Multilocus and morphological analysis of south-eastern Iberian Wall lizards (Squamata,Podarcis)

The phylogenetic relationships among the wall lizards of the Podarcis hispanicus complex that inhabit the south-east (SE) of the Iberian Peninsula and other lineages of the complex remain unclear. In this study, four mitochondrial and two nuclear markers were used to study genetic relationships within this complex. The phylogenetic analyses based on mtDNA gene trees constructed with ML and BI, and a species tree using *BEAST support three divergent clades in this region: the Valencia, Galera and Albacete/Murcia lineages. These three lineages were also corroborated in species delimitation analyses based on mtDNA using bPTP, mPTP, GMYC, ABGD and BAPS. Bayesian inference species delimitation method (BPP) based on both nuclear data and a combined data set (mtDNA + nuclear) showed high posterior probabilities for these three SE lineages (≥0.94) and another Bayesian analysis (STACEY) based on combined data set recovered the same three groups in this region. Divergence time dating of the species tree provided an estimated divergence of the Galera lineage from the other SE group (Podarcis vaucheri, (Albacete/Murcia, Valencia)) at 12.48 Ma. During this period, the Betic–Rifian arc was isolated, which could have caused the isolation of the Galera form distributed to the south of the Betic Corridor. Although lizards from the Albacete/Murcia and Galera lineage are morphologically similar, they clearly represent distinct genetic lineages. The noteworthy separation of the Galera lineage enables us to conclude that this lineage must be considered as a new full species.     

Construction of Aspergillus nidulans strains producing enzymes of potential use in enology

Two different Aspergillus nidulans recombinant strains producing either the Aspergillus nidulans a-L-arabinofuranosidase A or a Candida molischiana b-glucosidase have been constructed. Depending on the growing conditions, the modified strains produce up to 4 or 18 times more b-glucosidase or a-L-arabinofuranosidase activity levels, respectively, than the wild type strain.     

Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Novel Approach to Simulate Sleep Apnea Patients for Evaluating Positive Pressure Therapy Devices

Bench testing is a useful method to characterize the response of different automatic positive airway pressure (APAP) devices under well-controlled conditions. However, previous models did not consider the diversity of obstructive sleep apnea (OSA) patients’ characteristics and phenotypes. The objective of this proof-of-concept study was to design a new bench test for realistically simulating an OSA patient’s night, and to implement a one-night example of a typical female phenotype for comparing responses to several currently-available APAP devices. We developed a novel approach aimed at replicating a typical night of sleep which includes different disturbed breathing events, disease severities, sleep/wake phases, body postures and respiratory artefacts. The simulated female OSA patient example that we implemented included periods of wake, light sleep and deep sleep with positional changes and was connected to ten different APAP devices. Flow and pressure readings were recorded; each device was tested twice. The new approach for simulating female OSA patients effectively combined a wide variety of disturbed breathing patterns to mimic the response of a predefined patient type. There were marked differences in response between devices; only three were able to overcome flow limitation to normalize breathing, and only five devices were associated with a residual apnea-hypopnea index of <5/h. In conclusion, bench tests can be designed to simulate specific patient characteristics, and typical stages of sleep, body position, and wake. Each APAP device behaved differently when exposed to this controlled model of a female OSA patient, and should lead to further understanding of OSA treatment.     

Paracoccidioides brasiliensis Interferes on Dendritic Cells Maturation by Inhibiting PGE2 Production

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii. The mechanisms involved in the initial interaction of the fungus with cells of the innate immune response, as dendritic cells (DCs), deserve to be studied. Prostaglandins (PGs) are eicosanoids that play an important role in modulating functions of immune cells including DCs. Here we found that human immature DCs derived from the differentiation of monocytes cultured with GM-CSF and IL-4 release substantial concentrations of PGE₂, which, however, were significantly inhibited after challenge with P. brasiliensis. In vitro blocking of pattern recognition receptors (PRRs) by monoclonal antibodies showed the involvement of mannose receptor (MR) in PGE₂ inhibition by the fungus. In addition, phenotyping assays showed that after challenge with the fungus, DCs do not change their phenotype of immature cells to mature ones, as well as do not produce IL-12 p70 or adequate concentrations of TNF-α. Assays using exogenous PGE₂ confirmed an association between PGE₂ inhibition and failure of cells to phenotypically mature in response to P. brasiliensis. We conclude that a P. brasiliensis evasion mechanism exists associated to a dysregulation on DC maturation. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus.     

