A phenotypic screening bioassay for Escherichia coli stress and antibiotic responses based on Fourier-transform infrared (FTIR) spectroscopy and multivariate analysis.

AIMS: To develop and optimize a Fourier-transform infrared spectroscopy (FTIRS) phenotypic screening bioassay for stress responses, regarding the effect of nutrient content, bacterial growth phase and stress agent exposure time. METHODS AND RESULTS: A high-throughput FTIRS bioassay was developed to distinguish the stress responses of Escherichia coli to sodium hydroxide, hydrochloric acid, sodium chloride, sodium hypochlorite and ethanol. Principal component analysis and hierarchical clustering were used to quantify the effect of each parameter on bioassay performance, namely its reproducibility and metabolic resolution. Bioassay performance varied greatly, ranging from poor to very good. Spectra were partitioned into biologically relevant regions to evaluate their contributions to bioassay performance, but further improvements were not observed. Bioassay optimization was validated against empirical parameters, which confirmed a closer representation of known mechanisms on the antibiotic-induced stress responses. CONCLUSIONS: The optimized bioassay used standard nutrient content, cells in the late-stationary growth phase and a one-shift exposure duration. Only the optimized bioassay adequately and reproducibly distinguished the E. coli stress and antibiotic responses. The absence of performance improvements using partitioned spectra indicated that stress responses are imprinted on the whole-spectra metabolic signature. SIGNIFICANCE AND IMPACT OF THE STUDY: Highly optimized FTIRS bioassay parameters are vital in capturing whole-spectra metabolic signatures that can be used for satisfactory and reproducible phenotypic screening of stress and antibiotic responses.

A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors.

Nowadays, the dairy industry is continuously looking for new and more efficient clotting enzymes to create innovative products. Cyprosin B is a plant aspartic protease characterized by clotting activity that was previously cloned in Saccharomyces cerevisiae BJ1991 strain. The production of recombinant cyprosin B by a batch and fed-batch culture was compared using glucose and galactose as carbon sources. The strategy for fed-batch cultivation involved two steps: in the first batch phase, the culture medium presented glucose 1 % (w/v) and galactose 0.5 % (w/v), while in the feed step the culture medium was constituted by 5 % (w/v) galactose with the aim to minimize the GAL7 promoter repression. Based on fed-batch, in comparison to batch growth, an increase in biomass (6.6-fold), protein concentration (59 %) and cyprosin B activity (91 %) was achieved. The recombinant cyprosin B was purified by a single hydrophobic chromatography, presenting a specific activity of 6 × 10(4) U·mg(-1), corresponding to a purification degree of 12.5-fold and a recovery yield of 16.4 %. The SDS-PAGE analysis showed that recovery procedure is suitable for achieving the purified recombinant cyprosin B. The results show that the recombinant cyprosin B production can be improved based on two distinct steps during the fed-batch, presenting that this strategy, associated with a simplified purification procedure, could be applied to large-scale production, constituting a new and efficient alternative for animal and fungal enzymes widely used in cheese making.

Spintronic platforms for biomedical applications.

Since the fundamental discovery of the giant magnetoresistance many spintronic devices have been developed and implemented in our daily life (e.g. information storage and automotive industry). Lately, advances in the sensors technology (higher sensitivity, smaller size) have potentiated other applications, namely in the biological area, leading to the emergence of novel biomedical platforms. In particular the investigation of spintronics and its application to the development of magnetoresistive (MR) biomolecular and biomedical platforms are giving rise to a new class of biomedical diagnostic devices, suitable for bench top bioassays as well as point-of-care and point-of-use devices. Herein, integrated spintronic biochip platforms for diagnostic and cytometric applications, hybrid systems incorporating magnetoresistive sensors applied to neuroelectronic studies and biomedical imaging, namely magneto-encephalography and magneto-cardiography, are reviewed. Also lab-on-a-chip MR-based platforms to perform biological studies at the single molecule level are discussed. Overall the potential and main characteristics of such MR-based biomedical devices, comparing to the existing technologies while giving particular examples of targeted applications, are addressed.

Use of chemometrics in the selection of a Saccharomyces cerevisiae expression system for recombinant cyprosin B production.

