Human interferon B1 ser17: Coding DNA synthesis, expression, purification and characterization of bioactive recombinant protein

A protocol to produce large amounts of bioactive homogeneous human interferon β1 expressed in Escherichia coli was developed. Human interferon β1 ser17 gene was constructed, cloned and subcloned, and the recombinant protein expressed in E. coli cells. Solubilization of recombinant human interferon β1 ser17 (rhIFN-β1 ser17) was accomplished by employing a brief shift to high alkaline pH in the presence of non-ionic detergent. The recombinant protein was purifi ed by three chromatographic steps. N-terminal amino acid sequencing and mass spectrometry analysis provided experimental evidence for the identity of the recombinant protein. Reverse phase liquid chromatography demonstrated that the content of deamidates and sulphoxides was similar to a commercial standard. Size exclusion chromatography demonstrated the absence of high molecular mass aggregates and dimers. The protocol represents an effi cient and high-yield method to obtain bioactive homogeneous monomeric rhIFN-β1 ser17 protein. It may thus represent an important step towards scaling up for rhIFN-β1 ser17 large-scale production. © 2010 Villela AD, et al.

Immobilization and stabilization of a bimolecular aggregate of the lipase from Pseudomonas fluorescens by multipoint covalent attachment

The soluble lipase from Pseudomonas fluorescens (PFL) forms bimolecular aggregates in which the hydrophobic active centers of the enzyme monomers are in close contact. This bimolecular aggregate could be immobilized by multipoint covalent linkages on glyoxyl supports at pH 8.5. The monomer of PFL obtained by incubation of the soluble enzyme in the presence of detergent (0.5% TRITON X-100) could not be immobilized under these conditions. The bimolecular aggregate has two amino terminal residues in the same plane. A further incubation of the immobilized derivative under more alkaline conditions (e.g., pH 10.5) allows a further multipoint attachment of lysine (Lys) residues located in the same plane as the amino terminal residues. Monomeric PFL was immobilized at pH 10.5 in the presence of 0.5% TRITON X-100. The properties of both PFL derivatives were compared. In general, the bimolecular derivatives were more active, more selective and more stable both in water and in organic solvents than the monomolecular ones. The bimolecular derivative showed twice the activity and a much higher selectivity (100 versus 20) for the hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) in aqueous media at pH 5.0 compared to the monomeric derivative. In experiments measuring thermal inactivation at 75 °C, the bimolecular derivative was 5-fold more stable than the monomeric derivative (and 50-fold more stable than a one-point covalently immobilized PFL derivative), and it had a half-life greater than 4 h. In organic solvents (cyclohexane and tert-amyl alcohol), the bimolecular derivative was much more stable and more active than the monomeric derivative in catalyzing the transesterification of olive oil with benzyl alcohol. © 2012 Elsevier Ltd. All rights reserved.

Multi-choice mixed integer goal programming optimization for real problems in a sugar and ethanol milling company

Goal Programming (GP) is an important analytical approach devised to solve many realworld problems. The first GP model is known as Weighted Goal Programming (WGP). However, Multi-Choice Aspirations Level (MCAL) problems cannot be solved by current GP techniques. In this paper, we propose a Multi-Choice Mixed Integer Goal Programming model (MCMI-GP) for the aggregate production planning of a Brazilian sugar and ethanol milling company. The MC-MIGP model was based on traditional selection and process methods for the design of lots, representing the production system of sugar, alcohol, molasses and derivatives. The research covers decisions on the agricultural and cutting stages, sugarcane loading and transportation by suppliers and, especially, energy cogeneration decisions; that is, the choice of production process, including storage stages and distribution. The MCMIGP allows decision-makers to set multiple aspiration levels for their problems in which the more/higher, the better and the less/lower, the better in the aspiration levels are addressed. An application of the proposed model for real problems in a Brazilian sugar and ethanol mill was conducted; producing interesting results that are herein reported and commented upon. Also, it was made a comparison between MCMI GP and WGP models using these real cases. © 2013 Elsevier Inc.

