LAT1 overexpression and function compensates downregulation of ASCT2 in an in vitro model of renal proximal tubule cell ageing

Amino acid transporters provide cells neutral amino acids indispensable for growth and proliferation-dependent protein synthesis. This study evaluates whether prolonged serial cell passaging during 6 months (over 50 passages) may induce changes in amino acid transporters properties in Opossum kidney (OK) proximal tubular cells. High passage OK cells exhibit polyploidy, but no difference in the proliferation potential was observed when compared to low passage OK cells. Increased time in culture was accompanied by an increased total, membrane and cytosol protein content. The Na(+)-insensitive [(14)C]-L-leucine uptake was promoted almost exclusively thought LAT1 (~ 90 vs 80%, high versus low passage OK cells). The increased LAT1 protein abundance in high passage OK cells correlated positively with enhanced ability to take up [(14)C]-L-leucine, despite a 4.3-fold decrease in affinity for the substrate. The Na(+)-sensitive [(14)C]-L-alanine transport was decreased by 2.5-fold in high passage OK cells. However, no differences in ASCT2 expression were observed between high and low passage OK cells. It is concluded that OK cells show functional differences in both L-leucine and L-alanine uptake as a function of passage time in culture. The increased expression and activity of LAT1 in high passage OK cells may correspond to a mechanism enabling the cell to develop the hypertrophy response to prolonged cell passaging, when the function of ASCT2 is markedly depressed.     

Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.     

First isolation of Pseudogymnoascus destructans in bats from Portugal

The psychrophilic fungus Pseudogymnoascus destructans (formerly known as Geomyces destructans) is considered the etiological agent of white-nose disease (WND), an emerging disease which affects bats during their hibernation period. This disease is clinically characterized by the growth of a white fungus on muzzle, ears, and wings’ membranes of affected bats. This infection caused the death of several million bats in North America. Conversely, European bats show no evidence of significant mortality occurrences associated with P. destructans colonization. This fungus has been isolated from bats in at least 15 European countries since 2008, but was never before reported in the Iberian Peninsula. This study describes the first case report of P. destructans colonization in bats from Portugal. We isolated P. destructans from three hibernating Myotis blythii (lesser mouse-eared bat) with visual signs of P. destructans colonization, during a routine visit to a mine located in the Trás-os-Montes region, Northern Portugal. M. blythii is one of the rarest bat species in Europe, classified as critically endangered in Portugal. P. destructans was obtained from at least three different parts of the body of each specimen analyzed. The identification of the respective fungal isolates was based on the macroscopic and microscopic characterization of the cultures and confirmed by PCR-based analysis. All nucleotide sequences obtained showed 100 % identity with previous data reported for P. destructans. This new finding improves the current knowledge about the European distribution of P. destructans, which is of great interest for forthcoming studies on the fungus dispersion and impact among bat populations at regional and/or global level.     

Worldwide Occurrence of Integrative Conjugative Element Encoding Multidrug Resistance Determinants in Epidemic Vibrio cholerae O1

In the last decades, there has been an increase of cholera epidemics caused by multidrug resistant strains. Particularly, the integrative and conjugative element (ICE) seems to play a major role in the emergence of multidrug resistant Vibrio cholerae. This study fully characterized, by whole genome sequencing, new ICEs carried by multidrug resistant V. cholerae O1 strains from Nigeria (2010) (ICEVchNig1) and Nepal (1994) (ICEVchNep1). The gene content and gene order of these two ICEs are the same, and identical to ICEVchInd5, ICEVchBan5 and ICEVchHai1 previously identified in multidrug resistant V. cholerae O1. This ICE is characterized by dfrA1, sul2, strAB and floR antimicrobial resistance genes, and by unique gene content in HS4 and HS5 ICE regions. Screening for ICEs, in publicly available V. cholerae genomes, revealed the occurrence and widespread distribution of this ICE among V. cholerae O1. Metagenomic analysis found segments of this ICE in marine environments far from the direct influence of the cholera epidemic. Therefore, this study revealed the epidemiology of a spatio-temporal prevalent ICE in V. cholerae O1. Its occurrence and dispersion in V. cholerae O1 strains from different continents throughout more than two decades can be indicative of its role in the fitness of the current pandemic lineage.     

Liquid-liquid extraction of a Schistosoma mansoni recombinant protein from an Escherichia coli extract in aqueous two-phase system

This work describes the partition of a Schistosoma mansoni tegumental antigen produced by a recombinant Escherichia coli strain using an aqueous two-phase system composed of polyethylene glycol (PEG) and potassium phosphate. The effects of the polymer molecular weight, tie line length and pH on antigen partitioning were investigated. The detection of the antigen in both phases were determined by ELISA. The system composed of PEG 3550 (19.7% w/w) and potassium phosphate (17.7% w/w) led to a yield of 59% and an antigen purification factor of 3 in the PEG-rich phase. It was observed that the antigen partition in ATPS was strongly affected by the pH value and tie line length. In addition, it was possible in a single step, to remove the cell debris, that precipitated at the interface of the system.     

A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.     