Phototrophic utilization of toluene under anoxic conditions by a new strain of Blastochloris sulfoviridis

The capacity of anoxygenic phototrophic bacteria to utilize aromatic hydrocarbons was investigated in enrichment cultures with toluene. When mineral medium with toluene (provided in an inert carrier phase) was inoculated with activated sludge and incubated under infrared illumination (> 750 nm), a red-to-brownish culture developed. Agar dilution series indicated the dominance of two types of phototrophic bacteria. One type formed red colonies, had rod-shaped cells with budding division, and grew on benzoate but not on toluene. The other type formed yellow-to-brown colonies, had oval cells, and utilized toluene and benzoate. One strain of the latter type, ToP1, was studied in detail. Sequence analysis of the 16S rRNA gene and DNA-DNA hybridization indicated an affiliation of strain ToP1 with the species Blastochloris sulfoviridis, a member of the α-subclass of Proteobacteria. However, the type strain (DSM 729) of Blc. sulfoviridis grew neither on toluene nor on benzoate. Light-dependent consumption of toluene in the presence of carbon dioxide and formation of cell mass by strain ToP1 were demonstrated in quantitative growth experiments. Strain ToP1 is the first phototrophic bacterium shown to utilize an aromatic hydrocarbon. In the supernatant of toluene-grown cultures and in cell-free extracts incubated with toluene and fumarate, the formation of benzylsuccinate was detected. These findings indicate that the phototrophic bacterium activates toluene anaerobically by the same mechanism that has been reported for denitrifying and sulfate-reducing bacteria. The natural abundance of phototrophic bacteria with the capacity for toluene utilization was examined in freshwater habitats. Counting series revealed that up to around 1% (1.8 × 10⁵ cells per gram dry mass of sample) of the photoheterotrophic population cultivable with acetate grew on toluene. Received: 29 March 1999 / Accepted: 1 July 1999     

Detection of Atomic Scale Changes in the Free Volume Void Size of Three-Dimensional Colorectal Cancer Cell Culture Using Positron Annihilation Lifetime Spectroscopy

Positron annihilation lifetime spectroscopy (PALS) provides a direct measurement of the free volume void sizes in polymers and biological systems. This free volume is critical in explaining and understanding physical and mechanical properties of polymers. Moreover, PALS has been recently proposed as a potential tool in detecting cancer at early stages, probing the differences in the subnanometer scale free volume voids between cancerous/healthy skin samples of the same patient. Despite several investigations on free volume in complex cancerous tissues, no positron annihilation studies of living cancer cell cultures have been reported. We demonstrate that PALS can be applied to the study in human living 3D cell cultures. The technique is also capable to detect atomic scale changes in the size of the free volume voids due to the biological responses to TGF-β. PALS may be developed to characterize the effect of different culture conditions in the free volume voids of cells grown in vitro.     

Magneto-Chemotaxis in Sediment: First Insights

Magnetotactic bacteria (MTB) use passive alignment with the Earth magnetic field as a mean to increase their navigation efficiency in horizontally stratified environments through what is known as magneto-aerotaxis (M-A). Current M-A models have been derived from MTB observations in aqueous environments, where a >80% alignment with inclined magnetic field lines produces a one-dimensional search for optimal living conditions. However, the mean magnetic alignment of MTB in their most widespread living environment, i.e. sediment, has been recently found to be <1%, greatly reducing or even eliminating the magnetotactic advantage deduced for the case of MTB in water. In order to understand the role of magnetotaxis for MTB populations living in sediment, we performed first M-A observations with lake sediment microcosms. Microcosm experiments were based on different combinations of (1) MTB position with respect to their preferred living depth (i.e. above, at, and below), and (2) magnetic field configurations (i.e. correctly and incorrectly polarized vertical fields, horizontal fields, and zero fields). Results suggest that polar magnetotaxis is more complex than implied by previous experiments, and revealed unexpected differences between two types of MTB living in the same sediment. Our main findings are: (1) all investigated MTB benefit of a clear magnetotactic advantage when they need to migrate over macroscopic distances for reaching their optimal living depth, (2) magnetotaxis is not used by all MTB under stationary, undisturbed conditions, (3) some MTB can rely only on chemotaxis for macroscopic vertical displacements in sediment while other cannot, and (4) some MTB use a fixed polar M-A mechanisms, while other can switch their M-A polarity, performing what can be considered as a mixed polar-axial M-A. These observations demonstrate that sedimentary M-A is controlled by complex mechanical, chemical, and temporal factors that are poorly reproduced in aqueous environments.