From Caves to the Savannah, the Mitogenome History of Modern Lions (Panthera leo) and Their Ancestors.

Lions (Panthera leo) play a crucial ecological role in shaping and maintaining fragile ecosystems within Africa. Conservation efforts should focus on genetic variability within wild populations when considering reintroduction attempts. We studied two groups of lions from two conservation sites located in Zambia and Zimbabwe to determine their genetic make-up, information that is usually unknown to the sites. In this study, we analysed 17 specimens for cytb and seven microsatellite markers to ascertain family relationships and genetic diversity previously obtained by observational studies. We then produced a standardised haplogroup phylogeny using all available entire mitogenomes, as well as calculating a revised molecular clock. The modern lion lineage diverged ~151 kya and was divided into two subspecies, both containing three distinct haplogroups. We confirm that Panthera leo persica is not a subspecies, but rather a haplogroup of the northern P.l. leo that exited Africa at least ~31 kya. The progenitor to all lions existed ~1.2 Mya, possibly in SE Africa, and later exited Africa and split into the two cave lion lineages ~175 kya. Species demography is correlated to major climactic events. We now have a detailed phylogeny of lion evolution and an idea of their conservation status given the threat of climate change.

Tropical rainforests that persisted: inferences from the Quaternary demographic history of eight tree species in the Guiana shield.

How Quaternary climatic and geological disturbances influenced the composition of Neotropical forests is hotly debated. Rainfall and temperature changes during and/or immediately after the last glacial maximum (LGM) are thought to have strongly affected the geographical distribution and local abundance of tree species. The paucity of the fossil records in Neotropical forests prevents a direct reconstruction of such processes. To describe community-level historical trends in forest composition, we turned therefore to inferential methods based on the reconstruction of past demographic changes. In particular, we modelled the history of rainforests in the eastern Guiana Shield over a timescale of several thousand generations, through the application of approximate Bayesian computation and maximum-likelihood methods to diversity data at nuclear and chloroplast loci in eight species or subspecies of rainforest trees. Depending on the species and on the method applied, we detected population contraction, expansion or stability, with a general trend in favour of stability or expansion, with changes presumably having occurred during or after the LGM. These findings suggest that Guiana Shield rainforests have globally persisted, while expanding, through the Quaternary, but that different species have experienced different demographic events, with a trend towards the increase in frequency of light-demanding, disturbance-associated species.

G72 primate-specific gene: a still enigmatic element in psychiatric disorders.

Numerous studies have demonstrated a link between genetic markers on chromosome 13 and schizophrenia, bipolar affective disorder, and other psychiatric phenotypes. The G72/G30 genes (transcribed in opposite directions) are located on chromosome 13q33, a region demonstrating strong evidence for linkage with various neuropsychiatric disorders. G72/G30 was identified in 2002 as a schizophrenia susceptibility locus; however, subsequent association studies did not reach consensus on single SNPs within the locus. Simultaneously, a new vision for the genetic architecture of psychiatric disorders suggested that schizophrenia was a quantitative trait, therefore ascribable to potentially hundreds of genes and subjected to the vagaries of the environment. The main protein product of G72 gene is named pLG72 or D-amino acid oxidase activator DAOA (153 amino acids) and its function is still debated. Functional analyses, also showing controversial results, indicate that pLG72 contributes to N-methyl-D-aspartate receptor modulation by affecting activity of the flavoprotein D-amino acid oxidase, the enzyme responsible for degrading the neuromodulator D-serine. In this review we, for the first time, summarize findings from molecular genetic linkage and association studies concerning G72 gene, cellular and molecular studies on pLG72, and investigations performed on G72/G30 transgenic mice. This will help elucidate the role of psychosis susceptibility genes, which will have a major impact on our understanding of disease pathophysiology and thus change classification and treatment.

TAS2R38 taste receptor gene and chronic rhinosinusitis: new data from an Italian population.

