Loading...
20 results
Search Results
Now showing 1 - 10 of 20
- Characterization of gastric cells infection by diverse Helicobacter pylori strains through Fourier-transform infrared spectroscopyPublication . Marques, Vanda; Ribeiro Da Cunha, Bernardo; Couto, Andreia; Sampaio, Pedro; Fonseca, Luís P. P.; Aleixo, Sandra; Calado, CecíliaThe infection of Helicobacter pylori, covering 50% of the world-population, leads to diverse gastric diseases as ulcers and cancer along the life-time of the human host. To promote the discovery of biomarkers of bacterial infection, in the present work, Fourier-transform infrared spectra were acquired from adenocarcinoma gastric cells, incubated with H. pylori strains presenting different genotypes concerning the virulent factors cytotoxin associated gene A and vacuolating cytotoxin A. Defined absorbance ratios were evaluated by diverse methods of statistical inference, according to the fulfillment of the tests assumptions. It was possible to define from the gastric cells, diverse absorbance ratios enabling to discriminate: i) The infection; ii) the bacteria genotype; and iii) the gastric disease of the patients from which the bacteria were isolated. These biomarkers could fasten the knowledge of the complex infection process while promoting a platform for a new diagnostic method, rapid but also specific and sensitive towards the diagnosis of both infection and bacterial virulence.
- Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinasePublication . Calado, Cecília; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The effect of culture media composition, and fermentation conditions and strategy on the growth and cutinase production of recombinant Saccharomyces cerevisiae and subsequent cutinase purification by expanded bed adsorption (EBA) was studied. The reduction in the amount of yeast extract used as nitrogen source from 20 g dm−3 to 10 g dm−3 in batch cultures led to a 29% decrease in the heterologous cutinase production, while the 5% cutinase dynamic adsorption capacity (q5%) on the cation Streamline SP XL was increased 6.7‐fold. By dilution of the whole fermentation broth, performed with the lowest yeast extract content, which reduces conductivity, the q5% was additionally increased by 1.9‐fold. After implementation of a fed‐batch strategy the cutinase concentration, cutinase yield on carbon source, cutinase yield on nitrogen source and productivity were increased by 10.8‐, 2.9‐, 5.3‐ and 6.4‐fold, respectively, in relation to the previously‐mentioned batch fermentation. However, the increased cutinase production was compromised by heterologous protein loss during the EBA recovery operation and the cutinase dynamic adsorption capacity and purification productivity decreased by 90% and 75%, respectively. Thus, target protein production by S cerevisiae fermentation and a downstream process with EBA cannot be considered as separate entities, where the understanding of the factors that affect the interactions among them are crucial towards optimization of the overall production process of heterologous proteins.
- Micro-analytical GO/HRP bioreactor for glucose determination and bioprocess monitoringPublication . Vojinović, V.; Calado, Cecília; Silva, A. I.; Mateus, M.; Cabral, J. M. S.; Fonseca, Luís P. P.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 μl 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 °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 μl 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 10 mg/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).
- Optimisation of culture conditions and characterisation of cutinase produced by recombinant Saccharomyces cerevisiaePublication . Calado, Cecília; Taipa, M. Ângela; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The optimum culture dissolved oxygen concentration and culture pH for the production of cutinase from Fusarium solani pisi by the recombinant Saccharomyces cerevisiae SU50 strain was investigated. Dissolved oxygen concentrations in the fermentation culture of 5, 30 and 60% of air saturation were evaluated. A high cutinase production, specific cutinase activity and the highest cutinase yield on biomass and the highest specific cutinase production rate were obtained with a 5% of air saturation, which could have impact on process economics. Furthermore, at a low dissolved oxygen concentration, the specific growth rate, specific cutinase production rate, cutinase yield on biomass, cutinase activity and specific cutinase activity were increased when the pH control was changed from 5.25 to 6.25. The cutinase, accumulated in the yeast culture presents two glycosilated isoforms with molecular weights of 22.8 and 24.9 kDa measured by SDS-PAGE. Furthermore, cutinase in clarified culture samples presents a linear relationship between estereolytic and lypolitic activity and a high stability at room temperature.
