Prospects of barcoding the Italian wild dendroflora: oaks reveal severe limitations to tracking species identity

DNA barcoding may be particularly important in influencing ecology, economic issues, and the fundamental crisis facing biodiversity as a standardized, species-level identification tool for taxonomy assessment. Trees play important roles in the conservation of many land ecosystems, the wood trade, and the definition of biogeographical processes; nevertheless, peculiar biological, evolutionary and taxonomical features will probably constitute an intriguing challenge to barcoders. We examined whether four marker regions (trnh-psba, rbcL, rpoc1, matK) proposed by the Consortium for the Barcode of Life (CBOL) matched species taxonomy in a preliminary tree biodiversity survey of Italian forested land. Our objective was to provide a test of future in situ applications of DNA barcodes by evaluating the efficacy of species discrimination under the criteria of uniformity of methods and natural co-occurrence of the species in the main forest ecosystems. Fifty-two species were included in a floristic study. We obtained 73% total discrimination success, with trnH-psbA as the best performing marker and oaks as the least responsive plants to the markers used. A further taxon-based study of Quercus (thirty specimens, 12 species) revealed that this genus is refractory to barcoding (0% discrimination success), a probable consequence of low variation rate at the plastid genome level, hybridization, and the incidence of biogeography. We conclude that some species-rich tree genera in small geographical regions may prove exceptionally difficult to barcode. Until more efficient markers are developed, we recommend that improved and diversified sampling (multiple locations of sympatric and co-occurring congenerics) be embraced as a timely and important goal for the precise assessment of haplotype specificity to facilitate the productive application of barcoding in practice.     

Characterization of Salmonella occurring at high prevalence in a population of the land iguana Conolophus subcristatus in Galápagos Islands, Ecuador

The aim of the study was to elucidate the association between the zoonotic pathogen Salmonella and a population of land iguana, Colonophus subcristatus, endemic to Galápagos Islands in Ecuador. We assessed the presence of Salmonella subspecies and serovars and estimated the prevalence of the pathogen in that population. Additionally, we investigated the genetic relatedness among isolates and serovars utilising pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA and determined the antimicrobial susceptibility to a panel of antimicrobials. The study was carried out by sampling cloacal swabs from animals (n = 63) in their natural environment on in the island of Santa Cruz. A high prevalence (62/63, 98.4%) was observed with heterogeneity of Salmonella subspecies and serovars, all known to be associated with reptiles and with reptile-associated salomonellosis in humans. Serotyping revealed 14 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 48), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 1), and S. enterica subsp. houtenae (n = 7). Four serovars were predominant: S. Poona (n = 18), S. Pomona (n = 10), S. Abaetetuba (n = 8), and S.Newport (n = 5). The S. Poona isolates revealed nine unique XbaI PFGE patterns, with 15 isolates showing a similarity of 70%. Nine S. Pomona isolates had a similarity of 84%. One main cluster with seven (88%) indistinguishable isolates of S. Abaetetuba was observed. All the Salmonella isolates were pan-susceptible to antimicrobials representative of the most relevant therapeutic classes. The high prevalence and absence of clinical signs suggest a natural interaction of the different Salmonella serovars with the host species. The interaction may have been established before any possible exposure of the iguanas and the biocenosis to direct or indirect environmental factors influenced by the use of antimicrobials in agriculture, in human medicine or in veterinary medicine.     

PKCδ sensitizes neuroblastoma cells to L-buthionine-sulfoximine and etoposide inducing reactive oxygen species overproduction and DNA damage

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches.     