BACKGROUND: Chronic rhinosinusitis (CRS) is a frequent disease with high social impact and multifactorial pathogenesis. Recently, single nucleotide polymorphisms within the TAS2R38 gene have been implicated as possible contributors to the complex gene-environment interactions in CRS. The purpose of this study was to confirm the proposed correlation between TAS2R38 genotype, CRS and related comorbidities. METHODS: Fifty-three CRS patients and 39 healthy individuals were genotyped at the TAS2R38 locus. CRS patients were treated by endoscopic sinus surgery and medical therapies and subdivided in CRS with nasal polyps (CRSwNPs) and CRS without nasal polyps (CRSsNPs). The effect of genotype on CRS and CRS-related comorbidities was assessed. RESULTS: The distribution of the different genotypes at the TAS2R38 locus was not significantly different between CRS patients, either with or without nasal polyps, and controls. Besides, no association was found between the different genotypes at the TAS2R38 locus and CRS-related comorbidities. CONCLUSIONS: No association was found between TAS2R38 alleles or genotypes and CRS, thus questioning its role in the pathogenesis of CRS.

Genetic variants in apoptosis-related genes associated with colorectal hyperplasia.

Deregulation of apoptosis is a frequent alteration in early benign lesions of the colon mucosa and is thought to be a major contributor to tumor progression and cancer. Single nucleotide polymorphisms (SNPs) within apoptosis-related genes could affect apoptotic responses and their identification might provide a basis to assess individual risk for development of early lesions. To investigate a possible association between genetic polymorphisms and the occurrence of hyperplastic polyps (HP), we developed a custom DNA chip assay for 1,536 SNPs in the coding and flanking regions of 826 genes with known functional roles in apoptosis or apoptosis-associated (e.g., stress-related) pathways. During a first round of screening, genotypes were determined for 272 endoscopy patients harboring hyperplastic colorectal polyps and for 512 sex and aged-matched controls. A set of 14 candidate SNPs associated with HP (P < 0.01) was then evaluated in an independent cohort of patients (n = 38) and controls (n = 38). Following meta-analysis of Stages I and II, a false discovery rate approach was applied. Among the 14 candidate SNPs, eight showed significant association (combined P < 0.01) with the occurrence of HP. The SNPs rs4709583 (PARK2) and rs10476823 (HDAC3) were analyzed for potential functional effects on RNA splicing and RNA half-life. Despite its location near a splice site, alternative splicing was not detected for rs4709583 (PARK3). By contrast, cDNA analysis revealed use of a cryptic polyadenylation signal in the 3'UTR of HDAC3 mRNA and a longer mRNA half-life in a cell line heterozygous for rs10476823.

A Study of GUS Expression in Arabidopsis as a Tool for the Evaluation of Gene Evolution, Function and the Role of Expression Derived from Gene Duplication.

Gene duplication played a fundamental role in eukaryote evolution and different copies of a given gene can be present in extant species, often with expressions and functions differentiated during evolution. We assume that, when such differentiation occurs in a gene copy, this may be indicated by its maintenance in all the derived species. To verify this hypothesis, we compared the histological expression domains of the three ß-glucuronidase genes (AtGUS) present in Arabidopsis thaliana with the GUS evolutionary tree in angiosperms. We found that AtGUS gene expression overlaps in the shoot apex, the floral bud and the root hairs. In the root apex, AtGUS3 expression differs completely from AtGUS1 and AtGUS2, whose transcripts are present in the root cap meristem and columella, in the staminal cell niche, in the epidermis and in the proximal cortex. Conversely, AtGUS3 transcripts are limited to the old border-like cells of calyptra and those found along the protodermal cell line. The GUS evolutionary tree reveals that the two main clusters (named GUS1 and GUS3) originate from a duplication event predating angiosperm radiation. AtGUS3 belongs to the GUS3 cluster, while AtGUS1 and AtGUS2, which originate from a duplication event that occurred in an ancestor of the Brassicaceae family, are found together in the GUS1 cluster. There is another, previously undescribed cluster, called GUS4, originating from a very ancient duplication event. While the copy of GUS4 has been lost in many species, copies of GUS3 and GUS1 have been conserved in all species examined.

Oxidative stress enhances the therapeutic action of a respiratory inhibitor in MYC-driven lymphoma.

