Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error-prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three-dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility.

The Genome Sequence of Taurine Cattle: A Window to Ruminant Biology and Evolution

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.

Temporal evolution of epithelial, vascular and interstitial lung injury in an experimental model of idiopathic pulmonary fibrosis induced by butyl-hydroxytoluene

This study was undertaken to test whether the structural remodelling of pulmonary parenchyma can be sequentially altered in a model and method that demonstrate the progression of the disease and result in remodelling within the lungs that is typical of idiopathic pulmonary fibrosis. Three groups of mice were studied: (i) animals that received 3-5-di-tert-butyl-4-hydroxytoluene (BHT) and were killed after 2 weeks (early BHT = 9); (ii) animals that received BHT and were killed after 4 weeks (late BHT = 11); (iii) animals that received corn oil solution (control = 10). The mice were placed in a ventilated Plexiglas chamber with a mixture of pure humidified oxygen and compressed air. Lung histological sections underwent haematoxylin-eosin, immunohistochemistry (epithelial, endothelial and immune cells) and specific staining (collagen/elastic fibres) methods for morphometric analysis. When compared with the control group, early BHT and late BHT groups showed significant decrease of type II pneumocytes, lower vascular density in both and higher endothelial activity. CD4 was increased in late BHT compared with early and control groups, while CD8, macrophage and neutrophil cells were more prominent only in early BHT. The collagenous fibre density were significantly higher only in late BHT, whereas elastic fibre content in late BHT was lower than that in control group. We conclude that the BHT experimental model is pathologically very similar to human usual interstitial pneumonia. This feature is important in the identification of animal models of idiopathic pulmonary fibrosis that can accurately reflect the pathogenesis and progression of the human disease.

GALAXY EVOLUTION THROUGH THE COSMIC TIME

Elliptical galaxies are the best systems to study the early star formation activity in the universe. This work aims to understand the formation and evolution of these objects through the study of the integrated properties of their stellar populations. Here an evolutionary model is developed and their predicted spectrophotometric properties are presented.

Independent fusions and recent origins of sex chromosomes in the evolution and diversification of glass knife fishes (Eigenmannia)

The genus Eigenmannia comprises several species groups that display a surprising variety of diploid chromosome numbers and sex-determining systems. In this study, hypotheses regarding phylogenetic relationships and karyotype evolution were investigated using a combination of molecular and cytogenetic methods. Phylogenetic relationships were analyzed for 11 cytotypes based on sequences from five mitochondrial DNA regions. Parsimony-based character mapping of sex chromosomes confirms previous suggestions of multiple origins of sex chromosomes. Molecular cytogenetic analyses involved chromosome painting using probes derived from whole sex chromosomes from two taxa that were hybridized to metaphases of their respective sister cytotypes. These analyses showed that a multiple XY system evolved recently (<7 mya) by fusion. Furthermore, one of the chromosomes that fused to form the neo-Y chromosome is fused independently to another chromosome in the sister cytotype. This may constitute an efficient post-mating barrier and might imply a direct function of sex chromosomes in the speciation processes in Eigenmannia. The other chromosomal sex-determination system investigated is shown to have differentiated by an accumulation of heterochromatin on the X chromosome. This has occurred in the past 0.6 my, and is the most recent chromosomal sex-determining system described to date. These results show that the evolution of sex-determining systems can proceed very rapidly. Heredity (2011) 106, 391-400; doi:10.1038/hdy.2010.82; published online 23 June 2010

A GRADIENT-LIKE NONAUTONOMOUS EVOLUTION PROCESS

In this paper we consider a dissipative damped wave equation with nonautonomous damping of the form u(tt) + beta(t)u(t) - Delta u + f(u) (1) in a bounded smooth domain Omega subset of R(n) with Dirichlet boundary conditions, where f is a dissipative smooth nonlinearity and the damping beta : R -> (0, infinity) is a suitable function. We prove, if (1) has finitely many equilibria, that all global bounded solutions of (1) are backwards and forwards asymptotic to equilibria. Thus, we give a class of examples of nonautonomous evolution processes for which the structure of the pullback attractors is well understood. That complements the results of [Carvalho & Langa, 2009] on characterization of attractors, where it was shown that a small nonautonomous perturbation of an autonomous gradient-like evolution process is also gradient-like. Note that the evolution process associated to (1) is not a small nonautonomous perturbation of any autonomous gradient-like evolution processes. Moreover, we are also able to prove that the pullback attractor for (1) is also a forwards attractor and that the rate of attraction is exponential.