Soil erodibility assessment in a pasture and forest remnant using the inderbitzen device

Brazil has the largest cattle herd in the world with approximately 200 million head. An important feature of the Brazilian cattle industry is that most of its herd is raised on pasture, which constitutes one of the most economical and practical ways to produce and provide food for cattle. However, this production model is mishandled and can lead to soil degradation. Maintaining soil quality is essential for the conservation of natural ecosystems and the areas of production, thus soil quality improves the conditions for biogeochemical cycles. In this context, the objective of this study was to develop a device for testing the Inderbitzen way of assessing soil erodibility in two situations of usage and occupation. Therefore, one area was used as a sample collection occupied by grazing and the other as a forest fragment; both located in the city of Sorocaba in Sao Paulo State, Brazil. Thus, we concluded that the proposed device - the Inderbitzen - proved capable of assessing soil erodibility of the pasture and remnant forest. Accordingly, there was a tendency for a smaller loss of forest soils in the remnant when compared to the degraded pasture. The greatest resistance of the soil erosion in the forest remnant may be associated with the amount of organic matter released by the forest litter in all its diversity, influencing the quality of the structure of aggregates. © 2013 WIT Press.

Cover crops and no-till effects on physical fractions of soil organic matter

Physical fractions (free light fraction, intra-aggregate light fraction and heavy fraction) of soil organic matter (SOM) are good indicators of soil quality for sustainable land use. The objective of this study was to evaluate the effect of cover crops on total organic carbon (TOC) and physical fractions of soil organic matter in soil under a no-tillage system (NTS) and a conventional tillage system (CTS, one plowing and two disking). A three-year field experiment was carried out as a cover crop-rice (Oryza sativa)-cover crop-rice rotation. Treatments included cover crops (Panicum maximum, Brachiaria ruziziensis, Brachiaria brizantha, and pearl millet (Pennisetum glaucum), fallow, till or no till. The SOM was physically fractionated in free light fraction (FLF), intra-aggregates light fraction (IALF) and heavy fraction (HF). The levels of C in whole soil were also evaluated, as well as C in the light fractions (FLF+IALF) and in the HF. Results indicated that concentrations of C in the FLF and IALF in surface soils (0-0.05m) were much higher (10.8 and 1.95gkg-1, respectively) than that in the 0.05-0.1m soil depth (7.68 and 1.54gkg-1, respectively) and in the 0.1-0.2m soil depth (4.98 and 1.24gkg-1, respectively). The NTS resulted in higher levels of FLF (12.2gkg-1) and IALF (2.19gkg-1) than with CTS (1.37-7.30gkg-1). Millet had the highest C (19.5gkg-1) and N (1.1gkg-1) concentrations in soil. There was an accumulation of TOC and total N in the surface soil with cover crops, and concentrations of TOC were higher in the HF (79.0%) than in the light fractions (21.0%). Although SOM changed little during the two years of this experiment, the various C fractions were significantly affected by the tillage treatments. We conclude that SOM physical fractionation allowed seeing significant differences caused by the soil management in the organic matter dynamics in a short period of time. © 2013 Elsevier B.V.

Aggregate shape and tensile strength measurement

Tensile strength (TS) of soil aggregates is an important indicator of soil quality. However, TS varies with aggregate shape. Thus, the objective of this study was to quantify the influence of aggregate shape on TS and propose a shape standardization protocol to increase accuracy in the measurement of TS. The latter was determined on 7,560 aggregates divided into three shapes, (i) irregular shape (IS), (ii) spherical shape (SS), and (iii) flat surface (FS), while preserving the inherent structure of the aggregate. The aggregates with IS had a larger range in the TS (306 kPa) because of the shape variability when compared with SS (238 kPa) and FS (129 kPa). The TS determined in aggregates with FS had smaller coefficient of variation (46%) in comparison with those of IS (70%) and SS (66%), indicating that the aggregate uniformity reduced the influence of shape on the TS. A smaller force (42.12 kPa) was needed to rupture aggregates with FS than IS (58.43 kPa) and SS (56.89 kPa) because of better force distribution in causing the tensile stress. The use of aggregates with the FS enables an accurate assessment of TS in relation to a wide range of management treatments. Copyright © 2013 by Lippincott Williams & Wilkins.