Two multivariate statistical methods, factor analysis (FA) and hierarchical cluster analysis (HCA), were applied to experimental data set to evaluate their usefulness in selecting the adequate expression system and optimal growth parameters for recombinant cyprosin B production. Using FA, the large data set was reduced to two factors representing 73.4% of variability. Factor 1, with 53.5% of variability, corresponds to recombinant cyprosin B expression and efficient secretion, while factor 2, accounting for 19.9% of variability, represents cell growth and physiological characteristics. FA and HCA allowed the establishment of correlations among different variables and the clusters obtained providing clear identification of the experimental parameters related to cyprosin B production, which results on more accurate scientific output and time saving when selection of an adequate expression system is concerned.

Picomolar detection limit on a magnetoresistive biochip after optimization of a thiol-gold based surface chemistry.

The surface biochemistry plays a crucial role in the development of stable and reproducible bioanalytical devices. Very often, it represents the bottleneck of a successful integration of magnetoelectronic transducers with the biological receptors on its interface. Here is discussed how a thiolgold surface chemistry can be tailored and optimized in order to allow the biofunctionalization of a magnetoresistive biochip, preventing loss of viability by corrosion while improving its sensitivity. Two important parameters, type of buffer solution and salt concentration (globally ionic strength), were evaluated in the effectiveness of the sulfur-gold linkage and further influence on the biomolecular recognition between single stranded DNA molecules. A third, not less important variable under investigation was the blocking solution. Non-specific adsorption of magnetic labels to the sensing surface still is a major problem to be addressed. The effect of two well known blocking molecules (bovine serum albumin (BSA)) and thiolated polyethylene-glycol (SH-PEG)) on the prevention of non-specific adsorption of targets and labels are compared. The best conditions were selected using an optical microscopic characterization method. Optical images were analyzed for magnetic particles quantification and results presented as a percentage of surface coverage. The optimized protocol was further implemented on real magnetoresistive devices to assess its electric compatibility and bioassay performance. A good reproducibility (about 9% error) among different devices measuring the same target concentration was achieved. Also a reduced non-specific binding signal of 43 microV for non-complementary targets (30% complementarity) compares with a 500 microV for fully complementarity. A linear range on the biological detection of magnetically labeled target ssDNA oligonucleotides is demonstrated. Consequently, the limit of detection at the standard operational conditions of the device is situated at the picomolar range.

Quantitation of non-amplified genomic DNA by bead-based hybridization and template mediated extension coupled to alkaline phosphatase signal amplification.

Klenow I polymerase activity was combined with solid phase DNA hybridization to detect non-amplified genomic DNA (gDNA) sequences from Escherichia coli. Aminopropyl-controlled pore glass surface-bound oligonucleotides were hybridized to fragmented gDNA. The template-mediated extension at the 3'-terminus of the immobilized probe was then promoted in the presence of Klenow I polymerase and digoxigenin-labeled nucleotides. Detection of the extended probes was accomplished with an anti-digoxigenin alkaline phosphatase conjugate protocol coupled to colorimetric or fluorescent detection. Using the colorimetric protocol, the proof-of-concept was established. The fluorescence-based methodology, on the other hand, provided the basis for a quantitative interpretation of the data, affording a detection limit of 5 pM gDNA.

Application of central composite design for DNA hybridization onto magnetic microparticles.

Central composite face-centered (CCF) design and response surface methodologies were used to investigate the effect of probe and target concentration and particle number in immobilization and hybridization on a microparticle-based DNA/DNA hybridization assay. The factors under study were combined according to the CCF design matrix, and the intensity of the hybridization signal was quantified by flow cytometry. A second-order polynomial was fitted to data and validated by analysis of variance. The results showed a complex relationship between variables and response given that all factors as well as some interactions were significant, yet it could explain 95% of the data. Probe and target concentration had the strongest impact on hybridization signal intensity. Increments in initial probe concentration in solution positively affected the hybridization signal until a negative influence of a compact probe layer emerged. This trend was attributed to probe-probe interactions. By manipulating particle number on both immobilization and hybridization, enhancements on the assay sensitivity could be obtained. Under optimized conditions, the limit of detection (LOD) at the 95% confidence level was determined to be 2.3 nM of target solution concentration.

The role of probe-probe interactions on the hybridization of double-stranded DNA targets onto DNA-modified magnetic microparticles.

In this work, we have studied the effect of different probe lengths and surface densities on the hybridization of a 181-bp polymerase chain reaction product to probes tethered onto magnetic microparticles. Hybridization was shown to be favored by longer probes but only at probe surface densities where probe-to-probe interactions are absent. From these results, a simple rule was inferred for determining maximum surface densities above which hybridization signals decreased. According to this rule, if the average surface area occupied by an immobilized probe (Sigma) is larger than the projected surface area of each tethered probe molecule (S(ss)), hybridization efficiency increases with surface density, whereas the reverse occurs when Sigma-S(ss) < 0.

