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- D 2.5 Reports on the micorbiological characterisation of Black Sea area traditional foodsPublication . Levchuk, O.; Mudryk, M.; Bati, V.; Boyko, N.; Dolgikh, A.; Kutchak, I.; Pauk, A.; Boyko, N.
- Inherited Dyslipidemia VS Environmental Dyslipidemia in Portuguese ChildrenPublication . Medeiros, A.M.; Alves, A.C.; Bourbon, M.Hypercholesterolemia is an important cardiovascular risk (CV) factor that can be due to environmental or genetic causes. Identification of a young population with high CV risk allows early intervention and prevention, delaying or abolishing occurrence of CHD in adult life. Familial Hypercholesterolemia (FH) is associated with major risk of CV events and usually results from mutations in three different genes involved in lipid metabolism such as LDLR, APOB and PCSK9 genes. The Portuguese FH Study is established in the National Institute of Health, Lisbon, since 1999 where the biochemical characterization and DNA diagnosis have been performed for patients with clinical diagnosis of FH and cascade screening in relatives of the affected index patients.
- Transcription initiation arising from E-cadherin/CDH1 intron2: a novel protein isoform that increases gastric cancer cell invasion and angiogenesisPublication . Pinheiro, Hugo; Carvalho, Joana; Oliveira, Patrícia; Ferreira, Daniel; Teixeira Pinto, Marta; Osório, Hugo; Licastro, Danilo; Bordeira-Carriço, Renata; Jordan, Peter; Lazarevic, Dejan; Sanges, Remo; Stupka, Elia; Huntsman, David; Seruca, Raquel; Oliveira, CarlaDisruption of E-cadherin (CDH1 gene) expression, subcellular localization or function arises during initiation and progression of almost 90% of all epithelial carcinomas. Nevertheless, the mechanisms through which this occurs are largely unknown. Previous studies showed the importance of CDH1 intron 2 sequences for proper gene and protein expression, supporting these as E-cadherin cis-modulators. Through RACE and RT-PCR, we searched for transcription events arising from CDH1 intron 2 and discovered several new transcripts. One, named CDH1a, with high expression in spleen and absent from normal stomach, was demonstrated to be translated into a novel isoform, differing from canonical E-cadherin in its N-terminal, as determined by mass-spectrometry. Quantitative and functional assays showed that when overexpressed in an E-cadherin negative context, CDH1a replaced canonical protein interactions and functions. However, when co-expressed with canonical E-cadherin, CDH1a increased cell invasion and angiogenesis. Further, interferon-induced genes IFITM1 and IFI27 levels were increased upon CDH1a overexpression. Effects on invasion and IFITM1 and IFI27 expression were reverted upon CDH1a specific knockdown. Importantly, CDH1a was de novo expressed in gastric cancer cell lines. This study presents a new mechanism by which E-cadherin functions are impaired by cis-regulatory mechanisms possibly with the involvement of inflammatory machinery. If confirmed in other cancer models, our data encloses potential for designing targeted therapies to rescue E-cadherin function.
- Contaminação de água subterrânea por substâncias de limpeza da neve em estradas: o caso do sector de Nave de Santo António – Covão do Curral (Serra da Estrela, Centro de Portugal)Publication . Carvalho, A.; Espinha Marques, J.; Marques, J.M.; Carreira, P.M.; Moura, R.; Guerner Dias, A.; Chaminé, H.I.; Rocha, F.; Saraiva, R.; Tavares, M.; Mansilha, C.Num momento em que a gestão sustentável dos recursos hídricos se assume como uma das maiores preocupações à escala mundial, as regiões de montanha têm vindo a ser reconhecidas como Reservatórios de Água (PROGRAMA UNESCO IHP-VI). O facto de muitas destas áreas se localizarem a altitudes elevadas e serem recortadas por rodovias obriga a frequentes operações de limpeza de neve, especialmente no inverno. A aplicação de substâncias químicas como cloreto de sódio e cloreto de cálcio na promoção da fusão do gelo e na limpeza da neve das estradas constitui um problema ambiental de elevado interesse científico, social e económico. Devido às suas características geológicas, geomorfológicas e climáticas, a Serra da Estrela é considerada estratégica para a gestão da água em Portugal. No presente trabalho apresentam-se alguns resultados hidrogeoquímicos que indicam que a utilização dos referidos sais na limpeza do gelo e da neve das rodovias contribui para a contaminação dos recursos hídricos.