Semiclassical evolution of dissipative Markovian systems

A semiclassical approximation for an evolving density operator, driven by a `closed` Hamiltonian operator and `open` Markovian Lindblad operators, is obtained. The theory is based on the chord function, i.e. the Fourier transform of the Wigner function. It reduces to an exact solution of the Lindblad master equation if the Hamiltonian operator is a quadratic function and the Lindblad operators are linear functions of positions and momenta. Initially, the semiclassical formulae for the case of Hermitian Lindblad operators are reinterpreted in terms of a (real) double phase space, generated by an appropriate classical double Hamiltonian. An extra `open` term is added to the double Hamiltonian by the non-Hermitian part of the Lindblad operators in the general case of dissipative Markovian evolution. The particular case of generic Hamiltonian operators, but linear dissipative Lindblad operators, is studied in more detail. A Liouville-type equivariance still holds for the corresponding classical evolution in double phase space, but the centre subspace, which supports the Wigner function, is compressed, along with expansion of its conjugate subspace, which supports the chord function. Decoherence narrows the relevant region of double phase space to the neighbourhood of a caustic for both the Wigner function and the chord function. This difficulty is avoided by a propagator in a mixed representation, so that a further `small-chord` approximation leads to a simple generalization of the quadratic theory for evolving Wigner functions.

MODELING THE EVOLUTION OF COMPLEX NETWORKS THROUGH THE PATH-STAR TRANSFORMATION AND OPTIMAL MULTIVARIATE METHODS

The topology of real-world complex networks, such as in transportation and communication, is always changing with time. Such changes can arise not only as a natural consequence of their growth, but also due to major modi. cations in their intrinsic organization. For instance, the network of transportation routes between cities and towns ( hence locations) of a given country undergo a major change with the progressive implementation of commercial air transportation. While the locations could be originally interconnected through highways ( paths, giving rise to geographical networks), transportation between those sites progressively shifted or was complemented by air transportation, with scale free characteristics. In the present work we introduce the path-star transformation ( in its uniform and preferential versions) as a means to model such network transformations where paths give rise to stars of connectivity. It is also shown, through optimal multivariate statistical methods (i.e. canonical projections and maximum likelihood classification) that while the US highways network adheres closely to a geographical network model, its path-star transformation yields a network whose topological properties closely resembles those of the respective airport transportation network.

A quantitative and qualitative evaluation of reports of clinical trials published in six Brazilian dental journals indexed in the Scientific Electronic Library Online (SciELO)

INTRODUCTION: Open access publishing is becoming increasingly popular within the biomedical sciences. SciELO, the Scientific Electronic Library Online, is a digital library covering a selected collection of Brazilian scientific journals many of which provide open access to full-text articles.This library includes a number of dental journals some of which may include reports of clinical trials in English, Portuguese and/or Spanish. Thus, SciELO could play an important role as a source of evidence for dental healthcare interventions especially if it yields a sizeable number of high quality reports. OBJECTIVE: The aim of this study was to identify reports of clinical trials by handsearching of dental journals that are accessible through SciELO, and to assess the overall quality of these reports. MATERIAL AND METHODS: Electronic versions of six Brazilian dental Journals indexed in SciELO were handsearched at www.scielo.br in September 2008. Reports of clinical trials were identified and classified as controlled clinical trials (CCTs - prospective, experimental studies comparing 2 or more healthcare interventions in human beings) or randomized controlled trials (RCTs - a random allocation method is clearly reported), according to Cochrane eligibility criteria. CRITERIA TO ASSESS METHODOLOGICAL QUALITY INCLUDED: method of randomization, concealment of treatment allocation, blinded outcome assessment, handling of withdrawals and losses and whether an intention-to-treat analysis had been carried out. RESULTS: The search retrieved 33 CCTs and 43 RCTs. A majority of the reports provided no description of either the method of randomization (75.3%) or concealment of the allocation sequence (84.2%). Participants and outcome assessors were reported as blinded in only 31.2% of the reports. Withdrawals and losses were only clearly described in 6.5% of the reports and none mentioned an intention-to-treat analysis or any similar procedure. CONCLUSIONS: The results of this study indicate that a substantial number of reports of trials and systematic reviews are available in the dental journals listed in SciELO, and that these could provide valuable evidence for clinical decision making. However, it is clear that the quality of a number of these reports is of some concern and that improvement in the conduct and reporting of these trials could be achieved if authors adhered to internationally accepted guidelines, e.g. the CONSORT statement.

Survival and quality of life of patients with oral and oropharyngeal cancer at 1-year follow-up of tumor resection

OBJECTIVE: This study aimed to assess the survival and life quality evolution of patients subjected to surgical excision of oral and oropharyngeal squamous cell carcinoma. MATERIAL AND METHODS: Forty-seven patients treated at a Brazilian healthcare unit specialized in head and neck surgery between 2006 and 2007 were enrolled in the study. The gathering of data comprised reviewing hospital files and applying the University of Washington Quality of Life (UW-QOL) questionnaire previously and 1 year after the surgery. Comparative analysis used Poisson regression to assess factors associated with survival and a paired t-test to compare preoperative and 1-year postoperative QOL ratings. RESULTS: 1 year after surgery, 7 patients were not found (dropout of the cohort); 15 had died and 25 fulfilled the UW-QOL again. The risk of death was associated with having regional metastasis previously to surgery (relative risk=2.18; 95% confidence interval=1.09-5.17) and tumor size T3 or T4 (RR=2.30; 95%CI=1.05-5.04). Survivors presented significantly (p<0.05) poorer overall and domain-specific ratings of quality of life. Chewing presented the largest reduction: from 74.0 before surgery to 34.0 one year later. Anxiety was the only domain whose average rating increased (from 36.0 to 70.7). CONCLUSIONS: The prospective assessment of survival and quality of life may contribute to anticipate interventions aimed at reducing the incidence of functional limitations in patients with oral and oropharyngeal cancer.