Femtomolar limit of detection with a magnetoresistive biochip.

In this paper the biological limit of detection of a spin-valve-based magnetoresistive biochip applied to the detection of 20 mer ssDNA hybridization events is presented. Two reactional variables and their impact on the biomolecular recognition efficiency are discussed. Both the influence of a 250 nm diameter magnetic particle attached to the target molecule during the hybridization event and the effect of a magnetic focusing system in the hybridization of pre-labeled target DNA (assisted hybridization) are addressed. The particles carrying the target molecules are attracted to the probe active sensor sites by applying a 40 mA DC current on U-shaped aluminium current lines. Experiments comparing pre-hybridization versus post-hybridization magnetic labeling and passive versus magnetically assisted hybridization were conducted. The efficiency of a passive hybridization is reduced by about 50% when constrained to the operational conditions (sample volume, reaction time, temperature and magnetic label) of an on-chip real-time hybridization assay. This reduction has shown to be constant and independent from the initial target concentration. Conversely, the presence of the magnetic label improved the limit of detection when a magnetically assisted hybridization was performed. The use of a labeled target focusing system has permitted a gain of three orders of magnitude (from 1 pM down to 1 fM) in the sensitivity of the device, as compared with passive, diffusion-controlled hybridization.

Strongyloides stercoralis hyperinfection syndrome after liver transplantation: case report and literature review.

Strongyloides stercoralis is an intestinal nematode that causes human infections and whose life cycle has special features, including autoinfection. Strongyloides infection may be asymptomatic for years, owing to a low parasite load. During immunosuppressive therapy, however, if cellular immunity is depressed, autoinfection can occur at a higher rate, resulting in hyperinfection syndrome. In this specific circumstance, it can become a fatal illness. We describe a case of hyperinfection syndrome in a liver transplant recipient and also review the literature.

Chemiluminescent bead-based hybridization assay for the detection of genomic DNA from E. coli in purified plasmid samples.

A bead-based hybridization assay was developed for detection of traces of E. coli genomic DNA (gDNA) present in purified plasmid DNA (pDNA) samples. Standards of gDNA and pDNA samples were sheared by sonication and adsorbed onto aminopropyl controlled pore glass (CPG) particles (130 microm). A preliminary study was conducted to optimize the amount of DNA adsorbed on the particles. Results indicated that maximum attachment efficiency was obtained by adsorbing DNA for 2 h in 0.2 x SSC, pH 5.7. The DNA-bound particles were hybridized overnight with a 181-bp digoxigenin-labeled probe, specific for gDNA. Following a chemiluminescent detection protocol, signal intensities of the standards were plotted as a function of initial gDNA concentration. The calculated detection limit (LOD) was 1.4 pM of gDNA. The assay was able to detect gDNA in pure plasmid preparations at the 1% level even in the presence of 1,000-fold excess of noncomplementary target. Hybridization results were compared with a quantitative real-time PCR assay. Both methods afforded similar accurate results at the 95% confidence level.

Influence of tryptophan tags on the purification of cutinase, secreted by a recombinant Saccharomyces cerevisiae, using cationic expanded bed adsorption and hydrophobic interaction chromatography.

During cationic bed adsorption (EBA), with cutinase with varying length tryptophan tags (WP)(2)and (WP)(4), 33% and 10% of adsorption capacity and 80% and 32% eluted specific activity were observed in relation to wild type (wt)-cutinase in the conventional process. Therefore, as the hydrophobicity of the protein increases, it is important to integrate the EBA step with a hydrophobic interaction chromatography (HIC) process. As the length of the hydrophobic tag-(WP) increases from n = 2 to n = 4, the purification factor obtained by HIC was 1.8 and 2.2-fold higher than wt-cutinase. However, the recovery yield obtained in HIC decreases substantially as the length of hydrophobic tag increases (97%, 84% and 70% for wt-cutinase, cutinase-(WP)(2) and cutinase-(WP)(4)). The integration of two purification steps, EBA followed by HIC, resulted in the highest overall purity level for cutinase-(WP)(2), and the highest overall recovery yield for wt-cutinase. When optimizing the design of a hydrophobic tag fused to a protein secreted by Saccharomyces cerevisiae it must be considered that the cultivation parameters could impair the downstream process, and consequently the optimum tag is not necessarily the one that presents the highest purification factor in HIC.

