Use of chemometrics in the selection of a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expression system for recombinant cyprosin B production

Two multivariate statistical methods, factor analysis (FA) and hierarchical cluster analysis (HCA), were applied to experimental data set to evaluate their usefulness in selecting the adequate expression system and optimal growth parameters for recombinant cyprosin B production. Using FA, the large data set was reduced to two factors representing 73.4% of variability. Factor 1, with 53.5% of variability, corresponds to recombinant cyprosin B expression and efficient secretion, while factor 2, accounting for 19.9% of variability, represents cell growth and physiological characteristics. FA and HCA allowed the establishment of correlations among different variables and the clusters obtained providing clear identification of the experimental parameters related to cyprosin B production, which results on more accurate scientific output and time saving when selection of an adequate expression system is concerned.     

Yeasts from Macrobrachium amazonicum: a focus on antifungal susceptibility and virulence factors of Candida spp

In the present study, it was sought to compare yeast microbiota of wild and captive Macrobrachium amazonicum and evaluate the antifungal susceptibility and production of virulence factors by the recovered isolates of Candida spp. Additionally, cultivation water was monitored for the presence of fungi. Overall, 26 yeast isolates belonging to three genera and seven species were obtained, out of which 24 were Candida spp., with Candida famata as the most prevalent species for both wild and captive prawns. From cultivation water, 28 isolates of filamentous fungi were obtained, with Penicillium spp., Cladosporium spp. and Aspergillus spp. as the most frequent genera. Eight out of 24 Candida spp. isolates were resistant to azole derivatives, out of which four were recovered from wild-harvested prawns. As for production of virulence factors, three (12.5%) and eight (33.3%) isolates presented phospholipase and protease activity, respectively. This is the first comparative study between wild and captive prawns and the first report on yeast microbiota of M. amazonicum. The most relevant finding was the high percentage of resistant Candida spp., including from wild individuals, which suggests the occurrence of an environmental imbalance in the area where these prawns were captured.     

Location-specific Responsiveness to Environmental Perturbations in Wedge-capped Capuchins (Cebus olivaceus)

We recorded the responses of the members of a captive group of wedge-capped capuchins to novel and familiar objects placed in different parts of their cage in a study of the spatial dependency of activity with objects. We focused on behavioral pattern variability across subjects and across object location. Results show that, according to the location of the object, a great deal of within-subject response variability exists. The dominant male was slow to interact physically with objects and presented social-like behaviors—essentially grooming—towards objects in only one site. Implicit to the ethological approach is the assumption that consistent spatial location is irrelevant or, at best, of little importance to the definition of stimuli. Nevertheless, stimuli would be best considered as perturbations insofar as the significance of an object or event depends on where and when it is encountered. In order to evaluate how monkey cognition operates, it seems essential to investigate the role of the primate's own spatial structure. As a working hypothesis, we introduce the processes of spatial facilitation and inhibition and suggest that they affect how an individual interacts with objects and events.     

Indomethacin treatment reduces microglia activation and increases numbers of neuroblasts in the subventricular zone and ischaemic striatum after focal ischaemia

Neuroblasts from the subventricular zone (SVZ) migrate to striatum following stroke, but most of them die in the ischaemic milieu and this can be related to exacerbated microglial activation. Here, we explored the effects of the non-steroidal anti-inflammatory indomethacin on microglial activation, neuronal preservation and neuroblast migration following experimental striatal stroke in adult rats. Animals were submitted to endothelin-1 (ET-1)-induced focal striatal ischaemia and were treated with indomethacin or sterile saline (i.p.) for 7 days, being perfused after 8 or 14 days. Immunohistochemistry was performed to assess neuronal loss (anti-NeuN), microglial activation (anti-Iba1, ED1) and migrating neuroblasts (anti-DCX) by counting NeuN, ED1 and DCX-positive cells in the ischaemic striatum or SVZ. Indomethacin treatment reduced microglia activation and the number of ED1⁺ cells in both 8 and 14 days post injury as compared with controls. There was an increase in the number of DCX⁺ cells in both SVZ and striatum at the same survival times. Moreover, there was a decrease in the number of NeuN⁺ cells in indomethacin-treated animals as compared with the control group at 8 days but not after 14 days post injury. Our results suggest that indomethacin treatment modulates microglia activation, contributing to increased neuroblast proliferation in the SVZ and migration to the ischaemic striatum following stroke.     

A heterogeneous in vitro three dimensional model of tumour-stroma interactions regulating sprouting angiogenesis

Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model--a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre-capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis.     

