The colonization history of Juniperus brevifolia (Cupressaceae) in the Azores Islands

Background

A central aim of island biogeography is to understand the colonization history of insular species using current distributions, fossil records and genetic diversity. Here, we analyze five plastid DNA regions of the endangered Juniperus brevifolia, which is endemic to the Azores archipelago.

Methodology/Principal Findings

The phylogeny of the section Juniperus and the phylogeographic analyses of J. brevifolia based on the coalescence theory of allele (plastid) diversity suggest that: (1) a single introduction event likely occurred from Europe; (2) genetic diversification and inter-island dispersal postdated the emergence of the oldest island (Santa Maria, 8.12 Ma); (3) the genetic differentiation found in populations on the islands with higher age and smaller distance to the continent is significantly higher than that on the younger, more remote ones; (4) the high number of haplotypes observed (16), and the widespread distribution of the most frequent and ancestral ones across the archipelago, are indicating early diversification, demographic expansion, and recurrent dispersal. In contrast, restriction of six of the seven derived haplotypes to single islands is construed as reflecting significant isolation time prior to colonization.

Conclusions/Significance

Our phylogeographic reconstruction points to the sequence of island emergence as the key factor to explain the distribution of plastid DNA variation. The reproductive traits of this juniper species (anemophily, ornithochory, multi-seeded cones), together with its broad ecological range, appear to be largely responsible for recurrent inter-island colonization of ancestral haplotypes. In contrast, certain delay in colonization of new haplotypes may reflect intraspecific habitat competition on islands where this juniper was already present.     

Cytotoxicity of quinone drugs on highly proliferative human leukemia T cells: reactive oxygen species generation and inactive shortened SOD1 isoform implications

Drugs containing the quinone group were tested on hyperproliferative leukemia T cells (HLTC: Jhp and Jws) and parental Jurkat cells. Doxorubicin, menadione and adaphostin produced different effects on these cell lines. Rapid doxorubicin-induced cell death in Jurkat cells was mediated by caspase activation. Doxorubicin-induced cell death of HLTCs was delayed due to the absence of caspase-3 and -8 expression. Delayed HLTC cell death was mediated and triggered by the generation of reactive oxygen species (ROS). Other drugs containing quinone groups, such as menadione and adaphostin, were also tested on HLTC and both were toxic by a caspase-independent mechanism. The toxicity of these drugs correlated with the generation of the superoxide anion, which increased and was more effective in HLTCs than in parental Jurkat cells. Accordingly, SOD1 activity was much lower in HLTCs than in Jurkat cells. This lower SOD1 activity in HLTCs was associated not only with the absence of the wild-type (16 kDa) SOD1 monomer but also with the presence of a shortened (14 kDa) SOD1 monomer isoform. Moreover, the cytotoxicity of drugs containing the quinone group was prevented by incubation with manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable superoxide dismutase mimetic and a potent inhibitor of oxidation. These findings could explain the sensitivity of HLTCs to drugs containing the quinone group using a mechanism dependent on oxidative stress. These observations can also be useful to target hyperproliferative leukemias that are resistant to the classical caspase-dependent apoptotic pathway.     

Is livestock grazing compatible with amphibian diversity in the High Mountains of Córdoba, Argentina?

The High Mountains of Córdoba, Argentina have a long evolutionary history of grazing by large herbivores. However, about 400?years ago, European livestock were introduced and gradually replaced native herbivores. Since the 1920s, domestic herbivores have been the only large herbivores present in the area, causing severe soil erosion and a threat to the system diversity. The endemic fauna of the region includes four amphibian species. We evaluated the effect of livestock rearing on amphibian diversity and water bodies in woodlands and grasslands of the High Mountains of Córdoba. The work was conducted on stream stretches and ponds in two contrasting grazing situations: an area with livestock presence and another area where livestock was excluded 14?years ago. The following variables were recorded at each sampling site: amphibian richness and abundance, percentage of emergent, submerged and peripheral vegetation in areas surrounding the water bodies, water pH, and water dissolved O₂. No significant differences were detected in amphibian diversity between streams of both grazing situations, whereas a greater diversity (p?<?0.01) was observed in ponds in grazed grasslands. Our results suggest that livestock rearing, qualitatively measured as grazing and 14?years of livestock exclusion, in the study area would not have negative effects on amphibian diversity. This finding might be due to the long evolutionary grazing history of the area, large-scale livestock exclusion exhibiting a novel scenario. The ongoing reintroduction of native grazers may provide the benefits of grazing without the consequent soil erosion and habitat degradation associated with domestic livestock.     