MYC is a key oncogenic driver in multiple tumor types, but concomitantly endows cancer cells with a series of vulnerabilities that provide opportunities for targeted pharmacological intervention. For example, drugs that suppress mitochondrial respiration selectively kill MYC-overexpressing cells. Here, we unravel the mechanistic basis for this synthetic lethal interaction and exploit it to improve the anticancer effects of the respiratory complex I inhibitor IACS-010759. In a B-lymphoid cell line, ectopic MYC activity and treatment with IACS-010759 added up to induce oxidative stress, with consequent depletion of reduced glutathione and lethal disruption of redox homeostasis. This effect could be enhanced either with inhibitors of NADPH production through the pentose phosphate pathway, or with ascorbate (vitamin C), known to act as a pro-oxidant at high doses. In these conditions, ascorbate synergized with IACS-010759 to kill MYC-overexpressing cells in vitro and reinforced its therapeutic action against human B-cell lymphoma xenografts. Hence, complex I inhibition and high-dose ascorbate might improve the outcome of patients affected by high-grade lymphomas and potentially other MYC-driven cancers.

Human Adipose-Derived Stem Cell-Conditioned Medium Promotes Vascularization of Nanostructured Scaffold Transplanted into Nude Mice.

Several studies have been conducted on the interaction between three-dimensional scaffolds and mesenchymal stem cells for the regeneration of damaged tissues. Considering that stem cells do not survive for sufficient time to directly sustain tissue regeneration, it is essential to develop cell-free systems to be applied in regenerative medicine. In this work, by in vivo experiments, we established that a collagen-nanostructured scaffold, loaded with a culture medium conditioned with mesenchymal stem cells derived from adipose tissue (hASC-CM), exerts a synergic positive effect on angiogenesis, fundamental in tissue regeneration. To this aim, we engrafted athymic BALB-C nude mice with four different combinations: scaffold alone; scaffold with hASCs; scaffold with hASC crude protein extract; scaffold with hASC-CM. After their removal, we verified the presence of blood vessels by optical microscopy and confirmed the vascularization evaluating, by real-time PCR, several vascular growth factors: CD31, CD34, CD105, ANGPT1, ANGPT2, and CDH5. Our results showed that blood vessels were absent in the scaffold grafted alone, while all the other systems appeared vascularized, a finding supported by the over-expression of CD31 and CDH5 mRNA. In conclusion, our data sustain the capability of hASC-CM to be used as a therapeutic cell-free approach for damaged tissue regeneration.

Determination of cannabinoids in hair: Indicators for illegal vs CBD-rich cannabis use.

Cannabis products rich in cannabidiol (CBD) and low in Δ9-tetrahydrocannabinol (THC) (e.g., light cannabis in Italy) are becoming widely popular and available on the market as replacements for THC preparations and tobacco for their recreational and/or therapeutic benefits. In this paper, which aims to establish alternative discrimination parameters between hair samples from CBD-rich and THC-prevalent cannabis users, cannabinoid concentrations, such as THC, CBD, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) and 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) were quantified in 127 hair samples by a GC-MS/MS technique. Initially, this analysis was able to discriminate two cohorts: cohort 1 (individuals with THC values ≥ 0.05 ng/mg and THC-COOH ≥ 0.2 pg/mg or THC-positive users, n = 60) and cohort 2 (individuals with THC values ranging between 0.01 and 0.05 ng/mg and THC-COOH or 11-OH-THC ≥ LOQs, n = 67). The evaluation of CBD/THC ratio in cohort 2 identified two further sub-cohorts 2a (CBD/THC<<1 or ~ 1, THC-prevalent cannabis users) and 2b (CBD/THC>>1, suspected CBD-rich and THC-low cannabis users). The latter showed unusual profiles for THC metabolites, in particular for 11-OH-THC. Statistical evaluation of the data of cohort 1, cohort 2a and cohort 2b yielded significant differences in CBD/THC and THC/11-OH-THC. Based on the analysis of 337 seized cannabis samples and 630 CBD-rich/light cannabis samples by GC-FID and GC-MS, respectively, we also evaluated statistical differences in the CBD/THC ratio between biological (hair) and plant-derived samples. Considering the legal implications of a positive result, the obtained findings could be relevant for the interpretation of cannabinoid concentrations in hair. Further studies are necessary to elucidate the reason behind the unusual metabolic ratios.

