Mito-nuclear discordance in the degree of population differentiation in a marine goby.

An increasing number of phylogeographic studies on marine species shows discordant patterns in the degree of population differentiation between nuclear and mitochondrial markers. To understand better which factors have the potential to cause these patterns of discordance in marine organisms, a population genetic study was realized on the sand goby Pomatoschistus minutus (Pallas 1770; Gobiidae, Teleostei). Sand gobies from eight European locations were genotyped at eight microsatellite markers. Microsatellites confirmed the global phylogeographical pattern of P. minutus observed with mitochondrial DNA (mtDNA) markers and nuclear allozyme markers. Three groups consistent with the mitochondrial lineages were defined (the Mediterranean, Iberian and North Atlantic groups) and indications of a recent founder event in the northern Baltic Sea were found. Nevertheless, differences in the degree of population differentiation between the nuclear and mitochondrial markers were large (global F(ST)-values for microsatellites=0.0121; for allozymes=0.00831; for mtDNA=0.4293). Selection, sex-biased dispersal, homoplasy and a high effective population size are generally accepted as explanations for this mitonuclear discrepancy in the degree of population differentiation. In this study, selection on mtDNA and microsatellites, male-biased dispersal and homoplasy on microsatellite markers are unlikely to be a main cause for this discrepancy. The most likely reason for the discordant pattern is a recent demographical expansion of the sand goby, resulting in high effective population sizes slowing down the differentiation of nuclear DNA.

QTL for body weight, morphometric traits and stress response in European sea bass Dicentrarchus labrax.

Natural mating and mass spawning in the European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) complicate genetic studies and the implementation of selective breeding schemes. We utilized a two-step experimental design for detecting QTL in mass-spawning species: 2122 offspring from natural mating between 57 parents (22 males, 34 females and one missing) phenotyped for body weight, eight morphometric traits and cortisol levels, had been previously assigned to parents based on genotypes of 31 DNA microsatellite markers. Five large full-sib families (five sires and two dams) were selected from the offspring (570 animals), which were genotyped with 67 additional markers. A new genetic map was compiled, specific to our population, but based on the previously published map. QTL mapping was performed with two methods: half-sib regression analysis (paternal and maternal) and variance component analysis accounting for all family relationships. Two significant QTL were found for body weight on linkage group 4 and 6, six significant QTL for morphometric traits on linkage groups 1B, 4, 6, 7, 15 and 23 and three suggestive QTL for stress response on linkage groups 3, 14 and 23. The QTL explained between 8% and 38% of phenotypic variance. The results are the first step towards identifying genes involved in economically important traits like body weight and stress response in European sea bass.

Matrilinear phylogeography and demographical patterns of Rutilus rutilus: implications for taxonomy and conservation.

A phylogeographic analysis of mitochondrial DNA sequence variation was carried out to infer the geographical distribution of the genealogical lineages and the historical demography of roach Rutilus rutilus (L.). A total of 265 individuals from 52 sites covering most of the Eurasian distribution range were sequenced for a 475 bp fragment of the mitochondrial cytochrome b gene. The monophyletic roach contained two deep clades that dated back to the Pliocene. The Ponto-Caspian clade comprised populations from Greece to Siberia with a likely palaeorefugium at the west coast of the Caspian Sea. This clade largely corresponds to individuals with morphological features described as Rutilus heckelii. The west European clade included individuals from central and western Europe with the Danube and Dniester basins as possible palaeorefugia. This clade largely corresponds to individuals with morphological features described as R. rutilus. A suture-zone of the two main lineages was observed along the coastal region of the Black Sea. The neutrality tests and the mismatch distributions indicated a demographic expansion during the Middle-Pleistocene for both clades.

Positive non-linear capacitance: the origin of the steep subthreshold-slope in ferroelectric FETs.

We show that the non-linear positive capacitance (PC) of ferroelectrics (FE) can explain the steep subthreshold-slope (SS) observed in FE based MOSFETs and often attributed to the existence of a negative capacitance in FE capacitors. Physically attainable and unattainable regions of the S-shape curve used in the negative capacitance theory are investigated by self-consistently solving Landau-Khalatnikov and Maxwell equations and by experimental validation. Finally, the conditions for attaining a steep SS in FE based MOSFETs assuming only positive capacitances are discussed.

