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.

Nitrogen and Sulfur Fertilization and Dynamics in a Brazilian Entisol under Pasture

Nutrient dynamics in tropical soils sustaining forage grasses are still poorly understood. We conducted a study to evaluate the effect of combined N and S fertilizer rates on the growth of `Marandu` palisade grass [Brachiaria brizantha (Hochst. ex A. Rich.) Stapf], uptake of these elements from the soil by plants, soil organic matter concentration, soil pH, and the mineral and organic fractions of N and S in an Entisol. Combinations of five N rates (0, 100, 200, 300, and 400 g N m(-3)) with five S rates (0, 10, 20, 30, and 40 g S m(-3)) were evaluated in a partial 5 x 5 factorial in a pot experiment, with and without plants. Nitrogen and S were supplied as NH(4)NO(3) and CaSO(4)center dot 2H(2)O, respectively. The N addition in excess did not enhance the palisade grass production due to low plant-available Sin the soil. The supply of low rates of S with N greatly improved the overall N uptake efficiency by the forage plant. The contents of total N, NO(3)(-)-N, and NH(4)(+)-N in the soil varied with N rate and with N uptake by the plants. The association of palisade grass with S fertilization increased the ester-bonded S fraction in the soil. The results suggest that soil residual S could be a potential source of S for plants. Proper N and S fertilizer rates promoted increased grass production due to increased uptake of these nutrients and the dynamics of the organic N and S fractions and mineral fractions in this tropical soil.

Growth performance and body composition of pacu Piaractus mesopotamicus (Holmberg 1887) in response to dietary protein and energy levels

Improper dietary protein and energy levels and their ratio will lead to increased fish production cost. This work evaluated effects of dietary protein : energy ratio on growth and body composition of pacu, Piaractus mesopotamicus. Fingerling pacu (15.5 +/- 0.4 g) were fed twice a day for 10 weeks until apparent satiation with diets containing 220, 260, 300, 340 or 380 g kg-1 crude protein (CP) and 10.9, 11.7, 12.6, 13.4 or 14.2 MJ kg-1 digestible energy (DE) in a totally randomized experimental design, 5 x 5 factorial scheme (n = 3). Weight gain, specific growth rate increased and feed conversion ratio (FCR) decreased significantly (P < 0.05) when CP increased from 220 to 271, 268 and 281 g kg-1 respectively. Pacu was able to adjust feed consumption in a wide range of dietary DE concentration. Fish fed 260 CP diets showed best (P < 0.05) protein efficiency ratio and FCR with 11.7-12.6 MJ kg-1; but for the 380 CP-diets group, significant differences were observed only at 14.2 MJ kg-1 dietary energy level, suggesting that pacu favours protein as energy source. DE was the chief influence on whole body chemical composition. Minimum dietary protein requirement of pacu is 270 g kg-1, with an optimum CP : DE of 22.2 g MJ-1.

Dietary lysine requirement of juvenile pacu Piaractus mesopotamicus (Holmberg, 1887)

Lysine is the reference essential amino acid in fish feeds and usually the most limiting amino acid in feedstuffs. The dietary lysine requirement of juvenile pacu Piaractus mesopotamicus (4.3 g) was determined using five isonitrogenous (32% CP) test diets containing graded levels of lysine (0.9, 1.17, 1.44, 1.69 and 1.96% of dry diet) fed three times a day to four groups of 18 fish for 74 days. Growth, body composition, nutrient retention and hematological parameters of pacu were analyzed. Analysis of variance showed that all growth performance parameters were significantly affected by dietary treatments. The lysine requirements estimated using regression analysis for maximum weight gain and feed efficiency were 1.45 and 1.51% of dry diet, respectively. Nitrogen retention efficiency increased with increasing levels of dietary lysine up to 1.43% (p<0.05). Whole-body protein increased (p<0.05) and whole-body lipid decreased (p<0.05) with increasing dietary lysine level. Thus, the lysine requirement of juvenile pacu was estimated as being 1.4-1.5% of dry diet or 4.4-4.7% of dietary protein. (c) 2009 Elsevier B.V. All rights reserved.