- Preparation, characterization and evaluation by FTIR and NMR of antimicrobial activity of chitosan active filmsPublication . Lago, M.A.; Sendón, R.; Rodríguez-Bernaldo de Quirós, A.; Bueno, C.; Sanches-Silva, A.; Costa, H.S.; Sánchez-Machado, D.I.; Soto Valdez, H.; Angulo, I.; Aurrekoetxea, G.P.; López-Cervantes, J.; Paseiro, P.One of the main causes of food spoilage is the development of microorganisms. In order to inhibit or retard the growth of microorganisms and consequently, improve food security and extend the shelf life of food products, in the past years, active packaging and particularly films with antimicrobial properties have attracted the attention of the scientists. One of the approaches used is to add chitosan to the film. Due to their excellent properties, non-toxic, biodegradable, biofunctional and biocompatible with others antimicrobials, chitosan is one of the antimicrobial agents most appropriate for the development of active materials [1]. Chitosan (CAS nº 9012-76-4) is a polysaccharide, with the structure of a linear polymer of (1-4)-linked 2-amino-deoxy-β-D-glucan, obtained by the partial deacetylation of chitin, one of the most abundant polysaccharides in nature, found in shells of crustaceans [2]. Shrimp waste (heads and cephalothorax) samples were collected from local shrimp processing factories in South Sonora, Mexico. The waste was minced, fermented and centrifuged. After the treatment, three fractions were obtained: chitin rich fraction, protein rich liquor and lipid fraction. In this work, Fourier Transformed Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) were used to characterize three different samples of chitosan. Moreover, 100% chitosan-based films and films of polyamide with chitosan incorporated were also evaluated. The degree of acetylation (DA) values obtained may change, depending on the nature and level of impurities, source and polymer morphologies [3]. To evaluate the DA, FTIR and NMR were used. The samples were prepared as a thin pellet made from a mixture of KBr and the chitosan powder. To evaluate the DA in films, Fourier Transform Total Reflection infrared Spectroscopy (FTIR-ATR) was used. All spectra were recorded in the range of 400-4000 cm-1. Nuclear Magnetic Resonance (NMR) was also employed to evaluate the DA. Two experiments were tentatively carried out: 1H NMR and 13C NMR. In both cases approximately 5 mg of each sample were diluted in 1 % (v/v) CD3COOD in D2O. All data were compared with three commercially available standards submitted to the same experiments as samples. The sample 1 corresponding to chitosan obtained from shrimp waste and with high viscosity presented the lowest DA, therefore had higher antimicrobial activity. Both techniques; FTIR and NMR, led to the same conclusion.
- No ocular involvement in familial amyloidotic polyneuropathy ATTR V30M domino liver recipientsPublication . Melo Beirão, J.; Matos, E.; Beirão, I.; Costa, P.; Torres, P.In many transplantation centers domino liver transplantation is an established procedure, increasing the number of available liver grafts. Increasingly, grafts from familial amyloidotic polyneuropathy (FAP) patients are used. Ocular involvement is a well known manifestation of FAP, and can be vision-threatening. The aim of this study was to evaluate the risk of development of familial amyloidotic polyneuropathy ocular manifestations in domino liver recipients. Forty-four cirrhotic patients submitted to liver transplantation were studied, with an average of 6 years of follow up after the procedure. Twenty two patients had received a liver from a FAP donor (Group 1) and 22 had received a liver from a non-FAP cadaveric donor (Group 2). Both groups were similar for mean age and gender. Routine ophthalmological examinations with particular attention to amyloid deposition in the anterior segment and vitreous, peripheral retina state, lacrimal functions tests (Schirmer and tear break-up time) and pupillometry (dynamic and static) were performed. No statistically significant differences were observed in all studied ophthalmic parameters between the two groups. No FAP related ophthalmic manifestations were detected after 6 years of domino liver transplantation, but further prospective regular ophthalmological examinations are necessary to detect the eventual development of late ocular manifestations.