Innovation and entrepreneurship in scientific research

This research wants to lay emphasis on topics that historicize and rescue concepts, in addition, shows the relation between innovation and socioeconomic development resulted; reviews the legal framework to stimulate new innovation into Brazilian society; considers the definition and the entrepreneur university's new paradigm; and finally, this research highlights the importance of teaching entrepreneur at universities, with straight society benefits. The results suggests that innovation is directly enrolled with a successful development of certain society, as well, is actually the biggest business competitive differential into corporative universe. In one way of getting results of social advancement to inform entrepreneur research practice, it appears that entrepreneurs concepts, necessarily, must be incorporated into the array of Science/Technology/Innovation for the effective development of supported formula. This article develops a thought about actual scientific researches paradigm, the way that is built on today, and if it's sufficient to effectively get the results that society expects from main bodies to create human resource and researches, especially those with innovation aspects, at Brazilian economic improvement.

Time evolution of the South Atlantic Magnetic Anomaly

The South Atlantic Magnetic Anomaly (SAMA) is one of the most outstanding anomalies of the geomagnetic field. The SAMA secular variation was obtained and compared to the evolution of other anomalies using spherical harmonic field models for the 1590-2005 period. An analysis of data from four South American observatories shows how this large scale anomaly affected their measurements. Since SAMA is a low total field anomaly, the field was separated into its nondipolar, quadrupolar and octupolar parts. The time evolution of the non-dipole/total, quadrupolar/total and octupolar/total field ratios yielded increasingly high values for the South Atlantic since 1750. The SAMA evolution is compared to the evolution of other large scale surface geomagnetic features like the North and the South Pole and the Siberia High, and this comparison shows the intensity equilibrium between these anomalies in both hemispheres. The analysis of non-dipole fields in historical period suggests that SAMA is governed by (i) quadrupolar field for drift, and (ii) quadrupolar and octupolar fields for intensity and area of influence. Furthermore, our study reinforces the possibility that SAMA may be related to reverse fluxes in the outer core under the South Atlantic region.

Um retrato da área de Neurociência e comportamento no Brasil

A neurociência compreende o estudo do controle neural das funções vegetativas, sensoriais e motoras; dos comportamentos de locomoção, reprodução e alimentação; e dos mecanismos da atenção, memória, aprendizagem, emoção, linguagem e comunicação. Tem, portanto, uma importante área de interface com a Psicologia. Dentre seus objetivos, a neurociência busca esclarecer os mecanismos das doenças neurológicas e mentais por meio do estudo do sistema nervoso normal e patológico. Sua evolução no Brasil tem ocorrido desde meados do século passado, e seu desenvolvimento foi incentivado pela criação de sociedades científicas específicas. O presente artigo relata esse desenvolvimento e descreve os principais grupos atuantes na neurociência brasileira.

The Southern Brazilian shelf: general characteristics, quaternary evolution and sediment distribution

Extending from latitude 34ºS to 22ºS the Southern Brazilian shelf constitutes the only part of the Brazilian shelf with a subtropical to temperate environment. The studies on the different geological aspects of the area began in the 1960's and have recently been reassessed after studies related to the determination of the Economic Exclusive Zone. In terms of morphology, the Southern Brazilian shelf may be divided into three sectors, the São Paulo Bight, the Florianópolis-Mostardas Sector and the Rio Grande Cone, characterized by conspicuous differences in terms of geological determining factors, bathymetry, declivities and the presence of canyons and channels. Despite the existence of hundreds of radiocarbon datings the sea level changes curve of southern Brazil during the Last Glacial Cycle is still a matter of debate. A recent controversy on the Middle and late Holocene sea level changes curve raised the question of the amplitude of the oscillations which occurred in the period. Also, a few but relatively consistent radiocarbon datings suggest the occurrence of a high sea level during Isotope Stage 3. In terms of sedimentary cover the Southern Brazilian shelf exhibits a very strong hydrodynamic control, both latitudinal and bathymetrical. The sector southward from 25ºS is characterized by the influence of the plume of water carrying sediments originating from the Río de La Plata. Actually its presence is conspicuous up to 28ºS, with the area between this latitude and 25ºS constituting a transitional zone. In terms of bathymetry the outer shelf is marked by the "floor-polisher" effect of the Brazil Current, which is responsible for the maintenance of a relict facies in areas deeper than 100 meters.