SpectraClassifier 1.0: a user friendly,automated MRS-based classifier-development system

Background  

SpectraClassifier (SC) is a Java solution for designing and implementing Magnetic Resonance Spectroscopy (MRS)-based classifiers. The main goal of SC is to allow users with minimum background knowledge of multivariate statistics to perform a fully automated pattern recognition analysis. SC incorporates feature selection (greedy stepwise approach, either forward or backward), and feature extraction (PCA). Fisher Linear Discriminant Analysis is the method of choice for classification. Classifier evaluation is performed through various methods: display of the confusion matrix of the training and testing datasets; K-fold cross-validation, leave-one-out and bootstrapping as well as Receiver Operating Characteristic (ROC) curves.     

Synergistic effect of methyl jasmonate and cyclodextrins on anthraquinone accumulation in cell suspension cultures of Morinda citrifolia and Rubia tinctorum

Plant in vitro culture is a platform for producing secondary metabolites that combines safety, quality and low environmental impact. Besides, it is possible to increase the accumulation of these compounds by different strategies, such as elicitation. In this work, we analyzed the effects of the combination of methyl jasmonate (MeJ) and two cyclodextrins (CDs) on the production of anthraquinones (AQs) in cell cultures of Rubiaceae (Morinda citrifolia and Rubia tinctorum). These secondary metabolites have been traditionally used as dyes and have interesting therapeutic applications. The experiments were designed according to a full factorial design of two factors (MeJ and a CD) in two levels (0 and 0.1 mM for MeJ, and 0 and 20 mM of the CD). MeJ and (2-hydroxypropyl)-β-cyclodextrin (HPCD) synergistically increased intracellular AQ content in suspension cultures of R. tinctorum, and, to a lesser extent, in suspension cultures of M. citrifolia. Combination of MeJ with another CD, 2-methyl-β-cyclodextrin, led to a more intense and later increase in AQ content in cell cultures of R. tinctorum when compared to MeJ–HPCD treatment. However, the combination of CD and MeJ failed to induce a drastic AQ release to the culture media. Nevertheless, our results show that combination of strategies (using a CD and MeJ) was successful to increase secondary metabolite accumulation in suspension cultures. To our knowledge, this is the first report of synergistic effect of MeJ and CD on AQ accumulation in plant in vitro cultures.

     

Expression, Purification, and Initial Characterization of the Recombinant Storage Protein Precursor ofTheobroma cacao

The gene encoding the 67-kDa cocoa storage protein precursor has been cloned fromTheobroma cacaoand expressed inEscherichia coliusing the pET expression system. The recombinant storage protein has been renatured from inclusion bodies at 30°C using 20 m glycine–NaOH buffer, pH 10.0, containing 1 m oxidized glutathione and 0.1% Brij. The renatured protein was purified and demonstrated to adopt a stable native conformation by optical spectroscopy. Secondary structure analysis from circular dichroism indicated the protein to be 23% α-helix and 38% β-sheet, in close agreement with values obtained using a secondary structure prediction program.     

