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Pereira V.J.,IBET | Pereira V.J.,New University of Lisbon | Marques R.,IBET | Marques M.,IBET | And 3 more authors.
Water Research | Year: 2013

The effectiveness of free chlorine for the inactivation of fungi present in settled surface water was tested. In addition, free chlorine inactivation rate constants of Cladosporium tenuissimum, Cladosporium cladosporioides, Phoma glomerata, Aspergillus terreus, Aspergillus fumigatus, Penicillium griseofulvum, and Penicillium citrinum that were found to occur in different source waters were determined in different water matrices (laboratory grade water and settled water). The effect of using different disinfectant concentrations (1 and 3 mg/l), temperatures (21 and 4 °C), and pH levels (6 and 7) was addressed. The sensitivity degree of different fungi isolates to chlorine disinfection varied among different genera with some species showing a higher resistance to disinfection and others expected to be more prone to protection from inactivation by the water matrix components. When the disinfection efficiency measured in terms of the chlorine concentration and contact time (Ct) values needed to achieve 99% inactivation were compared with the Ct values reported as being able to achieve the same degree of inactivation of other microorganisms, fungi were found to be more resistant to chlorine inactivation than bacteria and viruses and less resistant than Cryptosporidium oocysts. © 2012 Elsevier Ltd.

Ribeiro T.,New University of Lisbon | Santos S.,IBET | Marques M.I.M.,Instituto Gulbenkian Of Ciencia | Gilmore M.,Massachusetts Eye and Ear Infirmary | De Fatima Silva Lopes M.,New University of Lisbon
International Journal of Antimicrobial Agents | Year: 2011

Subinhibitory concentrations of vancomycin are known to induce a cell-wall stimulon in some Gram-positive pathogens, but this has never been studied in the genus Enterococcus. In this study, Enterococcus faecalis V583 strain was submitted to a subinhibitory concentration of vancomycin. DNA microarray technology was used to analyse the transcriptomic changes induced by this antibiotic. EF2292, annotated as a hypothetical protein in the E. faecalis V583 genome, was highly induced in response to vancomycin exposure, to similar levels as the vanB operon genes. We investigated further and provide evidence for co-transcription of ef2292 with vanY BWH BBX B genes. It was also demonstrated that expression of ef2292 is under the control of vanR BS B and it is proposed to name it vanV. This gene was found not to be required for vancomycin resistance under the conditions tested, thus coding for another accessory protein in the vanB operon. vanV was detected in some, but not all, E. faecalis carrying the vanB operon, suggesting that this operon can have different composition amongst E. faecalis isolates. © 2011 Elsevier B.V. and the International Society of Chemotherapy.

Vicente T.,IBET | Mota J.P.B.,FCT Inc. | Peixoto C.,IBET | Alves P.M.,IBET | And 2 more authors.
Journal of Chromatography A | Year: 2010

Surface plasmon resonance (SPR) spectroscopy is used as a scaled-down, analytical, pseudo-chromatography tool for analyzing protein binding and elution over an ion-exchange surface under cyclic sorption conditions. A micrometric-scale adsorption surface was produced by immobilizing a typical ion exchange ligand - diethylaminoethyl (DEAE) - onto commercially available planar gold sensor chip surfaces pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid with known density. An explicit mathematical formulation is provided for the deconvolution and interpretation of the SPR sensorgrams. An adsorption rate model is proposed to describe the SPR sensorgrams for bovine serum albumin, used here as model protein, when the DEAE surface is subjected to a cyclic series of binding and elution steps. Overall, we demonstrate that the adsorption rate model is capable of quantitatively describing BSA binding and elution for protein titers from dilute conditions up to overloaded conditions and a broad range of salt concentrations. © 2010 Elsevier B.V. All rights reserved.

Vicente T.,IBET | Faber R.,Sartorius Stedim Biotech GmbH | Alves P.M.,IBET | Carrondo M.J.T.,IBET | And 2 more authors.
Biotechnology and Bioengineering | Year: 2011

The effect of ligand density on anion-exchange membrane chromatography (AEXmc) for the purification of recombinant baculoviruses (rBVs), potential viral vectors in clinical applications, is studied by surface plasmon resonance on customized AEX surfaces and gradient elution experiments on Sartobind D membrane prototypes with different diethylamine ligand densities, complemented by dynamic light scattering analysis for estimation of the hydrodynamic particle size of the various biologics. A chromatographic-column model based on the steric mass action model of ion exchange is employed to analyze the gradient-elution AEXmc experiments, extrapolate the results to other operating conditions, and provide directions for process improvement. Although counterintuitively, the experimental evidence provided in this study shows that the lowering of ligand density is beneficial for rBV purification by AEXmc in bind-and-elute-mode, because it decreases the residual concentrations of host cell protein, dsDNA, and non-infective rBVs in the eluted product cut, and increases the overall yield by roughly 20% over current standard values. Overall, we present a case study on how rational design can streamline downstream process development. © 2011 Wiley Periodicals, Inc.

