Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

Modified logit life table system: Principles, empirical validation, and application

Despite its widespread use, the Coale-Demeny model life table system does not capture the extensive variation in age-specific mortality patterns observed in contemporary populations, particularly those of the countries of Eastern Europe and populations affected by HIV/AIDS. Although relational mortality models, such as the Brass logit system, can identify these variations, these models show systematic bias in their predictive ability as mortality levels depart from the standard. We propose a modification of the two-parameter Brass relational model. The modified model incorporates two additional age-specific correction factors (gamma(x), and theta(x)) based on mortality levels among children and adults, relative to the standard. Tests of predictive validity show deviations in age-specific mortality rates predicted by the proposed system to be 30-50 per cent lower than those predicted by the Coale-Demeny system and 15-40 per cent lower than those predicted using the original Brass system. The modified logit system is a two-parameter system, parameterized using values of l(5) and l(60).

Optimizing electrode positioning in 3-lead ECG chest devices

Although the 12-lead electrocardiogram has become an essential medical and research tool, many current and envisaged applications would benefit from simpler devices, using 3-lead ECG configuration. This is particularly true for Ambient Assisted Living (in a broad perspective). However, the chest anatomy of female patients, namely during pregnancy, can hamper the adequate placement of a 3-lead ECG device and, very often, electrodes are placed below the chest rather than at the precise thoracic landmarks. Thus, the aim of this study was to compare the effect of electrode positioning on the ECG signal of pregnant women and provide guidelines for device development. The effect of breast tissue on the ECG signal was investigated by relating breast size with the signal-to-noise ratio, root mean square and R-wave amplitude. Results show that the 3-lead ECG should be placed on the breast rather than under the breast and indicate positive correlation between breast size and signal-to-noise ratio.

Neural Electrode Array Based on Aluminum: Fabrication and Characterization

A unique neural electrode design is proposed with 3 mm long shafts made from an aluminum-based substrate. The electrode is composed by 100 individualized shafts in a 10 × 10 matrix, in which each aluminum shafts are precisely machined via dicing-saw cutting programs. The result is a bulk structure of aluminum with 65 ° angle sharp tips. Each electrode tip is covered by an iridium oxide thin film layer (ionic transducer) via pulsed sputtering, that provides a stable and a reversible behavior for recording/stimulation purposes, a 40 mC/cm2 charge capacity and a 145 Ω impedance in a wide frequency range of interest (10 Hz-100 kHz). Because of the non-biocompatibility issue that characterizes aluminum, an anodization process is performed that forms an aluminum oxide layer around the aluminum substrate. The result is a passivation layer fully biocompatible that furthermore, enhances the mechanical properties by increasing the robustness of the electrode. For a successful electrode insertion, a 1.1 N load is required. The resultant electrode is a feasible alternative to silicon-based electrode solutions, avoiding the complexity of its fabrication methods and limitations, and increasing the electrode performance.

Bioactive albumin functionalized polylactic acid membranes for improved biocompatibility

Biocompatibility is a major challenge for successful application of many biomaterials. In this study the ability to coat chemically and enzymatically activated poly(L-lactic acid) (PLA) membranes with heat denatured human serum albumin to improve biocompatibility was investigated. PLA membranes hydrolyzed with NaOH or cutinase and then treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDAC) as a heterobifunctional cross-linker promoted the coupling single bondCOOH groups on PLA membranes and single bondNH2 groups of heat denatured human serum albumin. This resulted in increased hydrophilicity (lowest water contact angles of 43° and 35°) and highest antioxidant activity (quenching of 79 μM and 115 μM tetramethylazobisquinone (TMAMQ) for NaOH and cutinase pretreated membranes, respectively). FTIR analysis of modified PLA membranes showed new peaks attributed to human serum albumin (amide bond, NH2 and side chain stretching) appearing within 3600–3000 cm−1 and 1700–1500 cm−1 (Fig. 3). MTT studies also showed that osteoblasts-like and MC-3T3-E1 cells viability increased 2.4 times as compared to untreated PLA membranes. The study therefore shows that this strategy of modifying the surfaces of PLA polymers could significantly improve biocompatibility.