Impact of Holoshestes heterodon Eigenmann (Pisces: Characidae) on the plankton community of a subtropical reservoir: the importance of predation by Chaoborus larvae

We analyzed the effects of planktivorous Holeshestes heterodon Eigenmann (Characidae) predation on the plankton community of a small subtropical reservoir, using four enclosures (volume about 17.5 m³), open to the sediment, established in the littoral zone. Two enclosures were stocked with fish (mean TL 5.7 cm), at a density of about 4–5 fish m^–3 (approx. 8 g m^–3), whereas two remained fishless. The experiment lasted a little longer than one month. In the fish enclosures, most Crustacea and Chaoborus larvae remained scarce, probably as a result of visually selective fish predation. In both fishless enclosures, Chaoborus larvae became abundant. However, in only one of these did large individuals become relatively numerous; this discrepancy in the demographic structure of the Chaoborus populations between the two fishless enclosures is unexplained. Only in the fishless enclosure without appreciable numbers of large Chaoborus did densities of Crustacea increase greatly. It is suggested that in the enclosure containing large Chaoborus individuals, crustacean populations were prevented from developing due to predation pressure, while the small Chaoborus larvae of the other enclosure could not readily consume these prey. Rotifers were low in abundance in the absence of fish, probably as a consequence of Chaoborus predation. Phytoplankton density increased in all four enclosures, due probably to the lack of water flow. Only in the fishless enclosure with high densities of crustaceans did phytoplankton abundance decrease markedly at the end of the experiment, perhaps because of grazing losses.     

Survival strategies of a sterol auxotroph

The high sterol concentration in eukaryotic cell membranes is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila cannot synthesize sterols, but do require them for development. Does this simply reflect a requirement for sterols in steroid hormone biosynthesis, or is bulk membrane sterol also essential in Drosophila? If the latter is true, how do they survive fluctuations in sterol availability and maintain membrane homeostasis? Here, we show that Drosophila require both bulk membrane sterol and steroid hormones in order to complete adult development. When sterol availability is restricted, Drosophila larvae modulate their growth to maintain membrane sterol levels within tight limits. When dietary sterol drops below a minimal threshold, larvae arrest growth and development in a reversible manner. Strikingly, membrane sterol levels in arrested larvae are dramatically reduced (dropping sixfold on average) in most tissues except the nervous system. Thus, sterols are dispensable for maintaining the basic membrane biophysical properties required for cell viability; these functions can be performed by non-sterol lipids when sterols are unavailable. However, bulk membrane sterol is likely to have essential functions in specific tissues during development. In tissues in which sterol levels drop, the overall level of sphingolipids increases and the proportion of different sphingolipid variants is altered. These changes allow survival, but not growth, when membrane sterol levels are low. This relationship between sterols and sphingolipids could be an ancient and conserved principle of membrane homeostasis.     

Patterns of Genetic Variability in Island Populations of the Cane Toad (Rhinella marina) from the Mouth of the Amazon

The Amazonian coast has several unique geological characteristics resulting from the interaction between drainage pattern of the Amazon River and the Atlantic Ocean. It is one of the most extensive and sedimentologically dynamic regions of the world, with a large number of continental islands mostly formed less than 10,000 years ago. The natural distribution of the cane toad (Rhinella marina), one of the world’s most successful invasive species, in this complex Amazonian system provides an intriguing model for the investigation of the effects of isolation or the combined effects of isolation and habitat dynamic changes on patterns of genetic variability and population differentiation. We used nine fast-evolving microsatellite loci to contrast patterns of genetic variability in six coastal (three mainlands and three islands) populations of the cane toad near the mouth of the Amazon River. Results from Bayesian multilocus clustering approach and Discriminant Analyses of Principal Component were congruent in showing that each island population was genetically differentiated from the mainland populations. All F_ST values obtained from all pairwise comparisons were significant, ranging from 0.048 to 0.186. Estimates of both recent and historical gene flow were not significantly different from zero across all population pairs, except the two mainland populations inhabiting continuous habitats. Patterns of population differentiation, with a high level of population substructure and absence/restricted gene flow, suggested that island populations of R. marina are likely isolated since the Holocene sea-level rise. However, considering the similar levels of genetic variability found in both island and mainland populations, it is reliable to assume that they were also isolated for longer periods. Given the genetic uniqueness of each cane toad population, together with the high natural vulnerability of the coastal regions and intense human pressures, we suggest that these populations should be treated as discrete units for conservation management purposes.