Thermodynamic evaluation and modeling of proton and water exchange associated with benzamidine and berenil binding to ß-trypsin

Serine-proteases are involved in vital processes in virtually all species. They are important targets for researchers studying the relationships between protein structure and activity, for the rational design of new pharmaceuticals. Trypsin was used as a model to assess a possible differential contribution of hydration water to the binding of two synthetic inhibitors. Thermodynamic parameters for the association of bovine ß-trypsin (homogeneous material, observed 23,294.4 ± 0.2 Da, theoretical 23,292.5 Da) with the inhibitors benzamidine and berenil at pH 8.0, 25ºC and with 25 mM CaCl2, were determined using isothermal titration calorimetry and the osmotic stress method. The association constant for berenil was about 12 times higher compared to the one for benzamidine (binding constants are K = 596,599 ± 25,057 and 49,513 ± 2,732 M-1, respectively; the number of binding sites is the same for both ligands, N = 0.99 ± 0.05). Apparently the driving force responsible for this large difference of affinity is not due to hydrophobic interactions because the variation in heat capacity (DCp), a characteristic signature of these interactions, was similar in both systems tested (-464.7 ± 23.9 and -477.1 ± 86.8 J K-1 mol-1 for berenil and benzamidine, respectively). The results also indicated that the enzyme has a net gain of about 21 water molecules regardless of the inhibitor tested. It was shown that the difference in affinity could be due to a larger number of interactions between berenil and the enzyme based on computational modeling. The data support the view that pharmaceuticals derived from benzamidine that enable hydrogen bond formation outside the catalytic binding pocket of ß-trypsin may result in more effective inhibitors.

Optimization of a dynamic district heating power plant

The purpose of this Thesis is to find the most optimal heat recovery solution for Wärtsilä’s dynamic district heating power plant considering Germany energy markets as in Germany government pays subsidies for CHP plants in order to increase its share of domestic power production to 25 % by 2020. Different heat recovery connections have been simulated dozens to be able to determine the most efficient heat recovery connections. The purpose is also to study feasibility of different heat recovery connections in the dynamic district heating power plant in the Germany markets thus taking into consideration the day ahead electricity prices, district heating network temperatures and CHP subsidies accordingly. The auxiliary cooling, dynamical operation and cost efficiency of the power plant is also investigated.

Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus

Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.

Use of the Wasserman equation in optimization of the duration of the power ramp in a cardiopulmonary exercise test: a study of Brazilian men

This study aimed to analyze the agreement between measurements of unloaded oxygen uptake and peak oxygen uptake based on equations proposed by Wasserman and on real measurements directly obtained with the ergospirometry system. We performed an incremental cardiopulmonary exercise test (CPET), which was applied to two groups of sedentary male subjects: one apparently healthy group (HG, n=12) and the other had stable coronary artery disease (n=16). The mean age in the HG was 47±4 years and that in the coronary artery disease group (CG) was 57±8 years. Both groups performed CPET on a cycle ergometer with a ramp-type protocol at an intensity that was calculated according to the Wasserman equation. In the HG, there was no significant difference between measurements predicted by the formula and real measurements obtained in CPET in the unloaded condition. However, at peak effort, a significant difference was observed between oxygen uptake (V˙O2)peak(predicted)and V˙O2peak(real)(nonparametric Wilcoxon test). In the CG, there was a significant difference of 116.26 mL/min between the predicted values by the formula and the real values obtained in the unloaded condition. A significant difference in peak effort was found, where V˙O2peak(real)was 40% lower than V˙O2peak(predicted)(nonparametric Wilcoxon test). There was no agreement between the real and predicted measurements as analyzed by Lin’s coefficient or the Bland and Altman model. The Wasserman formula does not appear to be appropriate for prediction of functional capacity of volunteers. Therefore, this formula cannot precisely predict the increase in power in incremental CPET on a cycle ergometer.

Optimization of random amplified polymorphic DNA protocol for molecular identification of Lophius gastrophysus

Lophius gastrophysus has important commercial value in Brazil particularly for foreign trade. In this study, we described the optimization of Random Amplified Polymorphic DNA (RAPD) protocol for identification of L. gastrophysus. Different conditions (annealing temperatures, MgCl concentrations, DNA quantity) were tested to find reproducible and adequate profiles. Amplifications performed with primers A01, ² A02 and A03 generate the best RAPD profiles when the conditions were annealing temperature of 36ºC, 25 ng of DNA quantity and 2.5 mM MgCl2. Exact identification of the species and origin of marine products is necessary and RAPD could be used as an accurate, rapid tool to expose commercial fraud.

Optimization and validation of a methodology to determine total arsenic, As(III) and As(V), in water samples, through graphite furnace atomic absorption spectrometry

The Graphite furnace atomic absorption spectrometry (GF AAS) was the technique chosen by the inorganic contamination laboratory (INCQ/ FIOCRUZ) to be validated and applied in routine analysis for arsenic detection and quantification. The selectivity, linearity, sensibility, detection, and quantification limits besides accuracy and precision parameters were studied and optimized under Stabilized Temperature Platform Furnace (STPF) conditions. The limit of detection obtained was 0.13 µg.L-1 and the limit of quantification was 1.04 µg.L-1, with an average precision, for total arsenic, less than 15% and an accuracy of 96%. To quantify the chemical species As(III) and As(V), an ion-exchange resin (Dowex 1X8, Cl- form) was used and the physical-chemical parameters were optimized resulting in a recuperation of 98% of As(III) and of 90% of As(V). The method was applied to groundwater, mineral water, and hemodialysis purified water samples. All results obtained were lower than the maximum limit values established by the legal Brazilian regulations, in effect, 50, 10, and 5 µg.L-1 para As total, As(III) e As(V), respectively. All results were statistically evaluated.