Variation of magnetic properties in sedimentary rocks hosting the Foum Zguid dyke (southern Morocco): Combined effects of re-crystallization and Fe-metasomatism

The effects of dyke intrusion on the magnetic properties of host sedimentary rocks are still poorly understood. Therefore, we have evaluated bulk magnetic parameters of standard palaeomagnetic samples collected along several sections across the sediments hosting the Foum Zguid dyke in southern Morocco. The study has been completed with the evaluation of the magnetic fabric after laboratory application of sequential heating experiments. The present study shows that: (1) close to Fourn Zguid dykes, the variations of the bulk magnetic parameters and of the magnetic fabric is strongly related with re-crystallization and Fe-metasomatism intensity. (2) The thermal experiments on AMS of samples collected farther from the dyke and, thus, less affected by heating during dyke emplacement, indicate that 300-400 degrees C is the minimum experimental temperature necessary to trigger appreciable transformations of the pre-existing magnetic fabrics. For temperatures higher than ca. 580 degrees C, the magnetic fabric transformations are fully realized, with complete transposition of the initial fabric to a fabric similar to that of samples collected close to the dyke. Therefore, measured variations of the magnetic fabric can be used to evaluate re-crystallization temperatures experienced by the host sedimentary rock during dyke emplacement. The distinct magnetic behaviour observed along the cross-sections strongly suggests that samples collected farther from the dyke margins did not experience thermal episodes with temperatures higher than 300 degrees C after dyke emplacement. (3) AMS data shows a gradual variation of the magnetic fabric with distance from the dyke margin, from sub-horizontal K-3 away from the dyke to vertical K3 close to the dyke. Experimental heating shows that heat alone can be responsible for this strong variation. Therefore, such orientation changes should not be unequivocally interpreted as the result of a stress field (resulting from the emplacement of the dyke, for instance). (4) Magnetic studies prove to be a very sensitive tool to assess rock magnetic transformations, thermally and chemically induced by dyke intrusion in hosting sediments.

Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.

Numerical experiments with a modified regularization scheme for mathematical programs with complementarity constraints

On this paper we present a modified regularization scheme for Mathematical Programs with Complementarity Constraints. In the regularized formulations the complementarity condition is replaced by a constraint involving a positive parameter that can be decreased to zero. In our approach both the complementarity condition and the nonnegativity constraints are relaxed. An iterative algorithm is implemented in MATLAB language and a set of AMPL problems from MacMPEC database were tested.

The development and optimization of a modified single-drop microextraction method for organochlorine pesticides determination by gas chromatography-tandem mass spectrometry

We have developed a new method for single-drop microextraction (SDME) for the preconcentration of organochlorine pesticides (OCP) from complex matrices. It is based on the use of a silicone ring at the tip of the syringe. A 5 μL drop of n-hexane is applied to an aqueous extract containing the OCP and found to be adequate to preconcentrate the OCPs prior to analysis by GC in combination with tandem mass spectrometry. Fourteen OCP were determined using this technique in combination with programmable temperature vaporization. It is shown to have many advantages over traditional split/splitless injection. The effects of kind of organic solvent, exposure time, agitation and organic drop volume were optimized. Relative recoveries range from 59 to 117 %, with repeatabilities of <15 % (coefficient of variation) were achieved. The limits of detection range from 0.002 to 0.150 μg kg−1. The method was applied to the preconcentration of OCPs in fresh strawberry, strawberry jam, and soil.

Development of polyaniline microarray electrodes for cadmium analysis

Disposable screen-printed electrodes (SPCE) were modified using a cosmetic product to partially block the electrode surface in order to obtain a microelectrode array. The microarrays formed were electropolymerized with aniline. Scanning electron microscopy was used to evaluate the modified and polymerized electrode surface. Electrochemical characteristics of the constructed sensor for cadmium analysis were evaluated by cyclic and square-wave voltammetry. Optimized stripping procedure in which the preconcentration of cadmium was achieved by depositing at –1.20 V (vs. Ag/AgCl) resulted in a well defined anodic peak at approximately –0.7 V at pH 4.6. The achieved limit of detection was 4 × 10−9 mol dm−3. Spray modified and polymerized microarray electrodes were successfully applied to quantify cadmium in fish sample digests.

Desenvolvimento de um nanosensor para monitorizar a ciprofloxacina em águas superficiais