Flexible magnetic membranes based on bacterial cellulose and its evaluation as electromagnetic interference shielding material

Flexible magnetic membranes with high proportion of magnetite were successfully prepared by previous impregnation of the never dried bacterial cellulose pellicles with ferric chloride followed by reduction with sodium bisulfite and alkaline treatment for magnetite precipitation. Membranes were characterized by Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), vibrating magnetometer, field emission scanning electron microscopy (FEG-SEM) and impedance spectroscopy. Microwave properties of these membranes were investigated in the X-band (8.2 to 12.4 GHz). FEG-SEM micrographs show an effective coverage of the BC nanofibers by Fe 3O4 nanoparticles. Membranes with up to 75% in weight of particles have been prepared after 60 min of reaction. Magnetite nanoparticles in the form of aggregates well adhered to the BC fibers were observed by SEM. The average crystal sizes of the magnetic particles were in the range of 10 ± 1 to 13 ± 1 nm (estimated by XRD). The magnetic particles in the BC pellicles presented superparamagnetic behavior with a saturation magnetization in the range of 60 emu g- 1 and coercive force around 15 Oe. These magnetic pellicles also displayed high electrical permittivity and a potential application as microwave absorber materials. © 2013 Elsevier B.V.

Sodium dodecyl sulfate (SDS) effect on the thermal stability of oxy-HbGp: Dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) studies

Glossoscolex paulistus (HbGp) hemoglobin is an oligomeric protein, presenting a quaternary structure constituted by 144 globin and 36 non-globin chains (named linkers) with a total molecular mass of 3.6MDa. SDS effects on the oxy-HbGp thermal stability were studied, by DLS and SAXS, at pH 5.0, 7.0 and 9.0. DLS and SAXS data show that the SDS-oxy-HbGp interactions induce a significant decrease of the protein thermal stability, with the formation of larger aggregates, at pH 5.0. At pH 7.0, oxy-HbGp undergoes complete oligomeric dissociation, with increase of temperature, in the presence of SDS. Besides, oxy-HbGp 3.0mg/mL, pH 7.0, in the presence of SDS, has the oligomeric dissociation process reduced as compared to 0.5mg/mL of protein. At pH 9.0, oxy-HbGp starts to dissociate at 20°C, and the protein is totally dissociated at 50°C. The thermal dissociation kinetic data show that oxy-HbGp oligomeric dissociation at pH 7.0, in the presence of SDS, is strongly dependent on the protein concentration. At 0.5mg/mL of protein, the oligomeric dissociation is complete and fast at 40 and 42°C, with kinetic constants of (2.1±0.2)×10-4 and (5.5±0.4)×10-4s-1, respectively, at 0.6mmol/L SDS. However, at 3.0mg/mL, the oligomeric dissociation process starts at 46°C, and only partial dissociation, accompanied by aggregates formation is observed. Moreover, our data show, for the first time, that, for 3.0mg/mL of protein, the oligomeric dissociation, denaturation and aggregation phenomena occur simultaneously, in the presence of SDS. Our present results on the surfactant-HbGp interactions and the protein thermal unfolding process correspond to a step forward in the understanding of SDS effects. © 2013 Elsevier B.V.

Production of biofilm by Listeria monocytogenes in different materials and temperatures

Listeria monocytogenes, considered as one of the most important foodborne pathogens, is easily found on surfaces, particularly in the form of a biofilm. Biofilms are aggregates of cells that facilitate the persistence of these pathogens in food processing environments conferring resistance to the processes of cleaning and may cause contamination of food during processing, thus, representing a danger to public health. Little is known about the dynamics of the formation and regulation of biofilm production in L.monocytogenes, but several authors reported that the luxS gene may be a precursor in this process. In addition, the product of the inlA gene is responsible for facilitating the entry of the microorganism into epithelial cells that express the receptor E-cadherin, also participates in surface attachment. Thus, 32 strains of L.monocytogenes isolated from different foods (milk and vegetables) and from food processing environments were analyzed for the presence of these genes and their ability to form biofilms on three different surfaces often used in the food industry and retail (polystyrene, glass and stainless steel) at different temperatures (4, 20 and 30°C). All strains had the ilnA gene and 25 out of 32 strains (78.1%) were positive for the presence of the luxS gene, but all strains produced biofilm in at least one of the temperatures and materials tested. This suggests that genes in addition to luxS may participate in this process, but were not the decisive factors for biofilm formation. The bacteria adhered better to hydrophilic surfaces (stainless steel and glass) than to hydrophobic ones (polystyrene), since at 20°C for 24h, 30 (93.8%) and 26 (81.3%) produced biofilm in stainless steel and glass, respectively, and just 2 (6.2%) in polystyrene. The incubation time seemed to be an important factor in the process of biofilm formation, mainly at 35°C for 48h, because the results showed a decrease from 30 (93.8%) to 20 (62.5%) and from 27 (84.4%) to 12 (37.5%), on stainless steel and glass, respectively, although this was not significant (. p=0.3847). We conclude that L.monocytogenes is capable of forming biofilm on different surfaces independent of temperature, but the surface composition may be important factor for a faster development of biofilm. © 2013 Elsevier Ltd.

