Mitochondrial differentiation and biogeography of Hyla meridionalis (Anura: Hylidae): an unusual phylogeographical pattern

Aim To study the patterns of genetic variation and the historical events and processes that influenced the distribution and intraspecific diversity in Hyla meridionalis Boettger, 1874. Location Hyla meridionalis is restricted to the western part of the Mediterranean region. In northern Africa it is present in Tunisia, Algeria and Morocco. In south‐western Europe it is found in the south of France, north‐western Italy and north‐eastern and south‐western Iberian Peninsula. There are also insular populations, as in the Canaries and Menorca. Methods Sampling included 112 individuals from 36 populations covering the range of the species. We used sequences of mitochondrial DNA Cytochrome Oxidase I (COI) for the phylogeographical analysis (841 bp) and COI plus a fragment including part of tRNA lysine, ATP synthase subunits 6 and 8 and part of Cytochrome Oxidase III for phylogenetic analyses (2441 bp). Phylogenetic analyses were performed with paup *4.0b10 (maximum likelihood, maximum parsimony) and Mr Bayes 3.0 (Bayesian analysis). Nested clade analysis was performed using tcs 1.18 and Geo Dis 2.2. A dispersal‐vicariant analysis was performed with diva 1.0 to generate hypotheses about the geographical distribution of ancestors. Results We found little genetic diversity within samples from Morocco, south‐western Europe and the Canary Islands, with three well‐differentiated clades. One is distributed in south‐western Iberia and the High Atlas, Anti‐Atlas and Massa River in Morocco. The second is restricted to the Medium Atlas Mountains. The third one is present in northern Morocco, north‐eastern Iberia, southern France and the Canaries. These three groups are also represented in the nested clade analysis. Sequences from Tunisian specimens are highly divergent from sequences of all other populations, suggesting that the split between the two lineages is ancient. diva analysis suggests that the ancestral distribution of the different lineages was restricted to Africa, and that an explanation of current distribution of the species requires three different dispersal events. Main conclusions Our results support the idea of a very recent colonization of south‐western Europe and the Canary Islands from Morocco. South‐western Europe has been colonized at least twice: once from northern Morocco probably to the Mediterranean coast of France and once from the western coast of Morocco to southern Iberia. Human transport is a likely explanation for at least one of these events. Within Morocco, the pattern of diversity is consistent with a model of mountain refugia during hyperarid periods within the Pleistocene. Evaluation of the phylogenetic relationships of Tunisian haplotypes will require an approach involving the other related hylid taxa in the area.     

Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia

Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2′-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.     

EGFR kinase promotes acquisition of stem cell-like properties: a potential therapeutic target in head and neck squamous cell carcinoma stem cells

Members of the EGFR/ErbB family of tyrosine kinases are found to be highly expressed and deregulated in many cancers, including head and neck squamous cell carcinoma (HNSCC). The ErbB family, including EGFR, has been demonstrated to play key roles in metastasis, tumorigenesis, cell proliferation, and drug resistance. Recently, these characteristics have been linked to a small subpopulation of cells classified as cancer stem cells (CSCs) which are believed to be responsible for tumor initiation and maintenance. In this study, we investigated the possible role of EGFR as a regulator of "stemness" in HNSCC cells. Activation of EGFR by the addition of EGF ligand or ectopic expression of EGFR in two established HNSCC cell lines (UMSCC-22B and HN-1) resulted in the induction of CD44, BMI-1, Oct-4, NANOG, CXCR4, and SDF-1. Activation of EGFR also resulted in increased tumorsphere formation, a characteristic ability of cancer stem cells. Conversely, treatment with the EGFR kinase inhibitor, Gefinitib (Iressa), resulted in decreased expression of the aforementioned genes, and loss of tumorsphere-forming ability. Similar trends were observed in a 99.9% CD44 positive stem cell culture derived from a fresh HNSCC tumor, confirming our findings for the cell lines. Additionally, we found that these putative cancer stem cells, when treated with Gefitinib, possessed a lower capacity to invade and became more sensitive to cisplatin-induced death in vitro. These results suggest that EGFR plays critical roles in the survival, maintenance, and function of cancer stem cells. Drugs that target EGFR, perhaps administered in combination with conventional chemotherapy, might be an effective treatment for HNSCC.     