Genetic Variability and Admixture Zones in the Italian Populations of Turkey Oak (Quercus cerris L.).

The Turkey oak (Quercus cerris L.) is widely distributed in Italy, where it is the ecologically dominant oak on sandy and acidic soil. In this work, we analysed 23 natural populations by means of eight SSR (microsatellite) markers, to obtain the first synthetic map of genetic variability for this species and to study its dispersion during the Holocene, due to the possibility that at least one refugium during the Last Glacial Maximum was in Italy. The analyses showed a good amount of genetic variability together with fair differentiation between populations, as indicated by FST = 0.059. A Bayesian analysis of the amount of admixture among populations revealed the presence of four putative gene pools of origin and a rough subdivision of the populations according to their geographic location, as confirmed by the spatial analysis. No evidence for the existence of putative refugial populations was found; however, this study paves the way for the planning of conservation strategies also with regard to the relationship between Turkey oak and other oak species in Italy.

Aberrant activation of p53/p66Shc-mInsc axis increases asymmetric divisions and attenuates proliferation of aged mammary stem cells.

Aging is accompanied by the progressive decline in tissue regenerative capacity and functions of resident stem cells (SCs). Underlying mechanisms, however, remain unclear. Here we show that, during chronological aging, self-renewing mitoses of mammary SCs (MaSCs) are preferentially asymmetric and that their progeny divides less frequently, leading to decreased number of MaSCs and reduced regenerative potential. Underlying mechanisms are investigated in the p66Shc-/- mouse, which exhibits several features of delayed aging, including reduced involution of the mammary gland (MG). p66Shc is a mitochondrial redox sensor that activates a specific p53 transcriptional program, in which the aging-associated p44 isoform of p53 plays a pivotal role. We report here that aged p66Shc-/- MaSCs show increased symmetric divisions, increased proliferation and increased regenerative potential, to an extent reminiscent of young wild-type (WT) MaSCs. Mechanistically, we demonstrate that p66Shc, together with p53: (i) accumulates in the aged MG, (ii) sustains expression of the cell polarity determinant mInscuteable and, concomitantly, (iii) down-regulates critical cell cycle genes (e.g.,: Cdk1 and Cyclin A). Accordingly, overexpression of p53/p44 increases asymmetric divisions and decreases proliferation of young WT MaSCs in a p66Shc-dependent manner and overexpression of mInsc restores WT-like levels of asymmetric divisions in aged p66Shc-/- MaSCs. Notably, deletion of p66Shc has negligible effects in young MaSCs and MG development. These results demonstrate that MG aging is due to aberrant activation of p66Shc, which induces p53/p44 signaling, leading to failure of symmetric divisions, decreased proliferation and reduced regenerative potential of MaSCs.

Hyaluronan Regulates Neuronal and Immune Function in the Rat Small Intestine and Colonic Microbiota after Ischemic/Reperfusion Injury.

BACKGROUND: Intestinal ischemia and reperfusion (IRI) injury induces acute and long-lasting damage to the neuromuscular compartment and dysmotility. This study aims to evaluate the pathogenetic role of hyaluronan (HA), a glycosaminoglycan component of the extracellular matrix, as a modulator of the enteric neuronal and immune function and of the colonic microbiota during in vivo IRI in the rat small intestine. METHODS: mesenteric ischemia was induced in anesthetized adult male rats for 60 min, followed by 24 h reperfusion. Injured, sham-operated and non-injured animals were treated with the HA synthesis inhibitor, 4-methylumbelliferone (4-MU 25 mg/kg). Fecal microbiota composition was evaluated by Next Generation Sequencing. Neutrophil infiltration, HA homeostasis and toll like receptor (TLR2 and TLR4) expression in the small intestine were evaluated by immunohistochemical and biomolecular approaches (qRT-PCR and Western blotting). Neuromuscular responses were studied in vitro, in the absence and presence of the selective TLR2/4 inhibitor, Sparstolonin B (SsnB 10, 30 µM). RESULTS: 4-MU significantly reduced IRI-induced enhancement of potentially harmful Escherichia and Enterococcus bacteria. After IRI, HA levels, neutrophil infiltration, and TLR2 and TLR4 expression were significantly enhanced in the muscularis propria, and were significantly reduced to baseline levels by 4-MU. In the injured, but not in the non-injured and sham-operated groups, SsnB reduced both electrical field-stimulated (EFS, 0.1-40 Hz) contractions and EFS-induced (10 Hz) non-cholinergic non-adrenergic relaxations. CONCLUSIONS: enhanced HA levels after intestinal IRI favors harmful bacteria overgrowth, increases neutrophil infiltration and promotes the upregulation of bacterial target receptors, TLR2 and TLR4, in the muscularis propria, inducing a pro-inflammatory state. TLR2 and TLR4 activation may, however, underlay a provisional benefit on excitatory and inhibitory neuronal pathways underlying peristalsis.

