Presence of Cocaine in the Tissues of the European Eel, Anguilla anguilla, Exposed to Environmental Cocaine Concentrations

The presence of illicit drugs and their metabolites in surface waters has to be considered a new type of hazard, still unknown, for the aquatic ecosystem, due to the potent pharmacological activities of all the illicit drugs. Our research was therefore aimed at evaluating the impact of illicit drugs on the aquatic fauna, till now still undervalued. To this aim, we verified the ability of the European eel (Anguilla anguilla), a well-known biomonitor of environmental contamination, to bioaccumulate cocaine, one of the most abundant illicit drugs found in surface waters. Silver eels were exposed to a nominal cocaine concentration of 20?ng/l for 1?month; at the same time, control, carrier, and post-exposure recovery groups were made. Brains, gills, liver, kidney, muscle, gonads, spleen, digestive tract, and sections of dorsal skin were assayed by high-pressure liquid chromatography. Cocaine was found in the tissues of the treated eels and, at low concentrations, in almost all tissues of post-exposure recovery eels. These results indicate that cocaine is able to accumulate into the eel tissues; its presence suggests potential risks for eels since cocaine could affect their physiology and contribute to their decline, and for humans consuming contaminated fish.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

The mtDNA legacy of the Levantine early Upper Palaeolithic in Africa

Sequencing of 81 entire human mitochondrial DNAs (mtDNAs) belonging to haplogroups M1 and U6 reveals that these predominantly North African clades arose in southwestern Asia and moved together to Africa about 40,000 to 45,000 years ago. Their arrival temporally overlaps with the event(s) that led to the peopling of Europe by modern humans and was most likely the result of the same change in climate conditions that allowed humans to enter the Levant, opening the way to the colonization of both Europe and North Africa. Thus, the early Upper Palaeolithic population(s) carrying M1 and U6 did not return to Africa along the southern coastal route of the "out of Africa" exit, but from the Mediterranean area; and the North African Dabban and European Aurignacian industries derived from a common Levantine source.     

Measurement of condensed tannins and dry matter in red grape homogenates using near infrared spectroscopy and partial least squares

Samples (n = 620) of homogenized red grape berries were analyzed using a visible and near-infrared (NIR) spectrophotometer (400-2500 nm) in reflectance. The spectra and the analytical data were used to develop partial least-squares calibrations to predict dry matter (DM) content and condensed tannins (CT) concentrations. The coefficient of determination in cross-validation and the standard error of cross-validation were 0.92 and 0.83% w/w for DM and 0.86 and 0.46 mg/g epicatechin equivalents for CT, respectively. The standard error in prediction was 1.34% w/w for DM and 0.89 mg/g epicatechin equivalents for CT, respectively. By implementing a NIR spectroscopy method to measure DM and CT in red grape homogenates, we have developed an approach that is suited to large-scale compositional analysis in commercial wine production facilities, as it enables the analysis of large numbers of samples needed to stream batches of fruit. From an economical point of view, the calibration models could be achieved with relatively small data sets. Thus, NIR offers a suitable and efficient tool for the simultaneous measurement of DM and CT in addition to other important parameters in red grape homogenates such as total anthocyanins, total soluble solids, and pH, with minimal sample preparation and low cost.     

Comparative and collaborative studies for the validation of a nested PCR for the detection of Xanthomonas axonopodis pv. dieffenbachiae from Anthurium samples

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N‐PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N‐PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 90·6, 88·7 and 47·2% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 92·5% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N‐PCR assay. The N‐PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N‐PCR had undeniable advantages compared to the reference method (less labour‐intensive and less time‐consuming). In addition, post‐test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N‐PCR assay has since been included in a revised version of the EPPO detection protocol.     

Quantitative analysis of minerals and electric conductivity of red grape homogenates by near infrared reflectance spectroscopy

The use of near infrared (NIR) reflectance spectroscopy to measure the concentration of minerals and electric conductivity (EC) in red grape homogenates was investigated. Wine grape samples (n = 209) from two vintages, representing a wide range of varieties and regions were analysed by Inductively Coupled Plasma Optical Emission Spectrometry (ICPOES) for the concentrations of calcium (Ca), potassium (K), magnesium (Mg), phosphorus (P), sulphur (S), iron (Fe), and manganese (Mn) and scanned in reflectance in a NIR instrument (400-2500 nm). The spectra were pre-processed using multiple scatter correction (MSC) before developing the calibration models using partial least squares (PLS) regression and cross validation. Coefficients of determination in cross validation (R²) and the standard errors of cross validation (SECV) obtained were for Fe (0.60 and 1.49 mg kg⁻¹), Mn (0.71 and 0.41 mg kg⁻¹), Ca (0.75 and 60.89 mg kg⁻¹), Mg (0.84 and 12.93 mg kg⁻¹), K (0.78 and 285.34 mg kg⁻¹), P (0.70 and 40.19 mg kg⁻¹), S (0.88 and 14.45 mg kg⁻¹) and EC (0.87 and 7.66 mS). The results showed that Mg, S and EC in grape berries might be measured by NIR reflectance spectroscopy.     