No presente trabalho pretendeu-se estudar o comportamento da ciprofloxacina por técnicas voltamétricas e desenvolver novos sensores para monitorizar a ciprofloxacina em águas residuais. A investigação realizada contemplou essencialmente, os seguintes aspectos: estudo da influência do pH no comportamento voltamétrico da ciprofloxacina e comparação entre o eléctrodo de carbono vítreo e alguns eléctrodos modificados. O estudo foi efectuado em voltametria cíclica a diferentes velocidades de varrimento e também em voltametria de impulso diferencial. O estudo mostrou que o eléctrodo modificado com nanotubos de carbono permitiu a quantificação de níveis mais baixos de ciprofloxacina. O novo sensor desenvolvido foi utilizado em águas do rio Douro e rio Leça com o objectivo de monitorizar a concentração de ciprofloxacina. Traçaram-se curvas de calibração directa e por adição padrão de quantidades crescentes de ciprofloxacina. Os estudos efectuados com as águas do rio Douro e rio Leça foram recolhidos próximos da foz do rio estas amostras deveriam ser recolhidas em vários pontos do rio para se poder fazer uma comparação de resultados. Os estudos de recuperação permitiram verificar que a percentagem de recuperação para o rio Douro se situava nos 90% e as do rio Leça nos 75%, pelo método da calibração directa. Usando o método da adição padrão a recuperações foram de 99% para o rio Douro e 90% para o rio Leça. Os estudos em curso permitem concluir que este sensor poderá ser aplicado na monitorização da ciprofloxacina em amostras ambientais.

Square-wave voltametric method for determination of molinate concentration in a biological process using a hanging mercury drop electrode

A square-wave voltammetric (SWV) method using a hanging mercury drop electrode (HMDE) has been developed for determination of the herbicide molinate in a biodegradation process. The method is based on controlled adsorptive accumulation of molinate for 10 s at a potential of -0.8 V versus AgCl/Ag. An anodic peak, due to oxidation of the adsorbed pesticide, was observed in the cyclic voltammogram at ca. -0.320 V versus AgCl/Ag; a very small cathodic peak was also detected. The SWV calibration plot was established to be linear in the range 5.0x10-6 to 9.0x10-6 mol L-1; this corresponded to a detection limit of 3.5x10-8 mol L-1. This electroanalytical method was used to monitor the decrease of molinate concentration in river waters along a biodegradation process using a bacterial mixed culture. The results achieved with this voltammetric method were compared with those obtained by use of a chromatographic method (HPLC–UV) and no significant statistical differences were observed.

Multiresidue pesticides analysis in soils using modified QuEChERS with disposable pipette extraction and dispersive solid-phase extraction

QuEChERS original method was modified into a new version for pesticides determination in soils. The QuEChERS method is based on liquid–liquid portioning with ACN and was followed by cleanup step using dispersive SPE and disposable pipette tips. Gas chromatographic separation with MS detection was carried out for pesticides quantification. The method was validated using recovery experiments for 36 multiclass pesticides. Mean recoveries of pesticides at each of the four spiking levels between 10–300 µg/kg of soil ranged from 70–120% for 26 pesticides with RSD values less than 15%. The method achieved low limit of detection less than 7.6 µ g/kg. Matrix effects were observed for 13 pesticides. Matrix effects were compensated by using matrix-matched calibration. The method was applied successfully using d-SPE or DPX in the analysis of the pesticides in soils from organic farming and integrated pest management.

An electrochemical deamidated gliadin antibody immunosensor for celiac disease clinical diagnosis

The first electrochemical immunosensor (EI) for the detection of antibodies against deamidated gliadin peptides (DGP) is described here. A disposable nanohybrid screen-printed carbon electrode modified with DGP was employed as the transducer's sensing surface. Real serumsampleswere successfully assayed and the results were corroborated with an ELISA kit. The presented EI is a promising analytical tool for celiac disease diagnosis.

Application-specific modified particle swarm optimization for energy resource scheduling considering vehicle-to-grid

This paper presents a modified Particle Swarm Optimization (PSO) methodology to solve the problem of energy resources management with high penetration of distributed generation and Electric Vehicles (EVs) with gridable capability (V2G). The objective of the day-ahead scheduling problem in this work is to minimize operation costs, namely energy costs, regarding he management of these resources in the smart grid context. The modifications applied to the PSO aimed to improve its adequacy to solve the mentioned problem. The proposed Application Specific Modified Particle Swarm Optimization (ASMPSO) includes an intelligent mechanism to adjust velocity limits during the search process, as well as self-parameterization of PSO parameters making it more user-independent. It presents better robustness and convergence characteristics compared with the tested PSO variants as well as better constraint handling. This enables its use for addressing real world large-scale problems in much shorter times than the deterministic methods, providing system operators with adequate decision support and achieving efficient resource scheduling, even when a significant number of alternative scenarios should be considered. The paper includes two realistic case studies with different penetration of gridable vehicles (1000 and 2000). The proposed methodology is about 2600 times faster than Mixed-Integer Non-Linear Programming (MINLP) reference technique, reducing the time required from 25 h to 36 s for the scenario with 2000 vehicles, with about one percent of difference in the objective function cost value.