Performance and precision of double digestion RAD (ddRAD) genotyping in large multiplexed datasets of marine fish species.

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in "non-model" species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 1012 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies.

Identification of the cytochrome P450 enzymes involved in the metabolism of cisapride: in vitro studies of potential co-medication interactions.

Cisapride is a prokinetic drug that is widely used to facilitate gastrointestinal tract motility. Structurally, cisapride is a substituted piperidinyl benzamide that interacts with 5-hydroxytryptamine-4 receptors and which is largely without central depressant or antidopaminergic side-effects. The aims of this study were to investigate the metabolism of cisapride in human liver microsomes and to determine which cytochrome P-450 (CYP) isoenzyme(s) are involved in cisapride biotransformation. Additionally, the effects of various drugs on the metabolism of cisapride were investigated. The major in vitro metabolite of cisapride was formed by oxidative N-dealkylation at the piperidine nitrogen, leading to the production of norcisapride. By using competitive inhibition data, correlation studies and heterologous expression systems, it was demonstrated that CYP3A4 was the major CYP involved. CYP2A6 also contributed to the metabolism of cisapride, albeit to a much lesser extent. The mean apparent K(m) against cisapride was 8.6+/-3.5 microM (n = 3). The peak plasma levels of cisapride under normal clinical practice are approximately 0.17 microM; therefore it is unlikely that cisapride would inhibit the metabolism of co-administered drugs. In this in vitro study the inhibitory effects of 44 drugs were tested for any effect on cisapride biotransformation. In conclusion, 34 of the drugs are unlikely to have a clinically relevant interaction; however, the antidepressant nefazodone, the macrolide antibiotic troleandomycin, the HIV-1 protease inhibitors ritonavir and indinavir and the calcium channel blocker mibefradil inhibited the metabolism of cisapride and these interactions are likely to be of clinical relevance. Furthermore, the antimycotics ketoconazole, miconazole, hydroxy-itraconazole, itraconazole and fluconazole, when administered orally or intravenously, would inhibit cisapride metabolism.

Induction potential of antifungals containing an imidazole or triazole moiety. Miconazole and ketoconazole, but not itraconazole are able to induce hepatic drug metabolizing enzymes of male rats at high doses.

Male Wistar rats were dosed with miconazole, ketoconazole and itraconazole by gastric intubation once daily for up to 7 days. A dose- and time-dependent induction of the hepatic drug metabolizing enzyme system was observed for miconazole and ketoconazole, while itraconazole proved to be devoid of inductive properties even at the highest dose studied (160 mg/kg). No effect on drug metabolizing enzymes could be demonstrated for either drug at a dose level of 10 mg/kg, which is just above the antifungally active dose. At a dose of 40 mg/kg, miconazole, but not ketoconazole, significantly increased cytochrome P-450 content. At the highest dose of 160 mg/kg, both miconazole and ketoconazole increased the relative liver weight, the cytochrome P-450- and b5-content and NADPH-cyt c-reductase. Furthermore, miconazole, but not ketoconazole, increased specific microsomal aminopyrine and N,N-dimethylaniline N-demethylase activity, p-nitroanisole O-demethylase activity and UDP-glucuronyltransferase activity towards 4-nitrophenol while the specific aniline hydroxylase activity was unaffected. Ketoconazole at 160 mg/kg only induced O-demethylase activity and UDP-glucuronyltransferase activity, while it lowered the specific activities towards the other substrates. Miconazole was a relatively more potent inducer when compared to ketoconazole. Both drugs displayed biphasic effects on the mixed-function oxidase activities, which were lowered after acute administration (160 mg/kg, 1 hr before death) and were induced when determined after 23 hr had elapsed or after multiple dosage. Both drugs bound strongly to their respective induced cytochromes, giving rise to type II difference spectra, and inhibited the O-demethylase activity of the induced microsomes with an I50 of 5.2 microM for miconazole and 15.1 microM for ketoconazole. On the basis of a comparison of the enzymatic activities induced by both antimycotics with those induced by PB or 3-MC, it was concluded that miconazole behaved as a PB-type inducer, whereas ketoconazole did not belong to either category of inducers. A comparison of electrophoretograms of microsomes from different origins on SDS-PAGE revealed that miconazole increased the concentration of several proteins, whereas ketoconazole selectively induced a protein with Mr of 47,800. The protein pattern in the 50 kDa region of miconazole-induced microsomes resembled that of PB-microsomes qualitatively.