Serra H.,IBET | Serra H.,Laboratorio Medinfar S.A. | Bronze M.d.R.,IBET | Simplicio A.L.,IBET
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2010

A capillary electrophoresis method was developed and validated for the first time for the analysis of clopidogrel and its carboxylic acid metabolite. Prior to method optimization, the pH dependence of effective mobility of both compounds was determined in order to define the initial pH of the running buffer. The optimized method demonstrated to be selective, and linear in the concentration range of 2-100 μM for both compounds. The method limits of detection and quantification were, respectively, 1.2 and 3.7 μM for clopidogrel and 1.1 and 3.2 μM for the carboxylic acid metabolite. Moreover, method validation demonstrated acceptable results for method repeatability (RSD < 7%), intermediate precision (RSD < 7%) and accuracy (85-96%) and is suitable for the quantitative analysis of clopidogrel and its metabolite in serum samples. The validated method was also applied to the determination of the kinetic parameters of the enzymatic hydrolysis of clopidogrel. An apparent Km of 145 ± 30 μM and Vmax of 0.4, 1.5 and 3.4 μM/min, respectively for the enzyme concentrations 1.0, 2.0 and 4.0 U/ml, were obtained. © 2010 Elsevier B.V. All rights reserved.

Vicente T.,IBET | Mota J.P.B.,New University of Lisbon | Peixoto C.,IBET | Alves P.M.,IBET | And 2 more authors.
Biotechnology Advances | Year: 2011

The advent of advanced therapies in the pharmaceutical industry has moved the spotlight into virus-like particles and viral vectors produced in cell culture holding great promise in a myriad of clinical targets, including cancer prophylaxis and treatment. Even though a couple of cases have reached the clinic, these products have yet to overcome a number of biological and technological challenges before broad utilization. Concerning the manufacturing processes, there is significant research focusing on the optimization of current cell culture systems and, more recently, on developing scalable downstream processes to generate material for pre-clinical and clinical trials. We review the current options for downstream processing of these complex biopharmaceuticals and underline current advances on knowledge-based toolboxes proposed for rational optimization of their processing. Rational tools developed to increase the yet scarce knowledge on the purification processes of complex biologicals are discussed as alternative to empirical, "black-boxed" based strategies classically used for process development. Innovative methodologies based on surface plasmon resonance, dynamic light scattering, scale-down high-throughput screening and mathematical modeling for supporting ion-exchange chromatography show great potential for a more efficient and cost-effective process design, optimization and equipment prototyping. © 2011 Elsevier Inc.

Carvalho-Santos Z.,Instituto Gulbenkian Of Ciencia | Machado P.,Instituto Gulbenkian Of Ciencia | Alvarez-Martins I.,Instituto Gulbenkian Of Ciencia | Gouveia S.,Instituto Gulbenkian Of Ciencia | And 11 more authors.
Developmental Cell | Year: 2012

Cilia and flagella are involved in a variety of processes and human diseases, including ciliopathies and sterility. Their motility is often controlled by a central microtubule (MT) pair localized within the ciliary MT-based skeleton, the axoneme. We characterized the formation of the motility apparatus in detail in Drosophila spermatogenesis. We show that assembly of the central MT pair starts prior to the meiotic divisions, with nucleation of a singlet MT within the basal body of a small cilium, and that the second MT of the pair only assembles much later, upon flagella formation. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MTs and is required for the early steps of central MT pair formation. This work describes a genetically tractable system to study motile cilia formation and provides an explanation for BLD10/CEP135@s role in assembling highly stable MT-based structures, such as motile axonemes and centrioles.