Brain-derived antigens in lymphoid tissue of patients with acute stroke

In experimental animals, the presence of brain-derived constituents in cervical lymph nodes has been associated with the activation of local lymphocytes poised to minimize the inflammatory response after acute brain injury. In this study, we assessed whether this immune crosstalk also existed in stroke patients. We studied the clinical course, neuroimaging, and immunoreactivity to neuronal derived Ags (microtubule-associated protein-2 and N-methyl d-aspartate receptor subunit NR-2A), and myelin-derived Ags (myelin basic protein and myelin oligodendrocyte glycoprotein) in palatine tonsils and cervical lymph nodes of 28 acute stroke patients and 17 individuals free of neurologic disease. Stroke patients showed greater immunoreactivity to all brain Ags assessed compared with controls, predominantly in T cell zones. Most brain immunoreactive cells were CD68(+) macrophages expressing MHC class II receptors. Increased reactivity to neuronal-derived Ags was correlated with smaller infarctions and better long-term outcome, whereas greater reactivity to myelin basic protein was correlated with stroke severity on admission, larger infarctions, and worse outcome at follow-up. Patients also had more CD69(+) T cells than controls, indicative of T cell activation. Overall, the study showed in patients with acute stroke the presence of myelin and neuronal Ags associated with lymph node macrophages located near activated T cells. Whether the outcome of acute stroke is influenced by Ag-specific activation of immune responses mediated by CD69 lymphocytes deserves further investigation.     

High insulin levels are required for FAT/CD36 plasma membrane translocation and enhanced fatty acid uptake in obese Zucker rat hepatocytes

In myocytes and adipocytes, insulin increases fatty acid translocase (FAT)/CD36 translocation to the plasma membrane (PM), enhancing fatty acid (FA) uptake. Evidence links increased hepatic FAT/CD36 protein amount and gene expression with hyperinsulinemia in animal models and patients with fatty liver, but whether insulin regulates FAT/CD36 expression, amount, distribution, and function in hepatocytes is currently unknown. To investigate this, FAT/CD36 protein content in isolated hepatocytes, subfractions of organelles, and density-gradient isolated membrane subfractions was analyzed in obese and lean Zucker rats by Western blotting in liver sections by immunohistochemistry and in hepatocytes by immunocytochemistry. The uptake of oleate and oleate incorporation into lipids were assessed in hepatocytes at short time points (30-600 s). We found that FAT/CD36 protein amount at the PM was higher in hepatocytes from obese rats than from lean controls. In obese rat hepatocytes, decreased cytoplasmatic content of FAT/CD36 and redistribution from low- to middle- to middle- to high-density subfractions of microsomes were found. Hallmarks of obese Zucker rat hepatocytes were increased amount of FAT/CD36 protein at the PM and enhanced FA uptake and incorporation into triglycerides, which were maintained only when exposed to hyperinsulinemic conditions (80 mU/l). In conclusion, high insulin levels are required for FAT/CD36 translocation to the PM in obese rat hepatocytes to enhance FA uptake and triglyceride synthesis. These results suggest that the hyperinsulinemia found in animal models and patients with insulin resistance and fatty liver might contribute to liver fat accumulation by inducing FAT/CD36 functional presence at the PM of hepatocytes.     