- Occupational Exposure to toxigenic fungi from Aspergillus flavus complexPublication . Malta-Vacas, J.; Sabino, R.; Viegas, S.; Viegas, C.Bioaerosols are mainly composed of fungal particles, bacteria and plant spores, being fungi responsible for the release of VOCs and micotoxins into indoor environments. Aspergillus flavus is a common opportunistic pathogen causing human infections and is involved in the production of aflatoxin and other secondary metabolites associated with toxic and allergic reactions. Poultry workers are exposed to high concentrations of fungi and are therefore more prone to develop associated pathologies. To evaluate occupational exposure of the workers to Aspergillus flavus and aflatoxins, six animal production facilities were selected, including 10 buildings, from which indoor air samples and outdoor reference samples were obtained. Twenty-five duplicate samples were collected by two methodologies: impactation onto malt extract agar of 25L air samples using a Millipore Air Tester were used to evaluate quantitative (CFU/m3) and qualitative (species identification, whenever possible) sample composition; 300 L air samples collected with the Coriolis Air Sampler into phosphate–saline buffer were used to isolate DNA, following molecular identification of Aspergillus section flavi using nor-1 specific primers by real-time PCR. Overall, Aspergillus was the most frequent genus detected. Using conventional methodologies, A. flavus species were identified in five indoor samples belonging to three buildings and in two outdoor samples. Using real-time PCR, aflatoxigenic species were detected in two buildings, although only one was coincident with the ones identified by cultures. Using both methodologies we could quantify viable microorganisms and simultaneously identify potentially toxigenic species, resulting in complementary information useful in the adoption of strategies to minimize exposure to micotoxins.
- Characterization of chitosan intended to develop antimicrobial films: Microscopical StudiesPublication . Lago, M.A.; Sendón, R.; Rodríguez-Bernaldo de Quirós, A.; Sanches-Silva, A.; Costa, H.S.; Sánchez-Machado, D.I.; Soto Valdez, H.; Angulo, I.; Aurrekoetxea, G.P.; López-Cervantes, J.; Paseiro, P.Accumulation of organic wastes in intensive crustaceans culture ponds and nearby coastal waters has become a serious environmental and economical problem. For this reason, new ecofriendly and economically feasible products from agricultural wastes or byproducts for shrimp farms have been developed. This biowaste could be used as an important source of the useful biopolymer chitin and others components such as proteins or carotenoids like asthaxanthin [1]. Chitin is the most abundant polysaccharide after cellulose and the main source is the shell of crustaceans. Chitosan, derived from chitin, has proven useful for a wide range of applications due to its biodegradability, biocompatibility, antimicrobial activity, non-toxicity and versatile physicochemical properties. These properties make the chitosan an excellent candidate to use in food packaging [2]. The development of active packaging with antimicrobial and antioxidant activity based on chitosan and asthaxanthin obtained from shrimp waste is the main goal of the project: “Preparation of active packaging with antioxidant and antimicrobial activity based on asthaxanthin and chitosan” funded by FONCYCIT. The characterization of chitosan in the development of active materials is a key issue since their properties play an important role in its effectiveness as an antimicrobial agent. These properties are mainly molecular weight (Mw), acetylation degree (DA) and polymerization degree (PA). In addition, in mediums of low pH, the antimicrobial activity of chitosan increases [3]. The objective of the present study was characterized three different samples of chitosan obtained from shrimp waste by using two microscopy techniques, Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). Additionally, the films developed after the chitosan incorporation in the polyamide matrix were also characterized. Shrimp waste (heads and cephalotorax) samples were collected from local shrimp processing factories in South Sonora, Mexico. The waste was minced, fermented and centrifuged. After the treatment, three fractions were obtained: chitin-rich fraction, protein rich liquor and lipid fraction. Scanning Electron Microscopy was used to investigate the structure properties relationships of chitosan. Samples were spread on a carbon conducting adhesive tape pasted on a metallic stub, subjected to gold covering and observed. The samples for TEM observation were embedded in EPON resin and polymerized at 60 ºC; then were cut at (-120 ºC) using a Leica Ultracut crio-ultramicrotome. The images obtained showed the particle of chitosan embedded into the polyamide matrix.