- Fast identification of off-target liabilities in early antibiotic discovery with Fourier-transform infrared spectroscopyPublication . Ribeiro Da Cunha, Bernardo; Aleixo, Sandra M.; Fonseca, Luís P. P.; Calado, CecíliaStructural modifications of known antibiotic scaffolds have kept the upper hand on resistance, but we are on the verge of not having antibiotics for many common infections. Mechanism-based discovery assays reveal novelty, exclude off-target liabilities, and guide lead optimization. For that, we developed a fast and automatable protocol using high-throughput Fourier-transform infrared spectroscopy (FTIRS). Metabolic fingerprints of Staphylococcus aureus and Escherichia coli exposed to 35 compounds, dissolved in dimethyl sulfoxide (DMSO) or water, were acquired. Our data analysis pipeline identified biomarkers of off-target effects, optimized spectral preprocessing, and identified the top-performing machine learning algorithms for off-target liabilities and mechanism of action (MOA) identification. Spectral bands with known biochemical associations more often yielded more significant biomarkers of off-target liabilities when bacteria were exposed to compounds dissolved in water than DMSO. Highly discriminative models distinguished compounds with predominant off-target effects from antibiotics with well-defined MOA (AUROC > 0.87, AUPR > 0.79, F1 > 0.81), and from the latter predicted their MOA (AUROC > 0.88, AUPR > 0.70, F1 > 0.70). The compound solvent did not affect predictive models. FTIRS is fast, simple, inexpensive, automatable, and highly effective at predicting MOA and off-target liabilities. As such, FTIRS mechanism-based screening assays can be applied for hit discovery and to guide lead optimization during the early stages of antibiotic discovery.
- High-throughput bioassay for mechanism of action determination of antibacterial drugsPublication . Ribeiro Da Cunha, Bernardo; Fonseca, Luís P. P.; Calado, CecíliaWhile the ‘war’ on infectious diseases has been considered won, antibiotic-resistant bacteria are currently responsible for 25,000 death’s yearly in Europe. No new broadspectrum antibiotic has been introduced since the 1960s, and the last new class was discovered in 1986. As the antibiotic pipeline is clearly exhausted, new tools to advance antibiotic research are required. The current work explored Fourier-transform infrared spectroscopy to classify the mechanism of action of 13 antibiotics, acting by 3 distinct Mode-Of-Action (MOA) and belonging to 7 different classes. After optimization of a biological assay and pre-processing techniques, principal component analysis and partial least squares discriminant analysis were applied in a multi-level approach, including the MOA, antibiotic class and ultimately individual antibiotics acting on very specific molecular targets. Overall results indicate that the proposed method presents metabolic resolution to identify antibiotics at three levels of classification (i.e. different MOA, classes and even acting on specific targets). Interestingly, the resolution capacity obtained at these three levels of classification depended on the antibiotic type, which highlights the importance of the multilevel approach taken. Ultimately the present work reinforces the applicability of the method has a metabolic fingerprinting tool for antibiotic discovery.
- Metabolic fingerprinting with Fourier-Transform Infrared (FTIR) spectroscopy: towards a high-throughput screening assay for antibiotic discovery and mechanism-of-action elucidationPublication . Ribeiro Da Cunha, Bernardo; Fonseca, Luís P. P.; Calado, CecíliaThe discovery of antibiotics has been slowing to a halt. Phenotypic screening is once again at the forefront of antibiotic discovery, yet Mechanism-Of-Action (MOA) identification is still a major bottleneck. As such, methods capable of MOA elucidation coupled with the high-throughput screening of whole cells are required now more than ever, for which Fourier-Transform Infrared (FTIR) spectroscopy is a promising metabolic fingerprinting technique. A high-throughput whole-cell FTIR spectroscopy-based bioassay was developed to reveal the metabolic fingerprint induced by 15 antibiotics on the Escherichia coli metabolism. Cells were briefly exposed to four times the minimum inhibitory concentration and spectra were quickly acquired in the high-throughput mode. After preprocessing optimization, a partial least squares discriminant analysis and principal component analysis were conducted. The metabolic fingerprints obtained with FTIR spectroscopy were sufficiently specific to allow a clear distinction between different antibiotics, across three independent cultures, with either analysis algorithm. These fingerprints were coherent with the known MOA of all the antibiotics tested, which include examples that target the protein, DNA, RNA, and cell wall biosynthesis. Because FTIR spectroscopy acquires a holistic fingerprint of the effect of antibiotics on the cellular metabolism, it holds great potential to be used for high-throughput screening in antibiotic discovery and possibly towards a better understanding of the MOA of current antibiotics.
