Sturgeon conservation genomics: SNP discovery and validation using RAD sequencing.

Caviar-producing sturgeons belonging to the genus Acipenser are considered to be one of the most endangered species groups in the world. Continued overfishing in spite of increasing legislation, zero catch quotas and extensive aquaculture production have led to the collapse of wild stocks across Europe and Asia. The evolutionary relationships among Adriatic, Russian, Persian and Siberian sturgeons are complex because of past introgression events and remain poorly understood. Conservation management, traceability and enforcement suffer a lack of appropriate DNA markers for the genetic identification of sturgeon at the species, population and individual level. This study employed RAD sequencing to discover and characterize single nucleotide polymorphism (SNP) DNA markers for use in sturgeon conservation in these four tetraploid species over three biological levels, using a single sequencing lane. Four population meta-samples and eight individual samples from one family were barcoded separately before sequencing. Analysis of 14.4 Gb of paired-end RAD data focused on the identification of SNPs in the paired-end contig, with subsequent in silico and empirical validation of candidate markers. Thousands of putatively informative markers were identified including, for the first time, SNPs that show population-wide differentiation between Russian and Persian sturgeons, representing an important advance in our ability to manage these cryptic species. The results highlight the challenges of genotyping-by-sequencing in polyploid taxa, while establishing the potential genetic resources for developing a new range of caviar traceability and enforcement tools.

Phylogenetic relationships and demographic histories of the Atherinidae in the Eastern Atlantic and Mediterranean Sea re-examined by Bayesian inference.

The aim of our study is to examine the phylogenetic relationship, divergence times and demographic history of the five close-related Mediterranean and North-eastern Atlantic species/forms of Atherina using the full Bayesian framework for species tree estimation recently implemented in ∗BEAST. The inference is made possible by multilocus data using three mitochondrial genes (12S rRNA, 16S rRNA, control region) and one nuclear gene (rhodopsin) from multiple individuals per species available in GenBank. Bayesian phylogenetic analysis of the complete gene dataset produced a tree with strong support for the monophyly of each species, as well as high support for higher level nodes. An old origin of the Atherina group was suggested (19.2 MY), with deep split events within the Atherinidae predating the Messinian Salinity Crisis. Regional genetic substructuring was observed among populations of A. boyeri, with AMOVA and MultiDimensional Scaling suggesting the existence of five groupings (Atlantic/West Mediterranean, Adriatic, Greece, Black Sea and Tunis). The level of subdivision found might be consequence of the hydrographic isolation within the Mediterranean Sea. Bayesian inference of past demographic histories showed a clear signature of demographic expansion for the European coast populations of A. presbyter, possibly linked to post-glacial colonizations, but not for the Azores/Canary Islands, which is expected in isolated populations because of the impossibility of finding new habitats. Within the Mediterranean, signatures of recent demographic expansion were only found for the Adriatic population of A. boyeri, which could be associated with the relatively recent emergence of the Adriatic Sea.

Multilocus phylogenetic analysis of the genus Atherina (Pisces: Atherinidae).

Sand-smelts are small fishes inhabiting inshore, brackish and freshwater environments and with a distribution in the eastern Atlantic and Mediterranean Sea, extending south into the Indian Ocean. Here, we present a broad phylogenetic analysis of the genus Atherina using three mitochondrial (control region, 12S and 16S) and two nuclear markers (rhodopsin and 2nd intron of S7). Phylogenetic analyses fully support the monophyly of the genus. Two anti-tropical clades were identified, separating the South African Atherina breviceps from the north-eastern Atlantic and Mediterranean Atherina' species. In European waters, two groups were found. The first clade formed by a well supported species-pair: Atherina presbyter (eastern Atlantic) and Atherina hepsetus (Mediterranean), both living in marine waters; a second clade included Atherina boyeri (brackish and freshwater environments) and two independent lineages of marine punctated and non-punctated fishes, recently proposed as separate species. Sequence divergence values strongly suggest multiple species within the A. boyeri complex.

Rett syndrome: a wide clinical and autonomic picture.