- Diversity of β-lactamase-encoding genes in Escherichia coli strains isolated from food-producing, companion and zoo animals in PortugalPublication . Clemente, Lurdes; Correia, Ivone; Albuquerque, Teresa; Geraldes, Margarida; Matos, Filipa; Themudo, Patrícia; Manageiro, Vera; Jones-Dias, Daniela; Ferreira, Eugénia; Caniça, ManuelaA rapid development of plasmid-mediated resistance to extended-spectrum cephalosporins has been observed in Enterobacteriaceae worldwide, predominantly due to the dissemination of extended-spectrum beta-lactamases (ESBL) and plasmid-mediated AmpC beta-lactamases (PMAB). The aim of the present study was to evaluate the extension of ESBL- and PMAB-producing E. coli strains isolated from different animal origins in Portugal. For surveillance purposes, 376 E. coli isolates identified at National Laboratory of Veterinary Research (2009-2011) were submitted to antimicrobial susceptibility testing: 123, 51 and 202 were isolated from food-producing, companion and zoo animals, respectively. Minimum Inhibitory Concentrations (MIC) of 11 antimicrobials for all isolates was determined through agar dilution method. Susceptibility towards cefoxitina was determined through disk diffusion method. Breakpoints were interpreted accordingly to EUCAST epidemiological cut-off values. ‘Non-wild type’ (NWT) isolates for cefotaxime (MIC>0.25mg/L) and/or cefoxitina (<19mm) were screened for the presence of ESBL (blaTEM, blaOXA, blaSHV, blaCTX) and PMAB encoding genes, using PCR method. Sequencing was applied to fully identify beta-lactamases. Seventeen isolates (4.5%) were ‘NWT’ strains for cefotaxime, being 5 (29.4%) from companion animals, 4 (23.5%) from food-producing animals and 8 (47.1%) from zoo animals. We identified blaCTX-M-14 (n=1) in a dog and blaCTX-M-15-type genes (n=9) in 6 zoo animals and 3 in food-producing animals. We also identified blaCMY-type genes (n=3) in ‘NWT’ isolates for cefoxitin, one from each animal category. Other beta-lactamase encoding genes were identified: blaOXA in 5 strains (29.4%) isolated from dolphins, blaTEM in 7 strains (41.2%) isolated from 3 companion animals, 2 food-producing and 2 zoo animals, and blaSHV identified in one isolate (5.9%) from a zoo animal; 13 beta-lactamase-producing isolates (76.5%) were multidrug resistant. Among ‘NWT’ E. coli isolates for cefotaxime, we identified an important diversity of ESBL encoding genes, belonging to different families, being blaCTX-M-15-type gene the predominant. The spread of ESBL-producing bacteria among species from different origins, such as food-producing, companion and zoo animals, is a concern at public health level. Thus, it should be a priority to monitor and identify the reservoirs of antimicrobial resistance, contributing to a single health for all.
- Multilocus Sequence Typing for characterization of potential risk ESBLs-producing Escherichia coli isolated from pigs, including strains of new singletons ST2528, ST2524 and ST2525Publication . Ramos, Sónia; Caniça, Manuela; Igrejas, Gilberto; Jones-Dias, Daniela; Capelo-Martinez, José Luís; Poeta, PatríciaInfections caused by Escherichia coli harboring extended-spectrum beta-lactamases (ESBL) have a tremendous impact on public health, because of treatment complications. ESBL-producing E. coli are increasingly reported in healthy food-producing animals that can spread to humans either by direct contact or, more importantly, through the food chain. Here we describe a molecular survey aimed at determining the population structure and dynamics of ESBL-producing E. coli strains recovered from healthy pigs slaughtered for human consumption in Portugal. For this purpose, a total of 71 faecal samples from pigs were collected (2008 to 2009) in different geographical regions of Portugal. Susceptibility to 16 antibiotics was tested by disk-diffusion method in all recovered isolates and ESBL detection was carried out by double-disk test. PCR and sequencing methods characterized blaESBL genes responsible for the ESBL-phenotype. In addition, we used multilocus sequence typing (MLST) to identify the genetic lineages of all ESBL-producing E. coli strains, which were characterized by sequencing the internal fragments of 7 housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, recA); the MLST database was used to determine allelic profiles and for sequence type (ST) and clonal complex (CC) assignment. Among the 35 ESBL-producing strains, MLST analysis revealed 9 different STs under 6 CCs and 9 singletons STs. The CC10 and CC155 were the most common CCs, with 4 and 11 isolates, respectively. Two other isolates were assigned to the CC101. Moreover, 5 strains were included in 3 new STs; 3 of them were identified in a new allele for the fumC gene that originated the new ST2528; in addition, 2 isolates were registered as ST2524 and ST2525 through new combination of alleles. Through the MLST database we found that ST656 (CC10) and ST8 (CC165) have a higher homology to ST2524 and ST2525, respectively. However, by the definition of CCs, ST2524 and ST2525 most likely belong to CC10 and CC165, respectively. Our data shows the presence of ESBL producing E. coli isolates in pigs slaughtered for human consumption and raises important questions in the potential risk factors to public health due to the transmission of bacteria carrying resistance through the food chain, and spreading resistance to other bacteria of human clinical significance. A great heterogeneity of MLST types was observed, among which CC10, CC155 and CC101 have already been associated with human clinical isolates.