Cow/calf preweaning efficiency of Nellore and Bos taurus x Bos indicus crosses

The objectives of this study were to determine if percentage Bos taurus (0 or 50%) of the cow had an effect on ME requirements and milk production, and to compare cow/calf efficiency among 3 mating systems. Metabolizable energy requirements were estimated during a feeding trial that encompassed a gestation and lactation feeding trial for each of 2 groups of cows. Cows were 0 or 50% Bos taurus ( 100 or 50% Nellore) breed type: Nellore cows (NL; n = 10) mated to Nellore bulls, NL cows ( n = 9) mated to Angus bulls, Angus x Nellore (ANL; n = 10) and Simmental x Nellore (SNL; n = 10) cows mated to Canchim (5/ 8 Charolais 3/ 8 Zebu) bulls. Cows were individually fed a total mixed diet that contained 11.3% CP and 2.23 Mcal of ME/kg of DM. At 14-d intervals, cows and calves were weighed and the amount of DM was adjusted to keep shrunk BW and BCS of cows constant. Beginning at 38 d of age, corn silage was available to calves ad libitum. Milk production at 42, 98, 126, and 180 d postpartum was measured using the weigh-suckle-weigh technique. At 190 d of age, calves were slaughtered and body composition estimated using 9-10-11th-rib section to obtain energy deposition. Regression of BW change on daily ME intake (MEI) was used to estimate MEI at zero BW change. Increase in percentage Bos taurus had a significant effect on daily ME requirements (Mcal/d) during pregnancy (P < 0.01) and lactation (P < 0.01). Percentage Bos taurus had a positive linear effect on maintenance requirements of pregnant (P = 0.07) and lactating (P < 0.01) cows; during pregnancy, the ME requirements were 91 and 86% of those in lactation (131 +/- 3.5 vs. 145 +/- 3.4 Mcal.kg(-0.75).d(-1)) for the 0 and 50% B. taurus groups, respectively. The 50% B. taurus cows, ANL and SNL, suckling crossbred calves had greater total MEI (4,319 +/- 61 Mcal; P < 0.01) than 0% B. taurus cows suckling NL (3,484 +/- 86 Mcal) or ANL calves (3,600 +/- 91 Mcal). The 0% B. taurus cows suckling ANL calves were more efficient (45.3 +/- 1.6 g/Mcal; P = 0.03) than straightbred NL (35.1 +/- 1.5 g/Mcal) and ANL or SNL pairs (41.0 +/- 1.0 g/Mcal). Under the conditions of this study, crossbreeding improved cow/ calf efficiency and showed an advantage for cows that have lower energy requirements.

Parameterization and Evaluation of Predictions of DSSAT/CANEGRO for Brazilian Sugarcane

The DSSAT/CANEGRO model was parameterized and its predictions evaluated using data from five sugarcane (Sacchetrum spp.) experiments conducted in southern Brazil. The data used are from two of the most important Brazilian cultivars. Some parameters whose values were either directly measured or considered to be well known were not adjusted. Ten of the 20 parameters were optimized using a Generalized Likelihood Uncertainty Estimation (GLUE) algorithm using the leave-one-out cross-validation technique. Model predictions were evaluated using measured data of leaf area index (LA!), stalk and aerial dry mass, sucrose content, and soil water content, using bias, root mean squared error (RMSE), modeling efficiency (Eff), correlation coefficient, and agreement index. The Decision Support System for Agrotechnology Transfer (DSSAT)/CANEGRO model simulated the sugarcane crop in southern Brazil well, using the parameterization reported here. The soil water content predictions were better for rainfed (mean RMSE = 0.122mm) than for irrigated treatment (mean RMSE = 0.214mm). Predictions were best for aerial dry mass (Eff = 0.850), followed by stalk dry mass (Eff = 0.765) and then sucrose mass (Eff = 0.170). Number of green leaves showed the worst fit (Eff = -2.300). The cross-validation technique permits using multiple datasets that would have limited use if used independently because of the heterogeneity of measures and measurement strategies.

The development and production of leaves and tillers by Marandu palisadegrass fertilised with nitrogen and sulphur

The effects of combined nitrogen and sulphur fertilisation on the dynamics of leaf and tiller appearance in Marandu palisadegrass (Brachiaria brizantha cv. Marandu) and its impact on dry matter production were evaluated in a greenhouse study. Grass seedlings were grown in pots filled with a soil classified as an Entisol and were harvested after 43 days, a further 35 days and finally after 48 more days. Five rates of N (0, 100, 200, 300 and 400 mg/dm(3)) and 5 rates of S (0, 10, 20, 30 and 40 mg/dm(3)) were tested in an incomplete factorial design with 4 replications. Leaf and tiller development were monitored every 3 days by counting the appearance of recently expanded leaves and new basal tillers. The phyllochron and thermal time between appearance of tillers decreased as N and S fertiliser levels increased to about 300 and 25 mg/dm(3), respectively, then tended to increase. In contrast, leaf and tiller appearance rates increased with the supply of these nutrients to similar levels, then tended to decline. Leaf and tiller production and dry matter yields were affected by both N and S levels, with the role of S increasing as the growth phases increased.

