensities of peaks attributed to fulvic acid-like compounds were observed. In areas with minimal soil development, spatial and temporal variations in the fluorescence intensity of fulvic acid-like substances and other fluorophores derived from microbiological activity, together with other hydrochemical parameters, provide insights into the hydrogeological functioning of karst aquifers and the infiltration velocity of water from soil and facilitate assessment of contamination vulnerability in these aquifers.

Sperm segregation analysis of a (13;22) Robertsonian translocation carrier by FISH: a comparison of locus-specific probe and whole chromosome painting.

BACKGROUND: The t(13;22) Robertsonian translocation constitutes a rare form of rearrangement between acrocentric human chromosomes. Most of the meiotic segregation studies of human Robertsonian translocations have been performed on common t(13;14) and t(14;21) translocations. Analysis of the chromosomal constitution in sperm of Robertsonian translocation carriers is of great interest for assessing the risk of unbalanced forms and adapting genetic counselling. In the present study, we present the first meiotic segregation study of a t(13;22) Robertsonian translocation in human sperm. METHODS: A total of 11 787 sperm nuclei were scored using two distinct FISH labelling techniques, i.e. the locus-specific probes (LSI) method and the whole chromosome painting (WCP) technique. RESULTS: The frequency of normal or balanced sperm resulting from alternate meiotic segregation was 86%. Incidences of unbalanced complements resulting from adjacent segregation modes were 12.79% and 14.36% in LSI and WCP assays, respectively. No significant excess of nullisomy or disomy for the affected chromosomes was observed. CONCLUSIONS: Similar results in segregation were obtained with the two techniques, demonstrating the efficiency of the two strategies for the direct segregation analysis of Roberstsonian translocations. The results obtained indicated a moderate meiotic production of imbalance. This study shows that the rare Robertsonian translocation (13;22) displays a similar distribution of balanced and unbalanced sperm patterns as the common Robertsonian translocations previously studied. This suggests that the behaviour of acrocentric chromosomes was similar in all cases of centric fusion.

Effect of long abstinence periods on human sperm quality.

OBJECTIVE: To evaluate the effects of long abstinence periods on semen characteristics. DESIGN: Semen analysis was performed on six men after various sexual abstinence periods (from 2 to 18 days). The variations in semen parameters were analyzed statistically as a function of the duration of abstinence. SETTING: The Reproductive Biology Laboratory at the University of Montpellier Medical School. RESULTS: Lengthy sexual abstinence was found to affect all semen characteristics. Semen volume and concentration and total sperm count showed significant increases, whereas motility and normal morphology decreased significantly with duration of abstinence. Significant changes in the percentage of normal sperm forms were observed after more than seven days' abstinence. CONCLUSION: The study indicates that the influence of long sexual abstinence on semen quality varies with the variable considered. With regard to fertility, a long abstinence period might induce senescence of spermatozoa.

Assessment of chromosomal heterogeneity in tumoral cell lines using PRINS technique.

The primed in situ (PRINS) labeling technique has been used for the interphase cytogenetic investigation of 3 colon cancer cell lines (Caco-2, TC7 and PF1 1) derived from a same primary tumor. A panel of 10 chromosome-specific primers (for chromosomes 1, 2, 3, 7, 8, 9, 10, 13, 16 and 18) has been utilized in simple and double color PRINS reactions. Each cell line displayed a heterogeneous distribution of copy number for several chromosomes. The karyotypic heterogeneity was also significant between the 3 cell lines. These data indicate the common occurrence of chromosome heterogeneity in tumoral cell lines and demonstrate the feasibility of interphase PRINS procedure for analysis of numerical changes in tumoral cells.

A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligonucleotide primed in situ labelling (PRINS).

The centromeric alpha satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence and cannot be easily distinguished by mean of probes for Southern blot or in situ hybridisation. We have used the oligonucleotide-primed in situ (PRINS) labelling technique with primers defined from the alpha satellite sequence of chromosome 13. One primer was found to label specifically the centromeric region of chromosomes 13 and allowed the detection of a polymorphism between two chromosome 13 homologues in one individual.

Ultra-rapid multicolor PRINS protocol for chromosome detection in human sperm.

We report a new multicolor PRINS procedure for chromosome identification on human sperm. Based on the direct in-situ mixing of the colors of the fluorochromes (FITC, TRITC, Cascade Blue) incorporated in sequential PRINS reactions, this method facilitates rapid distinct labeling of 3 or 4 chromosomes. Each PRINS reaction consists of a unique 4 minute step for annealing and elongation. The method was successfully tested on lymphocytes and spermatozoa. Estimates of disomy were performed for chromosomes 7, 9 and 16 on sperm samples from 2 healthy donors. There was no significant difference between the disomy rates obtained with the conventional two-color PRINS technique and this new three-color procedure. By simplifying the multicolor PRINS protocol, this new protocol should facilitate the use and adaptation of PRINS to various cytogenetic applications.

Study of the occurrence of interchromosomal effect in spermatozoa of chromosomal rearrangement carriers by fluorescence in-situ hybridization and primed in-situ labelling techniques.

The possibility that a chromosomal rearrangement might disturb the meiotic behaviour of chromosomes not involved in the rearrangement and favour non-disjunction is a controversial issue in human cytogenetics. Using two-colour fluorescence in-situ hybridization and primed in-situ labelling techniques, we have investigated the segregation pattern of 10 chromosomes (chromosomes 1, 4, 9, 13, 15, 16, 20, 21, X and Y) in spermatozoa from nine carriers of balanced structural rearrangements and three normal men. The patients were divided into two groups according to their semen parameters. In rearrangement carriers and normal subjects, sex chromosomes and chromosome 21 displayed a higher rate of disomy than the other chromosomes. No evidence for the occurrence of interchromosomal effect was found in the spermatozoa of fertile rearrangement carriers, but significant variations were observed for all chromosomes tested in the group of infertile translocation carriers, suggesting a direct correlation between poor quality spermatozoa and increased aneuploidy rate in this group. In fertile carriers of chromosomal rearrangements, the occurrence of non-disjunction of chromosomes not involved in the rearrangement might therefore be considered as fortuitous, whereas in infertile carriers, the risk for interchromosomal effect appears to be real and should be taken into consideration in the genetic counselling of infertile couples with a male partner carrying a chromosomal rearrangement.