The First Occurrence in the Fossil Record of an Aquatic Avian Twig-Nest with Phoenicopteriformes Eggs: Evolutionary Implications

Background

We describe the first occurrence in the fossil record of an aquatic avian twig-nest with five eggs in situ (Early Miocene Tudela Formation, Ebro Basin, Spain). Extensive outcrops of this formation reveal autochthonous avian osteological and oological fossils that represent a single taxon identified as a basal phoenicopterid. Although the eggshell structure is definitively phoenicopterid, the characteristics of both the nest and the eggs are similar to those of modern grebes. These observations allow us to address the origin of the disparities between the sister taxa Podicipedidae and Phoenicopteridae crown clades, and traces the evolution of the nesting and reproductive environments for phoenicopteriforms.

Methodology/Principal Findings

Multi-disciplinary analyses performed on fossilized vegetation and eggshells from the eggs in the nest and its embedding sediments indicate that this new phoenicopterid thrived under a semi-arid climate in an oligohaline (seasonally mesohaline) shallow endorheic lacustine environment. High-end microcharacterizations including SEM, TEM, and EBSD techniques were pivotal to identifying these phoenicopterid eggshells. Anatomical comparisons of the fossil bones with those of Phoenicopteriformes and Podicipediformes crown clades and extinct palaelodids confirm that this avian fossil assemblage belongs to a new and basal phoenicopterid.

Conclusions/Significance

Although the Podicipediformes-Phoenicopteriformes sister group relationship is now well supported, flamingos and grebes exhibit feeding, reproductive, and nesting strategies that diverge significantly. Our multi-disciplinary study is the first to reveal that the phoenicopteriform reproductive behaviour, nesting ecology and nest characteristics derived from grebe-like type strategies to reach the extremely specialized conditions observed in modern flamingo crown groups. Furthermore, our study enables us to map ecological and reproductive characters on the Phoenicopteriformes evolutionary lineage. Our results demonstrate that the nesting paleoenvironments of flamingos were closely linked to the unique ecology of this locality, which is a direct result of special climatic (high evaporitic regime) and geological (fault system) conditions.     

A plate holder for non-destructive testing of mesophase crystallization assays

Here, we describe a goniometer holder to mount standard 96-well crystallization plates directly onto the goniometer head of an oscillation camera. This attachment was designed to check crystallization conditions straight from the crystallization plates under X-rays, and was proven to be useful for checking small crystals and solutions that destabilize monoolein-based lipidic cubic phase (LCP) crystallization experiments. A quick procedure for setting up LCP assays employing commercially available instruments is also reported.     

Using HPLC pigment analysis to investigate phytoplankton taxonomy: the importance of knowing your species

Phytoplankton microscopic enumerations and HPLC analyses of their pigments were performed weekly for a complete year at a coastal station in the English Channel. The taxonomic composition of the phytoplankton community was assessed using the HPLC results combined with the mathematical tool CHEMTAX in two different ways. Firstly, without using the species level taxonomic information obtained at the microscopic level (blind analyses), and secondly by including the information from the microscopic taxonomic analysis (directed analyses). The results indicate that, due to the particular pigment composition of some species (for example, the dinoflagellate, Karenia mikimotoi and the haptophyte, Phaeocystis pouchetii), a blind analysis would result in very significant errors in the taxonomic determination of the bloom events at this station. Major blooms of Karenia mikimotoi and P. pouchetii were mistaken for blooms of diatoms on the basis of a blind HPLC-CHEMTAX analysis. Only with the information from the microscopic observations was it possible to obtain an accurate representation of the phytoplankton community.Communicated by H.-D. Franke     

Mechanisms of membrane permeabilization by picornavirus 2B viroporin

Cell infection by picornaviruses leads to membrane permeabilization. Recent evidence suggests the involvement of the non-structural protein 2B in this process. We have recently reported the detection of 2B porin-like activity in isolated membrane-protein systems that lack other cell components. According to data derived from these model membranes, four self-aggregated 2B monomers (i.e. tetramers) would be sufficient to permeabilize a single lipid vesicle, allowing the free diffusion of solutes under ca. 1000 Da. Our findings also support a role for lipids in protein oligomerization and subsequent pore opening. The lipid dependence of these processes points to negatively charged cytofacial surfaces as 2B cell membrane targets.