Prediction of retention time of cutinases tagged with hydrophobic peptides in hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) is an important technique for protein purification, which exploits the separation of proteins based on hydrophobic interactions between the stationary phase ligands and hydrophobic regions on the protein surface. One way of enhancing the purification efficiency by HIC is the addition of short sequences of peptide tags to the target protein by genetic engineering, which could reduce the need for extra and expensive chromatographic steps. In the present work, a methodology for predicting retention times of cutinases tagged with hydrophobic peptides in HIC is presented. Cutinase from Fusarium solani pisi fused to tryptophan-proline (WP) tags, namely (WP)2 and (WP)4, and produced in Saccharomyces cerevisiae strains, were used as model proteins. From the simulations, the methodology based on tagged hydrophobic definition proposed by Simeonidis et al. (Phitagged), associated to a quadratic model for predicting dimensionless retention times, showed small differences (RMSE<0.022) between observed and estimated retention times. The difference between observed and calculated retention times being lower than 2.0% (RMSE<0.022) for the two tagged cutinases at three different stationary phases, except for the case of cut_(wp)2 in octyl sepharose-2 M ammonium sulphate. Therefore, we consider that the proposed strategy, based on tagged surface hydrophobicity, allows prediction of acceptable retention times of cutinases tagged with hydrophobic peptides in HIC.

Kinetic modelling of phenol co-oxidation using horseradish peroxidase.

Phenol is an industrial pollutant and its removal from industrial wastewaters is of great importance. In order to design optimised phenol removal procedures by using horseradish peroxidase-based systems, there are some points that have to be dealt with. One of the most important issues is the need for reliable kinetics as this is one of the difficulties found during process scale-up. Although simplified kinetics can be used for limited ranges of operating conditions, they are not usually reliable for the description of varying process conditions. The present work describes the implementation of a kinetic model, based on a mechanism, for the co-oxidation of phenol and 4-aminoantipyrine (Am-NH2), which is used as a chromogen agent, with hydrogen peroxide as the oxidant. The model covers not only the variation of the concentrations of all the species involved, but also the effect of temperature in the reaction. The estimation of kinetic rate constants and activation energies for the various steps in the mechanism is performed with a single optimisation procedure, and all the experimental results are described using a unique set of parameters, which, thus, is valid over an extended range of operating conditions. The mechanism allowed the determination of a reliable kinetic model which is appropriate for the range of experimental conditions used. The computational model was also tested with an independent set of experiments with different conditions from the ones for which the parameters were estimated.

Micro-analytical GO/HRP bioreactor for glucose determination and bioprocess monitoring.

A bi-enzymatic micro-analytical bioreactor integrated in a FIA system for glucose measurements is described. Its robustness and small dimensions (working volume of about 70 microl containing approximately 1.2 mg GO and 0.26 mg HRP) make it easy to operate. The column is based on immobilisation of glucose oxidase (GO) and horseradish peroxidase (HRP) on alkylamine controlled pore glass (CPG) beads. The column has excellent shelf life (no significant loss of activity after 1 year if kept at 4 degrees C), and a very high operational stability that was demonstrated through extensive usage for glucose determinations over 1 year period during which the column retained almost all of its activity. More importantly, this operational stability allows glucose monitoring in the culture media without a decay of signal over the experiment time and consequently no signal correction or re-calibration is needed. This high operational stability was also confirmed by continuous glucose conversion with 30% activity loss after converting quantity of glucose equivalent to 21600 FIA injections of 20 microl with 1.7 mM glucose. Such good performance is a result of an optimised immobilisation method and moreover of the implementation of in situ enzyme stabilisation strategy which consisted on promoting the instantaneous H2O2 consumption produced by the GO. This strategy has the additional advantage of allowing concomitant assay of the H2O2 based on the HAP catalysed co-oxidation of phenol-4-sulphonic acid (PSA) in the presence of 4-aminoantipyrine (4-AAP). The glucose measurements are reproducible with high precision against the standard HPLC method. Linear range and sensitivity depend on sample injection volume; the upper limit is about 1.1 g/l. Lower detection limit is 10mg/l. The column performance has been validated for E. coli and S. cerevisiae fermentation monitoring, and glucose measurements in an animal cell culture (rat Langerhans islets).

Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects.

Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell-required for the induction of cutinase production-and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.

Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi cutinase fusion proteins.