The release of mutagens from airborne particles in the presence of physiological fluids.

Airborne particulates collected indoors in residences and outdoors were extracted by soxhlet extraction and sonication with methanol. In a comparative study in which mutagenic activity was evaluated in the Salmonella typhimurium reversion assay both soxhlet extraction and sonication proved to be suitable extraction methods. First, the residue, obtained by sonication of loaded filters with methanol followed by evaporation to dryness (tar), was sonicated with newborn calf serum and lung lavage fluid from pigs. All serum extracts of the tar were mutagenic to Salmonella typhimurium TA98, and contained direct- and indirect-acting mutagens. However, the mutagenic activity recovered by serum was only about half of the total mutagenic activity of the tar. The other part of the mutagenic activity remained in the tar. Lung lavage fluid was only able to remove 5-10% of direct-acting mutagens from the tar of all samples. About 20% of indirect-acting mutagens from indoor air were recovered in lung lavage fluid, while the lung lavage fluid extract from outdoor air did not show indirect mutagenic activity. Second, mutagenic activity recovered by direct sonication of the filters with physiological fluids was comparable with the recovery obtained by sonication of the tar. However, after sonication of the filter with lung lavage fluid hardly any mutagenic activity remained on the filter, whereas after sonication of the tar a clear mutagenic activity was observed in the non-soluble residue.

Contribution of wood stoves and fire places to mutagenic activity of airborne particulate matter inside homes.

Wood combustion produces compounds that are mutagenic in the Salmonella/microsome assay. As combustion products can be emitted in the home and the use of wood as a residential energy source is growing, an impact on human health might be of concern. In this study experiments were carried out to determine the contribution of wood combustion in stoves and fire places to indoor mutagenic activity under normal living conditions. Airborne particles from living rooms which were heated by stoves, or by fire places, and from outdoors were collected simultaneously. In each room two samples were collected during two consecutive weeks: one week the room was heated by central heating, the other week by wood combustion. Sampling took place in a total of 24 homes. Methanol extracts of the samples were tested in the Salmonella/mammalian microsome assay. Results show that mutagenic activity of outdoor air exceeds indoor mutagenicity. At the same time a correlation is found between in- and out-door mutagenicity, both with and without S9. However, a large difference is found between the ratio -S9/+S9 of in- and out-door mutagenic activity. Systematic differences in the ratio -S9/+S9 between control and experimental conditions are not observed. The use of wood stoves caused an increase of indoor mutagenicity in 8 out of 12 homes. It could be concluded that the use of an open fire consistently leads to an increase of mutagenic activity. This increase was caused by wood combustion products.

Toxicity of airborne particulate matter to rat alveolar macrophages: A comparative study of five extracts collected indoors and outdoors.

The toxicity of indoor and outdoor air samples to rat alveolar macrophages was determined by studying the phagocytic activity of these cells in vitro. Clean air samples did not affect phagocytosis at concentrations up to 60 m(3)/plate, but polluted outdoor samples caused a dose-dependent inhibition of the phagocytosis at concentrations varying from 6 m(3) to 60 m(3)/plate. Indoor air samples, especially when polluted with wood smoke, affected phagocytosis at concentrations below 2 m(3)/plate.

Recovering full DNA barcodes from natural history collections of Tephritid fruitflies (Tephritidae, Diptera) using mini barcodes.