BACKGROUND: Rett Syndrome is a neurodevelopmental disorder almost exclusively affecting females, characterized by a broad clinical spectrum of signs and symptoms and a peculiar course. The disease affects different body systems: nervous, muscolo-skeletal, gastro-enteric. Moreover, part of the symptoms are related to the involvement of the autonomic nervous system. In the Tuscany Rett Center at Versilia Hospital, we collected data from 151 subjects with a clinical diagnosis of classical or variant RTT syndrome. For each subject, we assessed the severity of the condition with clinical-rating scales (ISS, PBZ), we quantified the performance of the autonomic nervous system, and we performed genetic analysis. We used multivariate statistical analysis of the data to evaluate the relation between the different clinical RTT forms, the cardiorespiratory phenotype, the different genetic mutations and the severity of the clinical picture. Individuals were classified according to existing forms: Classical RTT and three atypical RTT: Z-RTT, Hanefeld, Congenital. A correlation between C-Terminal deletions and lower severity of the clinical manifestations was evident, in the previous literature, but, considering the analysis of autonomic behaviour, the original classification can be enriched with a more accurate subdivision of Rett subgroups, which may be useful for early diagnosis. RESULTS: Present data emphasize some differences, not entirely described in the literature, among RTT variants. In our cohort the Z-RTT variant cases show clinical features (communication, growth, epilepsy and development), well documented by specific ISS items, less severe, if compared to classical RTT and show autonomic disorders, previously not reported in the literature. In this form epilepsy is rarely present. In contrast, Hanefeld variant shows the constant presence of epilepsy which has an earlier onset In Hanefeld variant the frequency of apneas was rare and, among the cardiorespiratory phenotypes, the feeble type is lacking. CONCLUSION: A quantitative analysis of the different autonomic components reveals differences across typical and atypical forms of RTT that leads to a more accurate classification of the groups. In our cohort of RTT individuals, the inclusion of autonomic parameter in the classification leads to an improved diagnosis at earlier stages of development.

Genomic Resources Notes accepted 1 April 2015 - 31 May 2015.

This article documents the public availability of transcriptomic resources for (i) the stellate sturgeon Acipenser stellatus, (ii) the flowering plant Campanula gentilis and (iii) two endemic Iberian fish, Squalius carolitertii and Squalius torgalensis.

Chromosomal changes in dysplastic nevi.

The dysplastic nevus is considered to be a precursor lesion of melanoma, representing one of the first steps in the progressive transformation from normal melanocyte to melanoma. Various risk degrees of developing cutaneous melanoma in patients with dysplastic nevi have been advanced, based on the presence of dysplastic nevi or melanoma or both in members of the patient's family. We report on the cytogenetic study of three nevi in a young patient with a family history of melanoma. Each nevus showed a simple clonal chromosome change. The t(6;15)(q13;q21) translocation found in one of them seems of particular significance in view of the fact that a similar one, with breakpoint at 6q13 was reported both in an acquired nevus from a patient with a family history of melanoma and in a case of cutaneous metastatic melanoma. These observations seem to support the hypothesis of the existence of a biological continuum between normal melanocyte and melanoma. Furthermore, the finding of chromosome changes similar to those associated with melanoma reinforces the need for a careful follow-up of patients with dysplastic nevi.

Bronchoalveolar glutathione and nitrite/nitrate in idiopathic pulmonary fibrosis and sarcoidosis.