Yield, Potassium Uptake, and Use Efficiency in Upland Rice Genotypes

Potassium (K) is an essential nutrient for higher plants. Information on K uptake and use efficiency of upland rice under Brazilian conditions is limited. A greenhouse experiment was conducted with the objective to evaluate influence of K on yield, K uptake, and use efficiency of six upland rice genotypes grown on Brazilian Oxisol. The K rate used was zero (natural soil level) and 200 mg K kg-1 of soil. Shoot dry weight and grain yield were significantly influenced by K level and genotype treatments. However, K x genotype interactions were not significant, indicating similar responses of genotypes at two K levels for shoot dry weight and grain yield. Genotypes produced grain yield in the order of BRS Primavera BRA 01596 BRSMG Curinga BRS 032033 BRS Bonanca BRA 02582. Potassium concentration in shoot was about sixfold greater compared to grain, across two K levels and six genotypes. However, K utilization efficiency ratio (KUER) (mg shoot or grain yield / mg K uptake in shoot or root) was about 6.5 times greater in grain compared to shoot, across two K level and six genotypes. Potassium uptake in shoot and grain and KUER were significantly and positively associated with grain yield. Soil calcium (Ca), K, base saturation, acidity saturation, Ca saturation, K saturation, Ca/K ratio, and magnesium (Mg)/K ratio were significantly influenced by K application rate.

X ray densitometry applied to technological evaluation of MDF panels of eucalyptus

MDF panels from eucalyptus wood fibers were manufactured in laboratory and industrial production and had their apparent density profile determined by X-ray densitometry. The MDF panels apparent density parameters (maximum density of the superior and inferior faces; medium and minimum density) were determined and compared. The results indicated that the density values of the MDF panels made in the laboratory and in industrial line did not show significant statistical differences, indicating the similarities in the pressing phase of the fibers of both kinds. However, for MDF panels of laboratory and production line, the values of maximum, mean and minimum densities showed statistically significant correlations. The determination of the density profile for MDF panels by X-ray densitometry is important for the evaluation of pressing phase and other variables of the industrial process of production, as well for the determination of the technological properties.

WATER USE EFFICIENCY OF HIDROPONIC MUSKMELON

With the aim to study the water efficiency on the muskmelon hydroponics during a long cycle of crop and with different intervals between irrigation was carried out an experiment in two season from October 2003 to January 2004 (season I) and from January to April (season II). The experiment was carried out on the Fitotecnia Department on the Universidade Federal of Santa Maria, Santa Maria, RS. Were determined the water consumptions on the growth of the plants to observe the water efficiency. The water efficiency was a maximum on the blossom phase (4.19g de FS m(-3)) on the season I and on the vegetative phase (8.22g de FS m(-3)) for season II, associated with an elevated growth rate and small water consumptions on these seasons.

Hemoglobin Regeneration Efficiency in Anemic Rats: Effects on Bone Mineral Composition and Biomechanical Properties

This study reports the effects of dietary iron (Fe) deficiency and recovery on bone mineral composition and strength in anemic rats submitted to a hemoglobin (Hb) repletion assay. Weanling male Wistar rats were fed a low-Fe diet (12 mg/kg) for 15 days followed by 2 weeks of Fe repletion with diets providing 35 mg Fe/kg as either ferrous sulfate (n = 8) or ferric pyrophosphate (FP; n = 12). At final day of each period (depletion and repletion), Fe-adequate animals were also euthanized. Iron status (blood Hb, Hb Fe pool, Hb regeneration efficiency), tibia mineral concentrations (Ca, Mg, Fe, Cu, and Zn) and biomechanical properties were evaluated. Iron-deficient rats had lower tibia Fe and Mg levels and bone strength when compared to controls. Yield load and resilience were positively related to tibia Mg levels (r = 0.47, P = 0.02 and r = 0.56, P = 0.004, respectively). Iron repletion did not recover tibia Mg concentrations impaired by Fe deficiency. Moreover, bone elastic properties were negatively affected by FP consumption. In conclusion, bone mineral composition and strength were affected by Fe deficiency, whereas dietary Fe source influenced tibia Mg and resistance in the period during which rats were recovering from anemia.

