2,335 research outputs found

    Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method

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    Despite the emergence of non-O157 shiga toxin-producing Escherichia coli (STEC) infections, E. coli O157 serotype is still the most commonly identified STEC in world. It causes high morbidity and mortality, and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain as the gold standard.In here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157.Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. Then, the method was optimized for the detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1×10-2 to 1×102 CFU per 25g or ml of food. Samples were then pre-enriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrixes with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% CI 82.83 - 100), a sensitivity of 97.22% (95% CI, 83.79 - 99.85) and an accuracy of 98,33% (CI 95%, 83.41 - 99.91). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.This work was supported by the Portuguese Institute Fundacao para a Ciencia e Tecnologia (FCT), project PIC/IC/82815/2007. C.A. acknowledges FCT for individual postdoctoral fellowship SFRH/BPD/74480/2010. We also acknowledge Biomode S.A. for providing some supplies for this project

    Development and characterization of an active chitosan-based film containing quercetin

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    This work aims at developing an active chitosan film through the incorporation of quercetin and the evaluation of physical and functional properties of the films made thereof. The addition of quercetin showed to influence films properties in terms of surface morphology, tensile strength, and opacity while elongation-at-break, thickness, water vapor, and oxygen permeability were not significantly affected with incorporation of quercetin. The color parameters of chitosan films were affected by quercetin incorporation with a decrease of the values of L* and a*. The film exhibited a high free-radical scavenging activity, showing antioxidant activity. The film-forming solutions of chitosan with or without quercetin showed antibacterial activity against four Gram-negative and three Gram-positive bacteria. These results showed that quercetin incorporation in chitosan-based films has potential to be used as a solution for active food packaging.Author Marthyna Pessoa de Souza thanks the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES/PDEE-Brazil) and Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE, Brazil) for fellowships. Miguel A. Cerqueira and Helder D. Silva (SFRH/BPD/72753/2010 and SFRH/BD/81288/2011, respectively) are recipients of a fellowship from the Fundacao para a Ciencia e Tecnologia (FCT, POPH-QREN and FSE Portugal). This research was financially supported by research grants and fellowships from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), as well as the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) and Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE). The authors also thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and the project "BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes," REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON. 2-O Novo Norte), QREN, FEDE

    Dielectric spectroscopy of melt-mixed polypropylene and pyrolytically stripped carbon nanofiber composites

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    In this work, pyrolytically stripped carbon nanofiber (CNF) polypropylene (PP) composites were synthesized following a scalable melt-mixing method, and the effects of CNF weight concentrations on the electrical conductivity, dielectric permittivity, electrical modulus and electrical impedance of PP/CNF composites were studied. Quite unexpectedly, the electrical conductivity of PP/CNF composites improved only slightly as the incorporation of CNFs was raised, yielding a maximum of ~10−10 S m−1 for PP/CNF 5 wt. % composites. The increase corresponded to a gradual improvement of the dielectric constant up to a maximum of ~9 for PP/CNF 5 wt. % composites at 1 MHz, which was attributed to the raise of interface polarization effect. Moreover, the Cole–Cole model was used to analyze the effects of CNF concentrations on the dielectric relaxation of PP/CNF composites, from which was deduced that the incorporation of CNFs increases their dielectric strength and relaxation times. The analysis gathered here aims to provide a better insight into the enhanced dielectric properties observed in low-conducting polymer composites filled with CNFs.A. J. Paleo gratefully acknowledges support from FCT-Foundation for Science and Technology by the project UID/CTM/00264/2021 of 2C2T under the COMPETE and FCT/MCTES (PIDDAC) co-financed by FEDER through the PT2020 program and “plurianual” 2020–2023 Project UIDB/00264/2020

    PNA-FISH as a new diagnostic method for the determination of clarithromycin resistance of Helicobacter pylori

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    <p>Abstract</p> <p>Background</p> <p>Triple therapy is the gold standard treatment for <it>Helicobacter pylori </it>eradication from the human stomach, but increased resistance to clarithromycin became the main factor of treatment failure. Until now, fastidious culturing methods are generally the method of choice to assess resistance status. In this study, a new genotypic method to detect clarithromycin resistance in clinical samples, based on fluorescent <it>in situ </it>hybridization (FISH) using a set of peptide nucleic acid probes (PNA), is proposed.</p> <p>Results</p> <p>The set of probes targeting the point mutations responsible for clarithromycin resistance was applied to <it>H. pylori </it>suspensions and showed 100% sensitivity and specificity (95% CI, 79.9-100 and 95% CI, 71.6-100 respectively). This method can also be amenable for application to gastric biopsy samples, as resistance to clarithromycin was also detected when histological slides were tested.</p> <p>Conclusions</p> <p>The optimized PNA-FISH based diagnostic method to detect <it>H. pylori </it>clarithromycin resistance shown to be a very sensitive and specific method for the detection of clarithromycin resistance in the <it>H. pylori </it>smears and also proved to be a reliable method for the diagnosis of this pathogen in clinical samples and an alternative to existing plating methods.</p

    The mechanism of formate oxidation by metal-dependent formate dehydrogenases

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    J Biol Inorg Chem (2011) 16:1255–1268 DOI 10.1007/s00775-011-0813-8Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C–H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results

    Molecular determinants of ligand specificity in family 11 carbohydrate binding modules - An NMR, X-ray crystallography and computational chemistry approach

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    12 pags, 6 figs, 1 tabThe direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi-subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate-binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X-ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 Å in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies. © 2008 The Authors.The authors would like to thank the research network REQUIMTE (Project Reqmol), as well as the Portuguese Science and Technology Foundation (FCT-MCTES), for financial support through projectPTDC⁄QUI⁄68286⁄2006 and scholarships SFRH⁄BPD⁄27237⁄2006 and SFRH⁄BD⁄31359⁄200

    Construction of a biocompatible and antioxidant multilayer coating by layer-by-layer assembly of -carrageenan and quercetin nanoparticles

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    The present work aimed at the construction and characterization of a multilayer coating based on -carrageenan and quercetin-loaded lecithin/chitosan nanoparticles (Np) by the layer-by-layer technique and the evaluation of its antioxidant capacity and potential cytotoxicity in vitro. The multilayered coating was successfully self-assembled, as confirmed by UV-Vis spectroscopy, contact angle, atomic force microscopy (AFM), and scanning electron microscopy (SEM). Multilayered coatings showed to have antioxidant capacity, with a DPPH radical scavenging activity of 31.32±3.13% and a result of the FRAP assay of 799.41±95.39 M of ferrous ion (Fe2+) equivalent. These coatings were also shown to be devoid of cell toxicity, as evaluated by determination of nitric oxide production and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. The alveolar macrophages culture was tested in the presence of the -carrageenan/quercetin-Np multilayer coating and showed a cell viability of 91.3±9.6%. These results suggest that this multilayered coating is adequate for surfaces modification in view of biomedical and food industry applications.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors would also like to thank the Brazilian Government for support given by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Carneiro-daCunha, M.G. expresses her gratitude to the CNPq for research grant.info:eu-repo/semantics/publishedVersio
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