Fibrinogen-Induced Streptococcus mutans Biofilm Formation and Adherence to Endothelial Cells

Streptococcus mutans, the predominant bacterial species associated with dental caries, can enter the bloodstream and cause infective endocarditis. The aim of this study was to investigate S. mutans biofilm formation and adherence to endothelial cells induced by human fibrinogen. The putative mechanism by which biofilm formation is induced as well as the impact of fibrinogen on S. mutans resistance to penicillin was also evaluated. Bovine plasma dose dependently induced biofilm formation by S. mutans. Of the various plasma proteins tested, only fibrinogen promoted the formation of biofilm in a dose-dependent manner. Scanning electron microscopy observations revealed the presence of complex aggregates of bacterial cells firmly attached to the polystyrene support. S. mutans in biofilms induced by the presence of fibrinogen was markedly resistant to the bactericidal effect of penicillin. Fibrinogen also significantly increased the adherence of S. mutans to endothelial cells. Neither S. mutans cells nor culture supernatants converted fibrinogen into fibrin. However, fibrinogen is specifically bound to the cell surface of S. mutans and may act as a bridging molecule to mediate biofilm formation. In conclusion, our study identified a new mechanism promoting S. mutans biofilm formation and adherence to endothelial cells which may contribute to infective endocarditis.

Cetyltrimethylammonium chloride (CTAC) effect on the thermal stability of oxy-HbGp: Dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) studies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Combining Experimental Evidence and Molecular Dynamic Simulations To Understand the Mechanism of Action of the Antimicrobial Octapeptide Jelleine-I

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of polymers and ceramics in new and discarded electrical insulators: reuse and recycling possibilities

The expansion and maintenance of electricity distribution networks generates large amounts of waste, much of it in the form of discarded insulators that are not reused or recycled. This paper describes the results of tests on used and new ceramic and polymeric insulators to verify if their exposure to weathering justifies their replacement. In new and used ceramic insulators, properties such as contact angle, relative density, porosimetry, dilatometry and X-ray diffraction patterns showed no differences or the differences that were found could not be related to their use. The discarded ceramic material showed high thermal stability, an interesting characteristic for application as chamotte. It can also be reused to replace gravel used in substations. In polymeric insulators, thermogravimetry, differential scanning calorimetry and relative density test results suggest degradation of used material compared to new. This would justify their replacement and discard as waste, but they show little recycling potential.

Rhamnolipids Production by a Pseudomonas eruginosa LBI Mutant: Solutions and Homologs Characterization

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Soil erodibility assessment in a pasture and forest remnant using the Indrbitzen device

Brazil has the largest cattle herd in the world with approximately 200 million head. An important feature of the Brazilian cattle industry is that most of its herd is raised on pasture, which constitutes one of the most economical and practical ways to produce and provide food for cattle. However, this production model is mishandled and can lead to soil degradation. Maintaining soil quality is essential for the conservation of natural ecosystems and the areas of production, thus soil quality improves the conditions for biogeochemical cycles. In this context, the objective of this study was to develop a device for testing the Inderbitzen way of assessing soil erodibility in two situations of usage and occupation. Therefore, one area was used as a sample collection occupied by grazing and the other as a forest fragment; both located in the city of Sorocaba in Sao Paulo State, Brazil. Thus, we concluded that the proposed device – the Inderbitzen – proved capable of assessing soil erodibility of the pasture and remnant forest. Accordingly, there was a tendency for a smaller loss of forest soils in the remnant when compared to the degraded pasture. The greatest resistance of the soil erosion in the forest remnant may be associated with the amount of organic matter released by the forest litter in all its diversity, influencing the quality of the structure of aggregates. Keywords: erosion, forest remnant, degraded pasture, Inderbitzen test.