Improvement of Brazilian bioethanol production – Challenges and perspectives on the identification and genetic modification of new strains of Saccharomyces cerevisiae yeasts isolated during ethanol process

In Brazil, bioethanol is produced by sucrose fermentation from sugarcane by Saccharomyces cerevisiae in a fed-batch process that uses high density of yeast cells (15–25 % of wet weight/v) and high sugar concentration (18–22 % of total sugars). Several research efforts have been employed to improve the efficiency of this process through the isolation of yeasts better adapted to the Brazilian fermentation conditions. Two important wild strains named CAT-1 and PE-2 were isolated during the fermentation process and were responsible for almost 60 % of the total ethanol production in Brazil. However, in the last decade the fermentative substrate composition was much modified, since new sugar cane crops were developed, the use of molasses instead of sugar cane juice increase and with the prohibition of burning of sugarcane prior harvest. As consequence, these previously isolated strains are being replaced by new wild yeasts in most of ethanol plants. In this new scenario the isolation of novel better adapted yeasts with improved fermentative characteristics is still a big challenge. Here, we discuss the main aspects of Brazilian ethanol production and the efforts for the selection, characterization and genetic modifications of new strains with important phenotypic traits such as thermotolerance.     

Efficient clustering in collaborative filtering recommender system: Hybrid method based on genetic algorithm and gravitational emulation local search algorithm

The ultimate goal of the Recommender System (RS) is to offer a proposal that is very close to the user's real opinion. Data clustering can be effective in increasing the accuracy of production proposals by the RS. In this paper, single-objective hybrid evolutionary approach is proposed for clustering items in the offline collaborative filtering RS. This method, after generating a population of randomized solutions, at each iteration, improves the population of solutions first by Genetic Algorithm (GA) and then by using the Gravitational Emulation Local Search (GELS) algorithm. Simulation results on standard datasets indicate that although the proposed hybrid meta-heuristic algorithm requires a relatively high run time, it can lead to more appropriate clustering of existing data and thus improvement of the Mean Absolute Error (MAE), Root Mean Square Error (RMSE) and Coverage criteria.     

一类捕食—食饵动力系统的定性分析

本文对一类形式广泛的捕食-食饵动力系统进行了定性研究,获得了确保此生态系统的所有解有界及极限环存在与唯一的若干充分条件,并给出了所获结论在生态学上的意义。     

Enrichment experiments and primary production at Sagres (SW Portugal)

Water was collected from the Sagres station (SW Portugal) in September 2002, at a site adjacent to the upwelling centre of Cabo São Vicente, during relaxation of upwelling conditions. Surface and depth samples were enriched with inorganic nutrients in order to evaluate their relative influence on the microalgal assemblage. Small-scale, short-term bioassays involved separate in vitro additions of nitrogen and phosphorus. Enrichments with nitrogen led to a general increase of primary production, suggesting nitrogen as the primary potential nutrient limiting microalgal growth during this period, as well as altering the relative microplanktonic composition in favour of diatoms.     

Substance P binding sites in the nucleus tractus solitarius of the cat

Substance P binding sites in the nucleus tractus solitarius were visualized with receptor autoradiography using Bolton-Hunter [¹²⁵I]substance P. Substance P binding sites were found to have distinct patterns within the cat nucleus tractus solitarius. The majority of substance P binding sites were present in the medial, intermediate and the peripheral rim of the parvocellular subdivisions. Lower amounts of substance P binding sites were present in the commissural, ventrolateral, interstitial and dorsolateral subdivisions. No substance P binding sites were present in the central region of the parvocellular subdivision or the solitary tract. The localization of substance P binding sites in the nucleus tractus solitarius is very similar to the patterns of substance P immunoreactive fibers previously described for this region. Results of this study add further support for a functional role of substance P in synaptic circuits of the nucleus tractus solitarius.     

Optimization of the Tet-On system for inducible expression of RAGE.

We have optimized a two-plasmid Tet-On system, the regulatory plasmid and the response plasmid, to produce tightly controlled inducible expression of the gene RAGE in cell-culture models. Two sets of plasmids were constructed: set 1 (universal; for broad range of cell types) and set 2 (neuron specific). For the response plasmid, the gene RAGE was cloned in pIRES2-EGFP plasmid (Clontech) and the CMV promoter replaced with TREtight (modified seven copies of Tet-operon fused with CMVm promoter). For the regulatory plasmid, rtTA (reverse tetracycline transactivator) was placed under either the CMV promoter or the cell-specific promoter neuronal specific enolase. Both plasmids have the mammalian selection marker neomycine; the EGFP reporter gene is only in the response plasmid and IRES is between the gene and EGFP. Following induction with doxycycline, cells expressing RAGE showed neomycine resistance and green fluorescence (EGFP). Our system has been tested in two different cell lines and showed negligible basal leakiness, high induction of the gene RAGE (142-fold), dose-dependent response to doxycycline, and strict cell-type specificity. This system is highly suitable for cell-specific expression of any gene of interest in primary cultures and mixed cell populations.     

Ethylene signaling: identification of a putative ETR1-AHP1 phosphorelay complex by fluorescence spectroscopy

The plant ethylene receptor ETR1, which shows substantial sequence homology to typical bacterial histidine kinases, is involved in the coordination of several growth and development processes. Fluorescence polarization studies presented here demonstrate a specific interaction of ETR1 with the histidine-containing transfer protein AHP1, supporting the idea that a phosphorelay module is involved in ethylene signaling. The sensitive assay employed in our studies allows analysis of protein-protein interactions in a homogenous aqueous environment, exact control of external parameters, and quantitative analysis of the affinity and stability of the complex. Thereby it provides the basics for a more quantitative elucidation of phosphorelay modules acquired in phytohormone signaling.     

HVJ-envelope vector for gene transfer into central nervous system

To overcome some problems of virus vectors, we developed a novel non-viral vector system, the HVJ-envelope vector (HVJ-E). In this study, we investigated the feasibility of gene transfer into the CNS using the HVJ-E both in vitro and in vivo. Using the Venus reporter gene, fluorescence could be detected in cultured rat cerebral cortex neurons and glial cells. In vivo, the reporter gene (Venus) was successfully transfected into the rat brain by direct injection into the thalamus, intraventricular injection, or intrathecal injection, without inducing immunological change. When the vector was injected after transient occlusion of the middle cerebral artery, fluorescence due to EGFP gene or luciferase activity could be detected only in the injured hemisphere. Finally, luciferase activity was markedly enhanced by the addition of 50 U/ml heparin (P<0.01). Development of efficient HVJ-E for gene transfer into the CNS will be useful for research and clinical gene therapy.