Genome-wide analysis of plastome sequence variation and development of plastidial CAPS markers in common potato and related <Emphasis Type="Italic">Solanum</Emphasis> species

The plastome sequence of the European cultivated potato, Solanum tuberosum subsp. tuberosum (tbr, GenBank accession no. DQ386163), was compared with that of S. bulbocastanum, a wild potato relative (blb, GenBank accession no. DQ347958), in order to characterize the degree and type of variability in different genomic regions, and develop molecular markers relevant to genetics, breeding and biotechnology of potato. One hundred forty-two and 251 PICs (Potentially Informative Characters) were found in coding and non-coding sequences (NCSs), respectively. Further, while variation in coding regions was almost exclusively due to nucleotide substitutions, 25% of PICs in NCSs of tbr and blb were due to indels, most of them mononucleotide or longer tandem repeats (micro and minisatellites). Four intergenic regions were selected for further analyses in other 16 tuber-bearing Solanum species. The rps16-trnQ ^UUG gene spacer was found to be the most variable, forty-six PICs in this region distinguishing 18 haplotypes. Analysis of haplotype relationships, based on variability in the four intergenic regions, confirmed that the most primitive species from Central America were the most distant to S. tuberosum. Finally, polymorphic sites in the same regions were used to develop a set of CAPS (Cleaved Amplified Polymorphic Sequences) markers for species/cytoplasm identification in Solanum spp.     

Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity

The present article reports the antimicrobial efficacy of four monoterpenes (thymol, carvacrol, p-cymene, and gamma-terpinene) against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. For a better understanding of their mechanism of action, the damage caused by these four monoterpenes on biomembranes was evaluated by monitoring the release, following exposure to the compounds under study, of the water-soluble fluorescent marker carboxyfluorescein (CF) from large unilamellar vesicles (LUVs) with different lipidic composition (phosphatidylcholine, PC, phosphatidylcholine/phosphatidylserine, PC/PS, 9:1; phosphatidylcholine/stearylamine, PC/SA, 9:1). Furthermore, the interaction of these terpenes with dimyristoylphosphatidylcholine multilamellar vesicles as model membranes was monitored by means of differential scanning calorimetry (DSC) technique. Finally, the results were related also with the relative lipophilicity and water solubility of the compounds examined. We observed that thymol is considerably more toxic against S. aureus than the other three terpenes, while carvacrol and p-cymene are the most inhibitory against E. coli. Thymol and carvacrol, but not gamma-terpinene and p-cymene, caused a concentration-dependent CF leakage from all kinds of LUVs employed; in particular, thymol was more effective on PC and PC/SA LUVS than on PC/PS vesicles, while carvacrol challenge evoked a CF leakage from PC/PS LUVs similar to that induced from PC/SA LUVs, and lower than that measured with PC vesicles. Concerning DSC experiments, these four terpenes caused a decrease in Tm and (especially carvacrol and p-cymene) DeltaH values, very likely acting as substitutional impurities. Taken together, our findings lead us to speculate that the antimicrobial effect of thymol, carvacrol, p-cymene, and gamma-terpinene may result, partially at least, from a gross perturbation of the lipidic fraction of the plasmic membrane of the microorganism. In addition to being related to the physicochemical characteristics of the compounds (such as lipophilicity and water solubility), this effect seems to be dependent on the lipidic composition and net surface charge of the microbic membranes. Furthermore, the compounds might cross the cell membranes, thus penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity.     

Geographic classification of spanish and Australian tempranillo red wines by visible and near-infrared spectroscopy combined with multivariate analysis

Visible (vis) and near-infrared (NIR) spectroscopy combined with multivariate analysis was used to classify the geographical origin of commercial Tempranillo wines from Australia and Spain. Wines (n = 63) were scanned in the vis and NIR regions (400-2500 nm) in a monochromator instrument in transmission. Principal component analysis (PCA), discriminant partial least-squares discriminant analysis (PLS-DA) and linear discriminant analysis (LDA) based on PCA scores were used to classify Tempranillo wines according to their geographical origin. Full cross-validation (leave-one-out) was used as validation method when PCA and LDA classification models were developed. PLS-DA models correctly classified 100% and 84.7% of the Australian and Spanish Tempranillo wine samples, respectively. LDA calibration models correctly classified 72% of the Australian wines and 85% of the Spanish wines. These results demonstrate the potential use of vis and NIR spectroscopy, combined with chemometrics as a rapid method to classify Tempranillo wines accordingly to their geographical origin.