The family of Tephritid fruit flies (Tephritidae, Diptera) is composed of more than 4000 species and more than 350 are of economic importance (EI). The Tephritid Barcoding Initiative (TBI) aims at obtaining DNA barcodes for all EI species and the majority of their congeners. Dry pinned specimens from natural history collections are an important resource for reference material, but were often collected decades ago. We observed a strong decrease in the success rate of obtaining a full COX1 DNA barcode (658 bp), with an increasing age of the specimens. Obtaining full barcodes is often not possible using standard protocols. We developed a universal Tephritid primer set for multiple overlapping mini-barcodes that allows reconstructing the full COX1 DNA barcode. These newly developed primers and the corresponding protocol will facilitate the utilization of the extensive natural history collection by the TBI consortium.

Isolation and characterization of expressed sequence tag-linked microsatellite loci for the European eel (Anguilla anguilla).

A European eel (Anguilla anguilla) expressed sequence tag database consisting of 795 contigs and 4008 singletons was screened for microsatellites sequences. Primers were designed to amplify 96 repeats, of which 86 gave good quality amplification products. Twenty-eight microsatellites were selected for further microsatellite genotyping. Only two loci were found to be monomorphic; out of the 26 polymorphic loci, number of alleles per locus ranged from two to 14, while the observed and expected heterozygosities ranged from 0.05 to 0.93, and from 0.05 to 0.95, respectively. All 28 primer sets tested revealed positive amplification in American eel (Anguilla rostrata).

Development and characterization of eight polymorphic microsatellite markers for Daphnia atkinsoni (Crustacea: Ctenodaphnia).

A microsatellite-enriched genomic library was developed for the water flea Daphnia atkinsoni Baird, 1859, a dominant species of intermittent wetlands in Europe. Eight polymorphic microsatellite markers were successfully optimized. Characterization of 77 individuals from Belgium and Spain showed moderate (in the former) to high (in the latter) levels of polymorphism with two to 11 alleles per locus and an observed heterozygosity ranging from 0 to 0.87. Some of these microsatellite markers were successfully amplified in three other Daphnia species (D. magna n = 4, D. similis n = 6; D. obtusa n = 6).

Development of nine polymorphic microsatellite loci in the spined loach, Cobitis taenia, and cross-species amplification in the related species C. elongatoides, C. taurica and C. tanaitica.

Nine polymorphic microsatellite loci were developed for the spined loach, Cobitis taenia (Teleostei: Cobitidae). The loci were validated using 50 individuals from a population in Belgium. Moderate to high levels of polymorphism were detected (two to 11 alleles). In addition, most markers amplified successfully in three closely related taxa that are known to hybridize with C. taenia: C. elongatoides, C. taurica and C. tanaitica. Some of the loci are most likely diagnostic among species. These markers will be valuable for the study of the historical and contemporary interactions within C. taenia and the Cobitis species complex.

A mitogenic view on the evolutionary history of the Holarctic freshwater gadoid, burbot (Lota lota).

Climatic oscillations during the Pleistocene epoch had a dramatic impact on the distribution of biota in the northern hemisphere. In order to trace glacial refugia and postglacial colonization routes on a global scale, we studied mitochondrial DNA sequence variation in a freshwater fish (burbot, Lota lota; Teleostei, Gadidae) with a circumpolar distribution. The subdivision of burbot in the subspecies Lota lota lota (Eurasia and Alaska) and Lota lota maculosa (North America, south of the Great Slave Lake) was reflected in two distinct mitochondrial lineages (average genetic distance is 2.08%). The lota form was characterized by 30 closely related haplotypes and a large part of its range (from Central Europe to Beringia) was characterized by two widespread ancestral haplotypes, implying that transcontinental exchange/migration was possible for cold-adapted freshwater taxa in recent evolutionary time. However, the derived mitochondrial variants observed in peripheral populations point to a recent separation from the core group and postglacial recolonization from distinct refugia. Beringia served as refuge from where L. l. lota dispersed southward into North America after the last glacial maximum. Genetic variation in the maculosa form consisted of three mitochondrial clades, which were linked to at least three southern refugia in North America. Two mitochondrial clades east of the Continental Divide (Mississippian and Missourian clades) had a distinct geographical distribution in the southern refuge zones but intergraded in the previously glaciated area. The third clade (Pacific) was exclusively found west of the Continental Divide.