- Quality monitoring of biodiesel and diesel/biodiesel blends: a comparison between benchtop FT-NIR versus a portable miniaturized NIR spectroscopic analysisPublication . Monteiro, Luísa; Zoio, Paulo; Carvalho, Bernardo B.; Fonseca, Luís P. P.; Calado, CecíliaA methodology such as near-infrared (NIR) spectroscopy, which enables in situ and in real-time analysis, is crucial to perform quality control of biodiesel, since it is blended into diesel fuel and the presence of contaminants can hinder its performance. This work aimed to compare the performance of a benchtop Fourier Transform (FT) NIR spectrometer with a prototype of a portable, miniaturized near-infrared spectrometer (miniNIR) to detect and quantify contaminants in biodiesel and biodiesel in diesel. In general, good models based on principal component analysis-linear discriminant analysis (PCA-LDA) of FT-NIR spectra were obtained, predicting with high accuracies biodiesel contaminants and biodiesel in diesel (between 75% to 95%), as well as good partial least square (PLS) regression models to predict contaminants concentration in biodiesel and biodiesel concentration in diesel/biodiesel blends, with high coefficients of determination (between 0.83 and 0.99) and low prediction errors. The miniNIR prototype’s PCA-LDA models enabled the prediction of target contaminants with good accuracies (between 66% and 86%), and a PLS model enabled the prediction of biodiesel concentration in diesel with a reasonable coefficient of determination (0.68), pointing to the device’s potential for preliminary analysis of biodiesel which, associated with its potential low cost and portability, could increase biodiesel quality control.
- Effect of pre-fermentation on the production of cutinase by a recombinant Saccharomyces cerevisiaePublication . Calado, Cecília; Monteiro, Sandra M. S.; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The importance of controlling the expression of heterologous cutinase in a recombinant Saccharomyces cerevisiae SU50 strain was investigated. Maximum specific growth rate and the biomass yield increased 1.91 and 1.16 fold, respectively, when cutinase production was induced by galactose in a pre-fermentation step. However, only 19% of specific cell activity was obtained in comparison to other fermentations following a pre-fermentation step without induction of cutinase expression. Thus, the pre-fermentation step was performed using a selective medium not containing galactose, and the fermentation was performed with a cheaper and complex non-selective medium containing galactose. Under these conditions, and with the aim of maximising the specific cutinase activity, a pre-fermentation with low volume and high density of viable cells must be used. However, due to the low pre-fermentation volume, low yeast cell concentrations and low specific cell activities were obtained after 96 h of fermentation. Otherwise, when the aim was to maximise cutinase yield and productivity, a pre-fermentation volume of 10% (v/v) in relation to fermentation and in the exponential growth phase with a cell concentration between 1.1 and 1.8 g dcw/l should be used. A higher pre-fermentation volume, such as 20% (v/v), would still be economical in the case of a pre-fermentation with low cell density or low cell viability.
- Optimization of the culture medium composition using response surface methodology for new recombinant cyprosin B production in bioreactor for cheese productionPublication . Sampaio, Pedro; Calado, Cecília; SOUSA, LISETE; Bressler, David C.; Pais, Maria Salomé; Fonseca, Luís P. P.The optimization of culture medium composition was carried out for improvement the recombinant cyprosin B production, an enzyme with high milk-clotting activity. Response surface methodology (RSM) was applied to evaluate the effect of variables namely glucose, yeast extract (YE) and bactopeptone present in the culture medium, used for recombinant cyprosin B production by transformed Saccharomyces cerevisiae BJ1991 strain in shake-flask and bioreactor culture conditions. The central composite experimental design (CCD) was adopted to derive a statistical model for optimizing the composition of the fermentation medium. The optimal concentration estimated for each variable related to a theoretical maximum of cyprosin B activity (488 U mL-1 ) was 30 g L-1 glucose, 15 g L-1 YE and 27 g L-1 bactopeptone. The optimized medium composition, based on empirical model, led to a cyprosin B activity of 519 U mL-1 , which corresponds to an increase of 46%. The fermentation using optimized culture medium in a 5-L bioreactor allowed a significant increase in biomass (82%) and recombinant cyprosin B production (139%). The improvement in the recombinant cyprosin B production after optimization process can be considered adequate for large-scale applications, and the clotting activity of cyprosin B account for their use in industrial cheese making.