The adsorption of Fusarium flocciferum spores on celite particles and their use in the degradation of phenol

Summary Spores of Fusarium flocciferum were inserted in porous celite beads. The effects of bead size, adsorption time course, washing cycle and spore concentration on spore loading were investigated. Cell loadings up to 50% (dry weight/beads) were obtained. The degradation of phenol using adsorbed cells was studied in batch experiments. The immobilized cell system was shown to efficiently degrade high concentrations of the substrate (up to 2.0 g/l) and to remain active for more than 2 motths. The oxygen uptake rate of free and immobilized cells was determined at various concentrations of phenol. The kinetic constants K _s=85 mg/l, K _i=345 mg/l and SMI=170 mg/l were estimated from the experimental data by linearization of the Haldane function for the free cells. The uptake rates exhibited by the confined cells were lower (30%) than those obtained for free cells and no significant differences were found for phenol concentrations between 150 and 1200 mg/l.     

Understanding extinction debts: spatio–temporal scales,mechanisms and a roadmap for future research

Extinction debt refers to delayed species extinctions expected as a consequence of ecosystem perturbation. Quantifying such extinctions and investigating long‐term consequences of perturbations has proven challenging, because perturbations are not isolated and occur across various spatial and temporal scales, from local habitat losses to global warming. Additionally, the relative importance of eco‐evolutionary processes varies across scales, because levels of ecological organization, i.e. individuals, (meta)populations and (meta)communities, respond hierarchically to perturbations. To summarize our current knowledge of the scales and mechanisms influencing extinction debts, we reviewed recent empirical, theoretical and methodological studies addressing either the spatio–temporal scales of extinction debts or the eco‐evolutionary mechanisms delaying extinctions. Extinction debts were detected across a range of ecosystems and taxonomic groups, with estimates ranging from 9 to 90% of current species richness. The duration over which debts have been sustained varies from 5 to 570 yr, and projections of the total period required to settle a debt can extend to 1000 yr. Reported causes of delayed extinctions are 1) life‐history traits that prolong individual survival, and 2) population and metapopulation dynamics that maintain populations under deteriorated conditions. Other potential factors that may extend survival time such as microevolutionary dynamics, or delayed extinctions of interaction partners, have rarely been analyzed. Therefore, we propose a roadmap for future research with three key avenues: 1) the microevolutionary dynamics of extinction processes, 2) the disjunctive loss of interacting species and 3) the impact of multiple regimes of perturbation on the payment of debts. For their ability to integrate processes occurring at different levels of ecological organization, we highlight mechanistic simulation models as tools to address these knowledge gaps and to deepen our understanding of extinction dynamics.     

Visitation rate of pollinators and nectar robbers to the flowers and inflorescences of Tabebuia aurea (Bignoniaceae): effects of floral display size and habitat fragmentation

Large floral displays favour pollinator attraction and the import and export of pollen. However, large floral displays also have negative effects, such as increased geitonogamy, pollen discounting and nectar/pollen robber attraction. The size of the floral display can be measured at different scales (e.g. the flower, inflorescence or entire plant) and variations in one of these scales may affect the behaviour of flower visitors in different ways. Moreover, the fragmentation of natural forests may affect flower visitation rates and flower visitor behaviour. In the present study, video recordings of the inflorescences of a tree species (Tabebuia aurea) from the tropical savannah of central Brazil were used to examine the effect of floral display size at the inflorescence and tree scales on the visitation rate of pollinators and nectar robbers to the inflorescence, the number of flowers approached per visit, the number of visits per flower of potential pollinators and nectar robbers, and the interaction of these variables with the degree of landscape disturbance. Nectar production was quantified with respect to flower age. Although large bees are responsible for most of the pollination, a great diversity of flower insects visit the inflorescences of T. aurea. Other bee and hummingbird species are highly active nectar robbers. Increases in inflorescence size increase the visitation rate of pollinators to inflorescences, whereas increases in the number of inflorescences on the tree decrease visitation rates to inflorescences and flowers. This effect has been strongly correlated with urban environments in which trees with the largest floral displays are observed. Pollinating bees (and nectar robbers) visit few flowers per inflorescence and concentrate visits to a fraction of available flowers, generating an overdispersed distribution of the number of visits per inflorescence and per flower. This behaviour reflects preferential visits to young flowers (including flower buds) with a greater nectar supply.     

Comparing the effect of immobilization methods on the activity of lipase biocatalysts in ester hydrolysis

The activity of various lipases was compared, in both free and immobilized forms, using the kinetics of the hydrolysis reaction of p-nitrophenyl butyrate, which was followed with in situ UV/Vis diode array spectrophotometry. Several enzymes were used to catalyze the reaction, namely Candida antarctica lipase B and Fusarium solani pisi cutinase wildtype and three single-mutation variants. The enzymes were tested in three different forms: free, immobilized as cross-linked aggregates and supported on zeolite NaY. A simple kinetic model was used to allow a quantitative comparison of the behavior of the different catalysts. It was concluded that although immobilization reduces the activity of the enzyme, the zeolite offers a much higher specific activity when compared to the cross-linked aggregates, thus supplying a heterogeneous catalyst with promising catalytic properties.