BACKGROUND AND AIM OF THE WORK: Bronchoalveolar lavage (BAL) is useful in diagnosis and management of interstitial lung diseases. Glutathione (GSH) represents an important defence molecule against reactive oxygen species produced during inflammation, which underlies both idiopathic pulmonary fibrosis (IPF) and sarcoidosis. Nitric oxide has been suggested as a marker of airway inflammation. The aim of this study was to examine the content of GSH and NO stable metabolites, nitrite/nitrate, in the BAL of a group of patients with IPF and sarcoidosis. METHODS: BAL was performed in 13 patients with newly diagnosed IPF, 13 patients with sarcoidosis and 4 controls. Total GSH and nitrite/nitrate were measured. BAL was repeated, in 5 patients with IPF, after 6 and 12 months and GSH was measured again. RESULTS: IPF patients had significantly lower levels of total GSH compared to sarcoidosis patients or controls (129 +/- 22 vs. 324 +/- 40 and 383 +/- 23 mM; p < 0.001) in the epithelial lining fluid (ELF). In patients with IPF (n = 5), total GSH levels in the ELF increased after 6 and 12 months (T0 = 98 +/- 19; T6 = 219 +/- 22; T12 = 301 +/- 34 microM; p < 0.05) following therapy with immunosuppressive drugs such as corticosteroids, methotrexate, cyclosporin A. Levels of nitrite/nitrate were found significantly elevated in IPF (n = 8) and sarcoidosis (n = 10) patients compared to controls (230 +/- 37 and 216 +/- 36 vs. 86 +/- 11 microM; p < 0.05). CONCLUSION: These observations support the role of inflammation and antioxidant defences in interstitial lung diseases and may provide insights into the pathogenesis of oxidant-induced interstitial disease and their therapy.

Lack of melatonin effect on hydrogen peroxide induced bronchoconstriction in isolated and perfused rat lung.

The effect of melatonin on hydrogen peroxide- induced broncho-and vasoconstriction was examined in vivo in the model of the isolated, perfused and ventilated lung. The administration of hydrogen peroxide (500 microM) to the perfusate caused a marked decrease in lung compliance, conductance and flow rate. The administration of melatonin (500 microM) to the perfusate 20 min before and during the hydroperoxide exposure did not cause any change in lung function. Exposure of lung microsomes to hydrogen peroxide (1-100 microM) did not induce any significant increase in malonaldehyde (MDA), an index of lipid peroxidation, and it was not affected by treatment with melatonin (500 microM). On the other hand, brain microsomes exposed to hydrogen peroxide (1-100 microM) give rise to increased levels of MDA, which were decreased by pre-treatment with melatonin (500 microM). The results suggest that melatonin may exert an antioxidant effect in conditions were lipid peroxidation is occurring. Its use may not be relevant in conditions where the mechanisms of the reactive oxygen species damage appears to be lipid peroxidation independent, such as the case of hydrogen peroxide induced broncho- and vasoconstriction.

Influence of lead nitrate on dimethylnitrosamine intoxication.

The effect of lead nitrate, an inhibitor of the hepatic drug-metabolizing enzyme system upon the acute, hepatotoxicity of dimethylnitrosamine (DMN) was studied. Lead pretreatment significantly prevented polysomal disaggregation induced by the nitrosamine. Cell necrosis, evaluated morphologically and by the release of serum glutamic-pyruvic transaminase (GPT), was also diminished. The metabolism of DMN in rats pretreated with lead nitrate was investigated by following its clearance from blood and by determining, in vitro the demethylation of the nitrosamine. Lead increased, although not significantly, the clearance of DMN from blood, but it lowered the activity of DMN-demethylase 24 h after its administration. Finally, lead lowered the lethal effects of DMN. The mechanism by which lead influenced DMN toxicity is discussed.

Involvement of capsaicin-sensitive nerves in paraquat-induced mortality.