IPHEN—a real-time network for phenological monitoring and modelling in Italy

This paper aims to describe the Italian PHEnology Network (IPHEN), a cooperative project started in 2006 with the aim of producing nationwide maps of analysis and forecast of plants phenological stages mainly used to satisfy the needs of agriculture, health and environmental care. Iphen is a data processing system composed of the following main segments (a) collection of atmospheric and phenological data, (b) processing of data with suitable phenological and geo-statistical models and (c) phenological maps of analysis and forecast. In more detail, IPHEN maps of analysis (featuring phenological stages reached at the date of processing) are produced with models based on a Normal Heat Hours approach which weighs hourly air temperature effectiveness for plant phenological progression applied to national grids of hourly temperature derived from the operational agro-meteorological network of CRA-CMA. A correction scheme based on phenological surveys provided by volunteer observers is applied to the first guess maps of analysis to obtain final maps. Forecast maps (prediction of the days of occurrence of relevant phenological stages) are produced on the basis of GFS model medium range forecasts and climatic data. Freeware IPHEN maps for grapevine, common and Arizona cypress, black elder, olive and locust trees are broadcasted weekly on the CRA-CMA website. The positive operational results of IPHEN are testified by 150 maps broadcasted during the 2011 season for the above-mentioned species. The system performances and reliability have been analysed focusing on the analysis of phenological simulation errors and on the sensitivity of phenological maps to anomalous atmospheric circulation patterns. The error analysis shows that phenological models are characterized by advances/delays that justify the adoption of an observation based correction scheme. The sensitivity analysis highlights that the system is responsive to the effects of circulation blocking systems leading to phenological advances and delays.     

Humpback whales washed ashore in southeastern Brazil from 1981 to 2011: stranding patterns and microbial pathogens survey

In the Southwest Atlantic Ocean the wintering breeding ground of Megaptera novaeangliae is located in northeastern coast of Brazil, mainly in the Abrolhos Bank (16°55′ S, 38°50′ W) and its surroundings. During migratory percussion, events of strandings are thought to occur. A total of 58 stranded humpback whales were recorded between 1981 and 2011 along the coast of the Rio de Janeiro state, Brazil. The number of strandings in 20 years (1992–2011) increased during the period of study, with a mean of 2.6 events per year. In 2010 a peak occurred with 13 records of strandings. Most of them occurred preferably in the southern half of the study area, or in the southeast area of Rio de Janeiro state. Three cases of entanglement were found, two of these involving calves with less than eight meters of body length. Stranding events were more frequent during winter and spring. No statistical differences were found between age categories, but 33% were classified as “dependent calf” (< 8 m length). Males stranded more often than females. Only one whale out of 16 specimens that had the stomach contents examined presented food remains, but comprising only two cephalopod beaks of the squid Doryteuthis sanpaulensis. Bacteriological survey of Vibrionaceae and Aeromonadaceae agents in three live stranded whales on the Brazilian coast indicated evidence of animal impairment that resulted or were associated with the cause of death and stranding event.     

Effect of ultraviolet-B radiation in laboratory on morphological and ultrastructural characteristics and physiological parameters of selected cultivar of Oryza sativa L.

Ultraviolet-B radiation (UVBR) affects plants in many important ways, including reduction of growth rate and primary productivity, and changes in ultrastructures. Rice (Oryza sativa) is one of the most cultivated cereals in the world, along with corn and wheat, representing over 50 % of agricultural production. In this study, we examined O. sativa plants exposed to ambient outdoor radiation and laboratory-controlled photosynthetically active radiation (PAR) and PAR + UVBR conditions for 2 h/day during 30 days of cultivation. The samples were studied for morphological and ultrastructural characteristics, and physiological parameters. PAR + UVBR caused changes in the ultrastructure of leaf of O. sativa and leaf morphology (leaf index, leaf area and specific leaf area, trichomes, and papillae), plant biomass (dry and fresh weight), photosynthetic pigments, phenolic compounds, and protein content. As a photoprotective acclimation strategy against PAR + UVBR damage, an increase of 66.24 % in phenolic compounds was observed. Furthermore, PAR + UVBR treatment altering the levels of chlorophylls a and b, and total chlorophyll. In addition, total carotenoid contents decreased after PAR + UVBR treatment. The results strongly suggested that PAR + UVBR negatively affects the ultrastructure, morphology, photosynthetic pigments, and growth rates of leaf of O. sativa and, in the long term, it could affect the viability of this economically important plant.     