Docosahexaenoic acid ethyl esther (DHAEE) microcapsule production by spray-drying: optimization by experimental design

Docosahexaenoic acid is an essential polyunsaturated fatty acid with important metabolic activities. Its conjugated double bonds make it susceptible to decomposition. Its stability may be improved through fatty acid entrapment with a spray-drying technique; however, the many parameters involved in this technique must be considered to avoid affecting the final product quality. Therefore, this study aimed to evaluate the entrapment conditions and yields of fish oil enriched with docosahexaenoic acid ethyl ester. Microcapsules were obtained from Acacia gum using a spray-drying technique. The experimental samples were analyzed by chromatography and delineated by Statistica software, which found the following optimum entrapment conditions: an inlet temperature of 188 °C; 30% core material; an N2 flow rate of 55 mm; and a pump flow rate of 12.5 mL/minute. These conditions provided a 66% yield of docosahexaenoic acid ethyl ester in the oil, corresponding to 19.8% of entrapped docosahexaenoic acid ethyl ester (w/w). This result was considered significant since 30% corresponded to wall material.

Optimization of extrusion process for production of nutritious pellets

A blend of 50% Potato Starch (PS), 35% Quality Protein Maize (QPM), and 15% Soybean Meal (SM) were used in the preparation of expanded pellets utilizing a laboratory extruder with a 1.5 × 20.0 × 100.0 mm die-nozzle. The independent variables analyzed were Barrel Temperature (BT) (75-140 °C) and Feed Moisture (FM) (16-30%). The effect of extrusion variables was investigated in terms of Expansion Index (EI), apparent density (ApD), Penetration Force (PF) and Specific Mechanical Energy (SME), viscosity profiles, DSC, crystallinity by X-ray diffraction, and Scanning Electronic Microscopy (SEM). The PF decreased from 30 to 4 kgf with the increase of both independent variables (BT and FM). SME was affected only by FM, and decreased with the increase in this variable. The optimal region showed that the maximum EI was found for BT in the range of 123-140 °C and 27-31% for FM, respectively. The extruded pellets obtained from the optimal processing region were probably not completely degraded, as shown in the structural characterization. Acceptable expanded pellets could be produced using a blend of PS, QPM, and SM by extrusion cooking.

Application of Doehlert experimental design in the optimization of experimental variables for the Pseudozyma sp. (CCMB 306) and Pseudozyma sp. (CCMB 300) cell lysis

This study aimed to verify the influence of pH and temperature on the lysis of yeast using experimental design. In this study, the enzymatic extract containing β-1,3-glucanase and chitinase, obtained from the micro-organism Moniliophthora perniciosa, was used. The experiment showed that the best conditions for lysis of Pseudozyma sp. (CCMB 306) and Pseudozyma sp. (CCMB 300) by lytic enzyme were pH 4.9 at 37 ºC and pH 3.9 at 26.7 ºC, respectively. The lytic enzyme may be used for obtaining various biotechnology products from yeast.

Optimization of the extraction of carrageenan from Kappaphycus alvarezii using response surface methodology

This study aims to optimize an alternative method of extraction of carrageenan without previous alkaline treatment and ethanol precipitation using Response Surface Methodology (RSM). In order to introduce an innovation in the isolation step, atomization drying was used reducing the time for obtaining dry carrageenan powder. The effects of extraction time and temperature on yield, gel strength, and viscosity were evaluated. Furthermore, the extracted material was submitted to structural analysis, by infrared spectroscopy and nuclear magnetic resonance spectroscopy (¹H-NMR), and chemical composition analysis. Results showed that the generated regression models adequately explained the data variation. Carrageenan yield and gel viscosity were influenced only by the extraction temperature. However, gel strength was influenced by both, extraction time and extraction temperature. Optimal extraction conditions were 74 ºC and 4 hours. In these conditions, the carrageenan extract properties determined by the polynomial model were 31.17%, 158.27 g.cm-2, and 29.5 cP for yield, gel strength, and viscosity, respectively, while under the experimental conditions they were 35.8 ± 4.68%, 112.50 ± 4.96 g.cm-2, and 16.01 ± 1.03 cP, respectively. The chemical composition, nuclear magnetic resonance spectroscopy, and infrared spectroscopy analyses showed that the crude carrageenan extracted is composed mainly of κ-carrageenan.

Thermodynamic properties of water sorption of jackfruit (Artocarpus heterophyllus Lam.) as a function of moisture content

The Jackfruit tree is one of the most significant trees in tropical home gardens and perhaps the most widespread and useful tree in the important genus Artocarpus. The fruit is susceptible to mechanical and biological damage in the mature state, and some people find the aroma of the fruit objectionable, particularly in confined spaces. The dehydration process could be an alternative for the exploitation of this product, and the relationship between moisture content and water activity provides useful information for its processing and storage. The aim of this study was to determine the thermodynamic properties of the water sorption of jackfruit (Artocarpus heterophyllus Lam.) as a function of moisture content. Desorption isotherms of the different parts of the jackfruit (pulp, peduncle, mesocarp, peel, and seed) were determined at four different temperatures (313.15, 323.15, 333.15, and 343.15 K) in a water activity range of 0.02-0.753 using the static gravimetric method. Theoretical and empirical models were used to model the desorption isotherms. An analytical solution of the Clausius-Clapeyron equation was proposed to calculate the isosteric heat of sorption, the differential entropy, and Gibbs' free energy using the Guggenhein-Anderson-de Boer and Oswin models considering the effect of temperature on the hygroscopic equilibrium.