Rubus rosaefolius Extract as a Natural Preservative Candidate in Topical Formulations

Even though the synthetic preservatives may offer a high antimicrobial efficacy, they are commonly related to adverse reactions and regarded as having potentially harmful effects caused by chronic consumption. The development of natural preservatives provides a way of reducing the amount of synthetic preservatives normally used in pharmaceutical and cosmetic preparations. In addition, these agents have less toxic effects and represent a possible natural and safer alternative of the preservatives. The purpose of this research was to evaluate the Rubus rosaefolius Smith extract efficiency as a natural preservative in base formulations. Of the extract, 0.2% (w/w) was assayed for its effectiveness of antimicrobial protection in two different base formulations (emulsion and gel). The microbial challenge test was performed following the standard procedures proposed by The United States Pharmacopoeia 33nd, European Pharmacopoeia 6th, Japanese Pharmacopoeia 15th, and the Cosmetics, Toiletries, and Fragrance Association using standardized microorganisms. The results demonstrated that R. rosaefolius extract at the studied concentration reduced the bacterial inocula, satisfying the criterion in all formulations, even though it was not able to present an effective preservative behavior against fungi. Thus, the investigation of new natural substances with preservative properties that could be applied in pharmaceutical and cosmetic products is relevant due to the possibility of substituting or decreasing the concentration of synthetic preservatives, providing a way for the development of safer formulas for the use of consumers.

Comparison of oxidation efficiency of disperse dyes by chemical and photoelectrocatalytic chlorination and removal of mutagenic activity

Degradation of Disperse Orange 1, Disperse Red 1 and Disperse Red 13 dyes has been performed using electrochemical oxidation on Pt electrode, chemical chlorination and photoelectrochemical oxidation on Ti/TiO(2) thin film electrodes in NaCl or Na(2)SO(4) medium. 100% discoloration was obtained for all tested methods after 1 h of treatment. Faster color removal was obtained by photoelectrocatalytic oxidation in 0.1 mol L(-1) NaCl pH 4.0 under UV light and an applied potential of +1.0V (vs SCE reference electrode), which indicates also values around 60% of TOC removal. The conventional chlorination method and electrochemical oxidation on Pt electrode resulted in negligible reduction of TOC removal. All dyes showed positive mutagenic activity in the Salmonella/microsome assay with the strain TA98 in the absence and presence of S9 (exogenous metabolic activation). Nevertheless, there is complete reduction of the mutagenic activity after 1 h of photoelectrocatalytic oxidation, suggesting that this process would be good option to remove disperse azo dyes from aqueous media. (C) 2008 Elsevier Ltd. All rights reserved.

Production efficiency and financing of public health: an analysis of small municipalities in the state of Sao Paulo - Brazil

This paper measured the variations in performance of small municipalities in the State of Sao Paulo, Brazil, regarding the technical efficiency in the use of public funds in public primary health care actions concerning the funding profile, in a scenario of fiscal federalism. Technical efficiency is one of the parameters of evaluation of public sector performance and was measured by means of Data Envelopment Analysis (DEA). The correlation analysis of DEA score was used to verify possible associations between technical efficiency and the funding profile of expenses with health care. The results showed that 6.41% of the municipalities were considered efficient. They also showed that the level of municipality dependence to inter-governmental general purpose grants and the national health funding specific purpose grants have negative correlation with efficiency scores.

Evaluating the efficiency of fractional integration parameter estimators

This article deals with the efficiency of fractional integration parameter estimators. This study was based on Monte Carlo experiments involving simulated stochastic processes with integration orders in the range]-1,1[. The evaluated estimation methods were classified into two groups: heuristics and semiparametric/maximum likelihood (ML). The study revealed that the comparative efficiency of the estimators, measured by the lesser mean squared error, depends on the stationary/non-stationary and persistency/anti-persistency conditions of the series. The ML estimator was shown to be superior for stationary persistent processes; the wavelet spectrum-based estimators were better for non-stationary mean reversible and invertible anti-persistent processes; the weighted periodogram-based estimator was shown to be superior for non-invertible anti-persistent processes.