Cannabinoid CB1 receptor antagonism prevents neurochemical and behavioural deficits induced by chronic phencyclidine.

Clinical and laboratory studies suggest that the endocannabinoid system is involved in schizophrenia disorders. Recent evidence indicates that cannabinoid receptor (CB1) antagonists have a pharmacological profile similar to antipsychotic drugs. We investigated the behavioural and biochemical effects of the CB1 antagonist AM251 in a phencyclidine (PCP) animal paradigm modelling the cognitive deficit and some negative symptoms of schizophrenia. Chronic AM251 (0.5 mg/kg for 3 wk) improved the PCP-altered recognition memory, as indicated by a significant amelioration of the discrimination index compared to chronic PCP alone (2.58 mg/kg for 1 month). AM251 also reversed the PCP-induced increase in immobility in the forced swim test resembling avolition, a negative sign of schizophrenia. In order to analyse the mechanisms underlying these behaviours, we studied the effects of AM251 on the endocannabinoid system (in terms of CB1 receptor density and functional activity and endocannabinoid levels) and c-Fos protein expression. The antagonist counteracted the alterations in CB1 receptor function induced by PCP in selected cerebral regions involved in schizophrenia. In addition, in the prefrontal cortex, the key region in the integration of cognitive and negative functions, AM251 markedly raised anandamide levels and reversed the PCP-induced increase of 2-arachidonoylglycerol concentrations. Finally, chronic AM251 fully reversed the PCP-elicited expression of c-Fos protein in the prefrontal cortical region. These findings suggest an antipsychotic-like profile of the CB1 cannabinoid receptor antagonist which, by restoring the function of the endocannabinoid system, might directly or indirectly normalize some of the neurochemical maladaptations present in this schizophrenia-like animal model.

PTCA (1-H-Pyrrole-2,3,5-Tricarboxylic Acid) as a Marker for Oxidative Hair Treatment: Distribution, Gender Aspects, Correlation with EtG and Self-Reports.

Hair analysis is an important and reliable resource for the assessment of alcohol or drug abstinence in both clinical and forensic toxicology. Recently, it has been demonstrated that hair oxidative cosmetic treatments lead to the reduction in incorporated xenobiotics in hair, such as ethyl glucuronide (EtG), a marker of alcohol abuse, and the formation of 1-H-pyrrole-2,3,5-tricarboxylic acid (PTCA), a degradation product of melanin. The aim of the present study was to investigate PTCA trends in a large number of samples in order to evaluate the reliability of this biomarker in recognizing previous cosmetic treatment in forensic analyses. Therefore, a single-step extraction followed by an high-performance liquid chromatography--tandem mass spectrometry (HPLC--MS-MS) method was established and validated for the simultaneous determination of EtG and PTCA. This method was applied to 1,219 scalp hair samples from two groups, namely self-reported untreated and in vivo treated hair, exhibiting a concentration range of 6.7 to 440.0 pg/mg for EtG (mean 26.8 pg/mg, median 14.6 pg/mg) and 0.009 to 49.8 ng/mg for PTCA (mean 0.66 ng/mg, median 0.02 ng/mg). The PTCA content was significantly different among the two experimental groups, with the in vivo treated group showing significantly higher levels of PTCA than the untreated group. Finally, an in vitro bleaching was performed and the results confirmed that a strong hair oxidative treatment may negatively affect EtG test results (false negative), whereas the mean PTCA content increased showing statistically significant differences between untreated and in vitro oxidative treated samples. The present study suggests that the determination of PTCA in routine hair analysis procedure could be useful in order to discover previous cosmetic treatment including oxidation.