Evaluation of Paracoccidioides brasiliensis Infection in Dairy Goats

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a systemic mycosis that affects mainly rural workers in Brazil and other Latin American countries. The participation of domestic and wild animal species in the ecoepidemiology of paracoccidioidomycosis is not well understood. The objective of this study was to evaluate P. brasiliensis infection in dairy goats. The humoral immune response to the gp43 antigen, the main antigen used for paracoccidioidomycosis serodiagnosis and seroepidemiology, was evaluated in two goats immunized with inactivated P. brasiliensis yeast cells. Both animals produced antibodies against the P. brasiliensis gp43 antigen, detected by ELISA, 2 weeks after immunization. A total of 202 goat serum samples were analyzed by ELISA and the immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens. The seropositivity observed by ELISA was 26.2 % although no reactivity was detected by immunodiffusion. The animals over 18 months of age showed significantly higher positivity (40 %) than animals aged 6–18 months (14.8 %) and 0–6 months (2.6 %). Taking into account that cross-reactivity may occur with other pathogens, the serum samples were also analyzed by ELISA using Histoplasma capsulatum exoantigen as antigen and the positivity observed was 14.3 %. The low correlation (0.267) observed between reactivity to P. brasiliensis gp43 and H. capsulatum exoantigen suggests co-infection rather than cross-reactivity. This is the first report showing serological evidence of P. brasiliensis infection in goats and reinforces that domestic animals are useful epidemiological markers of paracoccidioidomycosis.     

Hypoxia–Ischemia Alters Nucleotide and Nucleoside Catabolism and Na+,K+-ATPase Activity in the Cerebral Cortex of Newborn Rats

It is well known that the levels of adenosine in the brain increase dramatically during cerebral hypoxic-ischemic (HI) insults. Its levels are tightly regulated by physiological and pathophysiological changes that occur during the injury acute phase. The aim of the present study was to examine the effects of the neonatal HI event on cytosolic and ecto-enzymes of purinergic system––NTPDase, 5′-nucleotidase (5′-NT) and adenosine deaminase (ADA)––in cerebral cortex of rats immediately post insult. Furthermore, the Na⁺/K⁺-ATPase activity, adenosine kinase (ADK) expression and thiobarbituric acid reactive species (TBARS) levels were assessed. Immediately after the HI event the cytosolic NTPDase and 5′-NT activities were increased in the cerebral cortex. In synaptosomes there was an increase in the ecto-ADA activity while the Na⁺/K⁺ ATPase activity presented a decrease. The difference between ATP, ADP, AMP and adenosine degradation in synaptosomal and cytosolic fractions could indicate that NTPDase, 5′-NT and ADA were differently affected after insult. Interestingly, no alterations in the ADK expression were observed. Furthermore, the Na⁺/K⁺-ATPase activity was correlated negatively with the cytosolic NTPDase activity and TBARS content. The increased hydrolysis of nucleotides ATP, ADP and AMP in the cytosol could contribute to increased adenosine levels, which could be related to a possible innate neuroprotective mechanism aiming at potentiating the ambient levels of adenosine. Together, these results may help the understanding of the mechanism by which adenosine is produced following neonatal HI injury, therefore highlighting putative therapeutical targets to minimize ischemic injury and enhance recovery.     

Ghrelin and Nicotine Stimulate Equally the Dopamine Release in the Rat Amygdala

The orexigenic peptide ghrelin plays a prominent role in the regulation of energy balance and in the mediation of reward processes and reinforcement for addictive drugs, such as nicotine. Nicotine is the principal psychoactive component in tobacco, which is responsible for addiction and relapse of smokers. Ghrelin and nicotine activates the mesolimbicocortical dopaminergic pathways via growth hormone secretagogue receptors (GHS-R1A) and nicotinic acetylcholine receptors (nAchR), respectively, resulting in the release of dopamine in the nucleus accumbens, the amygdala and the prefrontal cortex. In the present study an in vitro superfusion of rat amygdalar slices was performed in order to investigate the direct action of ghrelin and nicotine on the amygdalar dopamine release. Ghrelin increased significantly the dopamine release from the rat amygdala following electrical stimulation. This effect was inhibited by both the selective GHS-R1A antagonist GHRP-6 and the selective nAchR antagonist mecamylamine. Under the same conditions, nicotine also increased significantly the dopamine release from the rat amygdala. This effect was antagonized by mecamylamine, but not by GHRP-6. Co-administration of ghrelin and nicotine induced a similar increase of amygdalar dopamine release. This stimulatory effect was partially reversed by both GHRP-6 and mecamylamine. The present results demonstrate that both ghrelin and nicotine stimulates directly the dopamine release in the amygdala, an important dopaminergic target area of the mesolimbicocortical pathway.