Paraquat (PQ), a broad spectrum herbicide, produces severe lung inflammation and necrosis resulting in pulmonary fibrosis and respiratory failure. Tachykinins are peptides released by sensory C fibers and have the ability of influencing respiratory functions and cellular proliferation. To examine whether the damage caused by PQ involves tachykinins, rats were depleted in their content of tachykinins by systemic treatment with capsaicin prior to PQ exposure. The animal subjected to this treatment showed a 3-fold higher viability compared to those treated with PQ alone (75 vs 27%). Depletion of reduced glutathione (GSH) is associated with oxidative stress produced by reactive oxygen intermediates during PQ metabolism. This is considered to be critical in the pathogenesis of lung damage by PQ. PQ treatment induced a significant depletion of GSH during the first days and a similar effect was also observed in the group of capsaicin-pretreated rats. Four weeks after PQ treatment the levels of GSH were similar to controls in rat pretreated or not with capsaicin plus PQ. This may indicate that the reduced levels of GSH may be associated to the toxicity observed in the acute phase, but not of importance in the final PQ-induced mortality. Neutral endopeptidase (NEP) is an enzyme considered to be critical in controlling the levels of tachykinins. Exposure of crude membrane preparations of rat lung to PQ resulted in a dose-dependent inhibition of NEP activity. Since NEP inactivation may occur in lung following a PQ exposure in vivo, the results indicate that during PQ intoxication a more sustained activity of tachykinins may be present, producing effects such as cell proliferation, fluid extravasation and bronchoconstriction. In conclusion, this finding supports the hypothesis that neuropeptides released from capsaicin-sensitive nerves could be involved in the modulation of PQ-induced lung damage.

Effect of verapamil on allyl alcohol hepatotoxicity.

The effects of verapamil, a calcium channel blocker, on allyl alcohol (AA) hepatotoxicity were studied in vivo. AA administration induced an increase of serum alanine aminotransferase (ALT) concentration and liver necrosis by means of glutathione (GSH) depletion. Pretreatment with verapamil reduced the increase of ALT in plasma and the morphological signs of necrosis induced by AA administration. Verapamil did not affect GSH levels by itself but prevented the decrease of the tripeptide by AA. In vitro, but not in vivo, verapamil inhibited the activity of alcohol dehydrogenase (ADH), the key enzyme in the conversion of AA into the toxic metabolite acrolein. These data indicate that verapamil protects against AA toxicity, probably by preventing the production of acrolein, its reactive metabolite.

Aspects of allyl alcohol toxicity.

The more recent experimental works on the chemistry, industrial uses and general toxicity (with particular reference to liver cell injury) of allyl alcohol (AA) have been briefly reviewed. AA is inactive per se and its toxic expression is modulated by its alcohol dehydrogenase (ADH) oxidation to form acrolein, which is responsible for the hepatotoxic action. The toxicity of the alcohol (or its metabolite acrolein) is dependent on the concentration of glutathione (GSH). After severe depletion of GSH, the reactive metabolite of AA can bind to essential sulfhydryl groups in the cellular macromolecules, leading to structural and functional modifications which can be responsible for cell death. In this case the appearance of lipid peroxidation could be merely the consequence of the death. GSH synthesis precursors exert a protective role in AA intoxication. The significance of calcium modifications in the course of AA toxicity is still under debate.

Mechanisms of PCBs mixture toxicity on isolated rat hepatocytes.

Exposure of isolated hepatocytes to a polychlorinated biphenyl mixture induced a rapid loss of cell viability. The effect was not dose-dependent. The biochemical effects in the cellular toxicity did not involve glutathione content, protein sulfhydryl groups and lipid peroxidation. A transient increase in cytosolic Ca2+ was observed after exposing the hepatocytes to the polychlorinated biphenyl mixture. Our findings indicate that polychlorinated biphenyls are able to kill hepatocytes and suggest that elevation of cytosolic Ca2+ concentration could be responsable of the toxicity.

[Mechanisms of tolerance to sulfur dioxide and sodium metabisulfite]. / Studio dei meccanismi della tolleranza al biossido di zolfo e al metabisolfito di sodio.