Discrepancies between genetic and ecological divergence patterns suggest a complex biogeographic history in a Neotropical genus.

Phylogenetic patterns and the underlying speciation processes can be deduced from morphological, functional, and ecological patterns of species similarity and divergence. In some cases, though, species retain multiple similarities and remain almost indistinguishable; in other cases, evolutionary convergence can make such patterns misleading; very often in such cases, the "true" picture only emerges from carefully built molecular phylogenies, which may come with major surprises. In addition, closely related species may experience gene flow after divergence, thus potentially blurring species delimitation. By means of advanced inferential methods, we studied molecular divergence between species of the Virola genus (Myristicaceae): widespread Virola michelii and recently described, endemic V. kwatae, using widespread V. surinamensis as a more distantly related outgroup with different ecology and morphology-although with overlapping range. Contrary to expectations, we found that the latter, and not V. michelii, was sister to V. kwatae. Therefore, V. kwatae probably diverged from V. surinamensis through a recent morphological and ecological shift, which brought it close to distantly related V. michelii. Through the modeling of the divergence process, we inferred that gene flow between V. surinamensis and V. kwatae stopped soon after their divergence and resumed later, in a classical secondary contact event which did not erase their ecological and morphological differences. While we cannot exclude that initial divergence occurred in allopatry, current species distribution and the absence of geographical barriers make complete isolation during speciation unlikely. We tentatively conclude that (a) it is possible that divergence occurred in allopatry/parapatry and (b) secondary contact did not suppress divergence.

Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model.

Cystic fibrosis (CF) is a hereditary disease due to mutations in the CFTR gene and causes mortality in humans mainly due to respiratory infections caused by Pseudomonas aeruginosa. In a previous work we used phage therapy, which is a treatment with a mix of phages, to actively counteract acute P. aeruginosa infections in mice and Galleria mellonella larvae. In this work we apply phage therapy to the treatment of P. aeruginosa PAO1 infections in a CF zebrafish model. The structure of the CFTR channel is evolutionary conserved between fish and mammals and cftr-loss-of-function zebrafish embryos show a phenotype that recapitulates the human disease, in particular with destruction of the pancreas. We show that phage therapy is able to decrease lethality, bacterial burden, and the pro-inflammatory response caused by PAO1 infection. In addition, phage administration relieves the constitutive inflammatory state of CF embryos. To our knowledge, this is the first time that phage therapy is used to cure P. aeruginosa infections in a CF animal model. We also find that the curative effect against PAO1 infections is improved by combining phages and antibiotic treatments, opening a useful therapeutic approach that could reduce antibiotic doses and time of administration.

Cannabidivarin completely rescues cognitive deficits and delays neurological and motor defects in male Mecp2 mutant mice.

BACKGROUND: Recent evidence suggests that 2-week treatment with the non-psychotomimetic cannabinoid cannabidivarin (CBDV) could be beneficial towards neurological and social deficits in early symptomatic Mecp2 mutant mice, a model of Rett syndrome (RTT). AIM: The aim of this study was to provide further insights into the efficacy of CBDV in Mecp2-null mice using a lifelong treatment schedule (from 4 to 9 weeks of age) to evaluate its effect on recognition memory and neurological defects in both early and advanced stages of the phenotype progression. METHODS: CBDV 0.2, 2, 20 and 200 mg/kg/day was administered to Mecp2-null mice from 4 to 9 weeks of age. Cognitive and neurological defects were monitored during the whole treatment schedule. Biochemical analyses were carried out in brain lysates from 9-week-old wild-type and knockout mice to evaluate brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) levels as well as components of the endocannabinoid system. RESULTS: CBDV rescues recognition memory deficits in Mecp2 mutant mice and delays the appearance of neurological defects. At the biochemical level, it normalizes BDNF/IGF1 levels and the defective PI3K/AKT/mTOR pathway in Mecp2 mutant mice at an advanced stage of the disease. Mecp2 deletion upregulates CB1 and CB2 receptor levels in the brain and these changes are restored after CBDV treatment. CONCLUSIONS: CBDV administration exerts an enduring rescue of memory deficits in Mecp2 mutant mice, an effect that is associated with the normalization of BDNF, IGF-1 and rpS6 phosphorylation levels as well as CB1 and CB2 receptor expression. CBDV delays neurological defects but this effect is only transient.