Genetic engineering was integrated with the production and purification of Fusarium solani pisi cutinases, in order to obtain the highest amount of enzyme activity units, after purification. An aqueous two-phase system (ATPS) of polyethylene glycol 3350, dipotassium phosphate and whole broth was used for the extraction of three extracellular cutinases expressed in Saccharomyces cerevisiae. The production/extraction process was evaluated regarding cutinases secretion in the medium, partition behaviour and extraction yields in the ATPS. The proteins studied were cutinase wild type and two fusion proteins of cutinase with the tryptophane-proline (WP) fusion tags, namely (WP)(2) and (WP)(4). The (WP)(4) fusion protein enabled a 300-fold increase of the cutinase partition coefficient when comparing to the wild type. However, the secretion of the fusion proteins was lower than of the wild type cutinase secretion. A batch extraction strategy was compared with a continuous extraction in a perforated rotating disc contactor (PRDC). The batch and continuous systems were loaded with as much as 60% (w/w) whole cultivation broth. The continuous extraction strategy provided a 2.5 higher separation capacity than the batch extraction strategy. Considering the integrated process, the cutinase-(WP)(2) proved to lead to the highest product activity, enabling five and six times more product activity than the wild type and the (WP)(4) fusion proteins, respectively.

Schistosomiasis mansoni is associated with pyogenic liver abscesses in the state of Minas Gerais, Brazil.

The association between pyogenic liver abscesses and schistosomiasis has been confirmed by clinical and experimental studies. In this retrospective study of 78 patients with pyogenic liver abscesses the association with schistosomiasis has been investigated. Pyodermitis, a known focus of bacteremia, was observed in 19 patients (24%). Blood eosinophilia was observed in 30 patients (39%). Staphylococcus aureus was cultured from abscesses in 17 out of 38 patients (45%). Forty-one out of 57 patients (53%) had stool examination. Schistosoma mansoni was the main parasite identified. Eggs of S. mansoni were also identified in liver biopsies in 7 out of 19 patients who did the exam. The large number of young patients with liver abscesses described here is different from what has been observed in developed countries. This clinical study provide support for the concept that granulomas of S. mansoni in the liver are foci for colonization with S. aureus, which in presence of staphylococcal bacteremia can form liver abscesses.

Downstream DNA sequences are required to modulate Pvlea-18 gene expression in response to dehydration.

We have previously shown that mRNA and protein encoded by the Pvlea-18 gene from Phaseolus vulgaris L., a member of a new family of late embryogenesis-abundant (LEA) proteins, accumulate in dark-grown bean seedlings not only in response to water deficit but also during optimal irrigation. In this work, we studied Pvlea-18 gene transcriptional regulation by using transgenic Arabidopsis thaliana plants containing a chimeric gene consisting of the Pvlea-18 promoter region and the 3'-nos terminator fused to the GUS gene-coding region. We demonstrate that the chimeric gene is active during Arabidopsis normal development under well-irrigated conditions, and that it is further induced in response to ABA and dehydration treatments. Replacing the 3'-nos terminator with the Pvlea-18 3' region led to an additional increase in expression during development and in response to dehydration, but not in response to exogenous ABA. These results reveal an enhancer effect of the Pvlea-18 3' region, which showed to be higher specifically under dehydration. The small decrease in Pvlea-18 promoter expression observed when transgenic plants treated with fluridone (an ABA biosynthesis inhibitor) were subjected to dehydration suggests that the Pvlea-18 gene dehydration response is predominantly ABA-independent. Finally, we present evidence indicating that Pvlea-18 gene expression is negatively regulated during etiolated growth, particularly in roots, in contrast to the expression pattern observed during normal development.

Direct product sequestration of a recombinant cutinase from batch fermentations of Saccharomyces cerevisiae.

The recovery of cutinase of Fusarium solani pisi produced by the yeast Saccharomyces cerevisiae was studied in a fluidised bed adsorption system directly integrated with a productive fermenter (so-called direct product sequestration; DPS). The relative efficiency of this system was compared with the one of a conventional purification process by discrete sequences of fermentation, broth clarification, ultrafiltration and fixed bed anion exchange chromatography. By direct product sequestration of the extracellular heterologous cutinase it was possible, through only one unit operation: (i) to perform broth clarification, (ii) to obtain a high cutinase concentration factor, and (iii) to recover cutinase with a specific activity that equalled that obtained with the conventional purification process. It was also possible (iv) to substantially reduce the total process time, (v) to improve the overall yield, and (vi) to increase cutinase productivity. Furthermore, the procedure outlined is suitable for large scale bioprocess exploitation.