Phylogenetic relationships among Palearctic and Nearctic burbot (Lota lota): Pleistocene extinctions and recolonization.

The burbot (Lota lota Linnaeus, 1758) is the only freshwater species from the cod family. Various taxonomic hypotheses were tested against molecular data by sequencing the mitochondrial cytochrome b locus of 120 burbot from 41 populations together with the related species Molva molva (ling) and Brosme brosme (tusk), which represented the other Lotinae genera. Within the genus Lota two distinct phylogroups were observed: one in North America south of the Great Slave Lakes (Lota lota maculosa) and one in Eurasia and the remainder of the Nearctic region (Lota lota lota). The burbot lineage separated 10 Myr BP from the other Lotinae, while the genetic variation within burbot appeared to be approximately 1 Myr old. However, fossil evidence suggested that burbot already existed in the Early Pliocene in Europe, from were it probably colonized North America in the Early Pleistocene. While Nearctic burbot survived climatic oscillations and diverged in several refugia, the Eurasian form became extinct or was reduced to a very small population. In the Late Pleistocene the species recolonized the Palearctic region to establish its present distribution range.

Interaction of miconazole, ketoconazole and itraconazole with rat-liver microsomes.

The interaction of the antimycotics miconazole, ketoconazole and itraconazole with liver microsomes from untreated rats or from rats pretreated with phenobarbital or 3-methylcholanthrene, gave rise to type II difference spectra. The interactions of the antimycotics with control, phenobarbital-induced or 3-methylcholanthrene-induced microsomes were biphasic, except for the monophasic binding of ketoconazole to phenobarbital-induced microsomes. The N-demethylation of N,N-dimethylaniline, the O-demethylation of p-nitroanisole and the hydroxylation of aniline in microsomes from untreated and inducer-treated rats were lowered by miconazole and ketoconazole, the former being the more potent inhibitor. Control microsomes were less sensitive than induced microsomes. Itraconazole was almost devoid of inhibitory properties. The three antimycotics were non-competitive (mixed) inhibitors of enzyme activities in phenobarbital-induced microsomes. The Ki values were of the same order of magnitude as the Ks values, except for itraconazole. For the latter drug, Ki values were much greater than could be expected from the spectral studies. It is concluded that the antimycotics affect microsomal enzyme activities via a direct interaction of an azole-nitrogen with the haem group of cytochrome P-450. The interaction with mammalian cytochrome P-450 decreases from miconazole greater than ketoconazole much greater than itraconazole and is much weaker than the interaction of the antimycotics with yeast cytochrome P-450.

Plasma protein binding of ketanserin and its distribution in blood.

The in vitro plasma protein binding and distribution in blood of ketanserin ((+/-)-3-[2-[4-(4-fluorobenzoyl)-1- piperidinyl]ethyl]-2,4(1H,3H)-quinazolinedione, R 41 468), a novel serotonin S2-receptor antagonist used in hypertension, was studied in rats, dogs and humans. Plasma protein binding of ketanserin amounted to 95.1% in healthy subjects, 88.1% in dogs and 98.8% in rats. Its blood to plasma concentration ratio was 0.70 in humans, 0.78 in dogs and 0.65 in rats. Plasma protein binding of ketanserin-ol, the main plasma metabolite of ketanserin, was 81.2% in humans and its blood to plasma concentration ratio was 1.04. The plasma protein binding of both ketanserin and ketanserin-ol was highly dependent on pH. Albumin was by far the main binding protein for ketanserin in human plasma and binding was independent of the ketanserin concentration within a very wide range. Plasma protein binding of ketanserin in elderly hypertensive patients was not significantly different from that in healthy adults. In chronic renal failure patients, whether on haemodialysis or not, the free ketanserin fraction was 40% higher than in healthy subjects. High therapeutic levels of ketanserin (0.25 microgram/ml) did not influence the plasma protein binding of diphenylhydantoin, hydrochlorothiazide, imipramine, ketoconazole, propranolol or warfarin. Out of 12 drugs, only tolbutamide at therapeutic concentrations decreased significantly the plasma protein binding of ketanserin. However, the resulting 5-20% increase of the free ketanserin fraction is hardly clinically relevant.