The genetic structure of the remnant populations of Centaurea horrida in Sardinia and associated islands.

BACKGROUND AND AIMS: The Mediterranean region is of prime importance to biodiversity at a global level, mainly due to the abundance of endemic plant species. However, information about these species is still scarce, especially at the genetic level. In this paper the first assessment is reported of the genetic structure of Centaurea horrida (Asteraceae), an endemic, sea-cliff-dwelling plant from Sardinia. METHODS: The study was conducted on seven populations covering the entire natural range of the species by means of SSR (microsatellite) markers. KEY RESULTS: A considerable amount of genetic variation was found (average H(e) = 0.603-0.854), together with a medium-high differentiation among populations, as estimated both by F(ST) (0.123) and R(ST) (0.158). Both Bayesian analysis and AMOVA were employed to detect genetic structuring in this species. The results suggest that the origins of the current populations of C. horrida lie in two gene pools. CONCLUSIONS: Despite the restricted range, C. horrida displays high levels of genetic diversity, structured in such a way that three management units could be deemed viable for its conservation. The protected status of the species will probably suffice to prevent the impoverishment of its genetic resources.

cDNA encoding sequences for myostatin and FGF6 in sea bass (Dicentrarchus labrax, L.) and the effect of fasting and refeeding on their abundance levels.

Fish have the ability to compensate for set-backs in growth as a result of fasting. When food levels are restored, growth in these fish can increase over and above normal rates. This phenomenon, known as "compensatory growth", has been studied with respect to enhancing food conversion efficiency. However, the mechanisms by which food intake activates an increase in somatic growth, and especially in muscle growth, are not well understood. In this study, we report first on the isolation of two complete cDNAs sequences encoding sea bass (Dicentrarchus labrax) myostatin and fibroblast growth factor 6 (FGF6), which have been shown to be major genetic determinants of skeletal muscle growth. The open reading frames of myostatin (376 amino acids) and FGF6 (209 amino acids) showed 97-63% and 87-62% sequence identity with other vertebrate myostatins and FGF6s, respectively. We also report on the expression profile of myostatin and FGF6 in sea bass skeletal muscle in response to different feeding regimens, as quantified by real-time RT-PCR. Nutritional status significantly influenced the myostatin expression levels in muscle, inducing an up-regulation during fasting and a down-regulation during the recovery from fasting, whereas the muscular FGF6 mRNA levels were not significantly affected by the feeding status of the animals. These findings suggest that myostatin has an inhibitory role in muscle growth in response to different feeding regimens, whereas FGF6 is not involved in the muscle compensatory growth induced by refeeding.

Different real time PCR approaches for the fine quantification of SNP's alleles in DNA pools: assays development, characterization and pre-validation.

Single nucleotide polymorphisms (SNPs) are becoming the most common type of markers used in genetic analysis. In the present report a SNP has been chosen to test the applicability of Real Time PCR to discriminate and quantify SNPs alleles on DNA pools. Amplification Refractory Mutation System (ARMS) and Mismatch Amplification Mutation Assay (MAMA) has been applied. Each assay has been pre-validated testing specificity and performances (linearity, PCR efficiency, interference limit, limit of detection, limit of quantification, precision and accuracy). Both the approaches achieve a precise and accurate estimation of the allele frequencies on pooled DNA samples in the range from 5 % to 95 % and don't require standard curves or calibrators. The lowest measurement that could be significantly distinguished from the background noise has been determined around the 1 % for both the approaches, allowing to extend the range of quantifications from 1 % to 99 %. Furthermore applicability of Real Time PCR assays for general diagnostic purposes is discussed.