Inhalation of sulphur dioxide (250 ppm), (SO2) or sodium metabisulfite (80 mM) (MBS) aerosol or perfusion with MBS (3 mM) induced a reduction in compliance and conductance in the isolated, perfused and ventilated guinea pig lung. Pretreatment of the lung with sodium sulfite (3 mM), a dissolution product of SO2 and MBS, reduced the bronchoconstriction induced by SO2 and MBS. Bronchoconstriction induced by SO2 and MBS in associated to increased levels of Calcitonin gene-Related Peptide (CGRP) in the perfusate effinent, indicating activation of sensory nerves. The release of CGRP induced by SO2 and MBS was not affected by sodium sulfite. Sulfite treatment did not modify lung reactivity towards acethylcholine, bradykinin, serotonin, histamine and substance P (fragment 5-11). An inhibitory effect by sulfite was observed on bronchoconstriction induced by neurokinin A (fragment 4-10). Since bronchoconstriction induced by SO2 and MBS appears to be mediated by neurokinin A release and action, sulfite may act by affecting its signal transduction pathway. In conclusion, the results indicate that during exposure to some environmental and occupational pollutants, e.g. SO2 and MBS, critical modifications of sulfhydryl groups on smooth muscle receptors may occur. We hypothesise this as a possible step in the development of tolerance and hyperreactivity.

Species and hybrid identification of sturgeon caviar: a new molecular approach to detect illegal trade.

Overexploitation of wild populations due to the high economic value of caviar has driven sturgeons to near extinction. The high prices commanded by caviar on world markets have made it a magnet for illegal and fraudulent caviar trade, often involving low-value farmed caviar being sold as top-quality caviar. We present a new molecular approach for the identification of pure sturgeon species and hybrids that are among the most commercialized species in Europe and North America. Our test is based on the discovery of species-specific single nucleotide polymorphisms (SNPs) in the ribosomal protein S7, supplemented with the Vimentin gene and the mitochondrial D-loop. Test validations performed in 702 specimens of target and nontarget sturgeon species demonstrated a 100% identification success for Acipenser naccarii, A. fulvescens, A. stellatus, A. sinensis and A. transmontanus. In addition to species identification, our approach allows the identification of Bester and AL hybrids, two of the most economically important hybrids in the world, with 80% and 100% success, respectively. Moreover, the approach has the potential to identify many other existing sturgeon hybrids. The development of a standardized sturgeon identification tool will directly benefit trade law enforcement, providing the tools to monitor and regulate the legal trade of caviar and protect sturgeon stocks from illicit producers and traders, hence contributing to safeguarding this group of heavily threatened species.

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2010-31 January 2011.

This article documents the addition of 238 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alytes dickhilleni, Arapaima gigas, Austropotamobius italicus, Blumeria graminis f. sp. tritici, Cobitis lutheri, Dendroctonus ponderosae, Glossina morsitans morsitans, Haplophilus subterraneus, Kirengeshoma palmata, Lysimachia japonica, Macrolophus pygmaeus, Microtus cabrerae, Mytilus galloprovincialis, Pallisentis (Neosentis) celatus, Pulmonaria officinalis, Salminus franciscanus, Thais chocolata and Zootoca vivipara. These loci were cross-tested on the following species: Acanthina monodon, Alytes cisternasii, Alytes maurus, Alytes muletensis, Alytes obstetricans almogavarii, Alytes obstetricans boscai, Alytes obstetricans obstetricans, Alytes obstetricans pertinax, Cambarellus montezumae, Cambarellus zempoalensis, Chorus giganteus, Cobitis tetralineata, Glossina fuscipes fuscipes, Glossina pallidipes, Lysimachia japonica var. japonica, Lysimachia japonica var. minutissima, Orconectes virilis, Pacifastacus leniusculus, Procambarus clarkii, Salminus brasiliensis and Salminus hilarii.

Lipid peroxidation induced by N-acetylcysteine in isolated rat hepatocytes.

In isolated rat hepatocytes N-acetylcysteine induces an increase of lipid peroxidation, as evaluated by the malondialdehyde production and diene conjugation. Lipid peroxidation did not result in increased cell mortality. Antioxidants and free radicals scavengers completely protect toward lipid peroxidation induced by N-acetylcysteine.

[Reduced glutathione in rat liver intoxicated with allyl alcohol]. / Centenuto di glutatione ridotto nel fegato di ratto intossicato con alcool allilico.

Within 24 h after administration of allyl alcohol the livers showed periportal necrosis and elevation of hepatic glutathione. After 48 h no necrosis was observed in spite of level of hepatic glutathione. The relationship between the hepatic glutathione and liver regeneration is discussed.