Teixeira A.P.,IBET | Duarte T.M.,IBET | Carrondo M.J.T.,IBET | Alves P.M.,IBET
Biotechnology and Bioengineering | Year: 2011

In this work, synchronous fluorescence spectroscopy (SFS) is evaluated as a new tool for real-time bioprocess monitoring of animal cell cultures. This technique presents several advantages over the traditional two-dimensional (2D) fluorometry since it provides data on various fluorescent compounds in a single spectrum, showing improved peak resolution and recording speed. Bioreactor cultures of three monoclonal antibody-producing CHO cell lines were followed in situ by both 2D and synchronous fluorometry techniques. The time profiles of the main spectral features in each data type present some differences, but principal component analysis indicated both as containing enough information to distinguish the cultures. Partial least squares regression models were then independently developed for viable cell density and antibody levels on the basis of the different fluorescence signals recorded, hiding half of the dataset for subsequent validation purposes. Regardless of the signal used, model predictions fit very well the off-line measurements; still, the synchronous spectra collected at a wavelength difference of 20nm allowed comparable and superior performances for cell density and antibody titer, respectively, with validation accuracies higher than 91%. Therefore, SFS compares favorably with the traditional 2D approach, becoming an improved, faster option for real-time monitoring of cells and product titer over culture time. The readiness in data acquisition facilitates the design of process control strategies meeting the requirements of a PAT application. © 2011 Wiley Periodicals, Inc.

Vicente T.,IBET | Peixoto C.,IBET | Alves P.M.,IBET | Carrondo M.J.T.,IBET | Carrondo M.J.T.,New University of Lisbon
Journal of Chromatography A | Year: 2010

Product-related impurities constitute a major burden in the production of recombinant viral vectors for gene therapy and vaccination; it impairs not only the biological efficacy of the preparation but the process yield/productivity. Recombinant baculovirus was used as an enveloped virus model to address this issue. Given that ion-exchange chromatography is a process of choice for purification of viral vectors, the analysis of the electrostatic behavior can be instrumental for the improvement of impurity removal. The main species, product (infective virus particle) and product-derived impurities (dsDNA-, glycoprotein-, and envelope-deprived baculovirus particles), were isolated and correspondent ζ potentials were analyzed through dynamic light scattering. A model of the virus based on the viral components critical for biological function is proposed. The contribution of these viral components to the overall particle electrostatic interaction energy profile (calculated between the particle and a putative ion-exchange surface) was assessed as a function of ionic strength and pH. This resulted in a deterministic tool capable of distinguishing the electrostatic properties of the infective virus particle from the major virus-related impurities. Within an ion-exchange bind-elute process, this knowledge helps narrow the optimization space in early stage process development for viral vectors by predicting the best selectivity conditions. © 2010 Elsevier B.V.

Braga T.M.,New University of Lisbon | Marujo P.E.,IBET | Pomba C.,The Interdisciplinary Center | Lopes M.F.S.,New University of Lisbon
Journal of Antimicrobial Chemotherapy | Year: 2011

Objectives: To investigate the role of a putative small multidrug resistance transporter, annotated in Enterococcus faecalis V583 genome as EFA0010 (we will refer to this gene as qacZ), in decreased susceptibility to biocides. Methods: A derivative strain of V583, susceptible to erythromycin (V583ErmS), was complemented with pORI23 carrying the qacZ gene (strain EF-SAVE1). MICs of benzalkonium chloride, chlorhexidine and ethidium bromide were determined for the complemented strain and wild-type. RT-PCR and ethidium bromide efflux assays were performed in order to fully understand the role and specificity of the qacZ gene. The presence of qacZ in 73 enterococcal strains from different origins was investigated by PCR, and MICs of benzalkonium chloride and chlorhexidine were determined for the same strains. Results: The complemented strain, EF-SAVE1, presented a higher MIC of benzalkonium chloride (8 mg/L) than V583ErmS (4 mg/L); the MICs of chlorhexidine and ethidium bromide were the same for both strains, 4 mg/L and 16 mg/L, respectively. Expression of qacZ was found to be higher in EF-SAVE1 and constitutive, i.e. not inducible by any of the three tested biocides. Overexpression of qacZ was not responsible for changes in ethidium bromide efflux. This gene was present in 52% of the enterococcal isolates studied and the MICs of benzalkonium chloride and chlorhexidine ranged between 2 and 8 mg/L. Conclusions: We demonstrate the involvement of the qacZ gene in tolerance to the quaternary ammonium compound benzalkonium chloride, but not ethidium bromide. This work constitutes the first report of a biocide resistance mechanism in E. faecalis, and reveals its dissemination amongst the genus Enterococcus. © The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

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