Validation of the Diagnostic Interview for Genetic Studies (DIGS) in Colombia

An interview tool, Diagnostic Interview for Genetic Studies (DIGS 3.0), was translated into Spanish for application in studies of psychiatric disorders in Colombia. Two Spanish translations of the original English version of DIGS were prepared and back-translated into English. A review committee verified the linguistic and cultural equivalence of the translations. The evaluator and test-retest reliability were assessed calculating Cohen's kappa for samples of 65 and 91 patients respectively. DIGS proved valid in both appearance and content. The confidence interval (C.I.) was excellent for schizophrenia (kappa = 0.81, C.I. 95% = 0.68-0.93), bipolar disorder (kappa = 0.87, C.I. 95% = 0.75-0.99), major depressive disorder (kappa = 0.86, C.I. 95% = 0.70-1.00), and for a normal diagnosis (kappa = 0.65, C.I. 95% = 0.41-0.89); it was good for other psychiatric diagnosis (kappa = 0.65, C.I. 95% = 0.41-0.89) and poor for schizoaffective disorder (kappa = 0.37, C.I. 95% = -0.02-0.76). Test-retest reliability was excellent for all diagnoses (kappa > 0.8), except for "other psychiatric diagnoses" (kappa = 0.64, C.I. 95% = 0.31-0.96). The Spanish translation of the DIGS was comprehensible, with face and content validity, and good test-retest and evaluator reliability. This translation will be a useful tool for genetic studies of psychiatric disorders in Latin America, particularly where schizophrenia and affective disorders are involved.     

The influence of the Miocene Mediterranean desiccation on the geographical expansion and genetic variation of Androcymbium gramineum (Cav.) McBride (Colchicaceae)

Chloroplast DNA (cpDNA) restriction site and isozyme data were combined to explore the spatial-temporal influence of the Messinian desiccation in the Mediterranean on the disjunct distribution of Androcymbium gramineum in Almería and Morocco (north and south of the straits of Gibraltar, respectively). Lack of evidence for different selective pressures, divergence time estimates based on the calibration of the isozyme molecular clock with the cpDNA data, the basal position of Almerian populations in the A. gramineum clade, and the much higher isozyme polymorphism in Almería suggest that (i) only a southern European range of A. gramineum existed before the Messinian [ approximately 11.2 million years ago (Ma), in the middle Miocene] and (ii) the desiccation of the Mediterranean basin about 5.5-4.5 Ma induced the migration of A. gramineum from Almería to Morocco (between 4.9 and 4.6 Ma, according to our time estimates). After the split into two allopatric units following the refilling of the Mediterranean, the major influence of drift associated with Plio-Pleistocene recurrent glaciation cycles and range expansions/contractions probably fostered the substantial interpopulation genetic differentiation observed within Almería (CGST = 0.41, average DNei = 0.185) and, to a lesser extent, within Morocco (CGST = 0.24, average DNei = 0.089), but did not hinder the maintenance of considerable levels of genetic variation in either geographical area (A = 2.14, HE = 0.230 and A = 1.90, HE = 0.213, respectively).     

The S. cerevisiae architectural HMGB protein NHP6A complexed with DNA: DNA and protein conformational changes upon binding

NHP6A is a non-sequence-specific DNA-binding protein from Saccharomyces cerevisiae which belongs to the HMGB protein family. Previously, we have solved the structure of NHP6A in the absence of DNA and modeled its interaction with DNA. Here, we present the refined solution structures of the NHP6A-DNA complex as well as the free 15bp DNA. Both the free and bound forms of the protein adopt the typical L-shaped HMGB domain fold. The DNA in the complex undergoes significant structural rearrangement from its free form while the protein shows smaller but significant conformational changes in the complex. Structural and mutational analysis as well as comparison of the complex with the free DNA provides insight into the factors that contribute to binding site selection and DNA deformations in the complex. Further insight into the amino acid determinants of DNA binding by HMGB domain proteins is given by a correlation study of NHP6A and 32 other HMGB domains belonging to both the DNA-sequence-specific and non-sequence-specific families of HMGB proteins. The resulting correlations can be rationalized by comparison of solved structures of HMGB proteins.