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- Cytogenetic and DNA damage on workers exposed to styrenePublication . Teixeira, João Paulo; Gaspar, Jorge; Coelho, Patrícia; Costa, Carla; Pinho-Silva, Susana; Costa, Solange; Da Silva, Susana; Laffon, Blanca; Pássaro, Eduardo; Rueff, José; Farmer, PeterStyrene is a commercially important chemical widely used in the manufacture of synthetic rubber, resins, polyesters and plastics. The highest levels of human exposure to styrene occur during the production of reinforced plastic products. The objective of this work was to evaluate both DNA and cytogenetic damage in styrene-exposed workers, analysing only non-smoker individuals. Environmental levels of styrene and urinary concentrations of mandelic and phenylglyoxylic acids were determined, and genetic damage was studied by means of micronucleus (MN) test, sister chromatid exchanges (SCEs) and comet assay. Fifty-two fibreglass-reinforced plastics workers and 54 controls took part in the study. The mean air concentration of styrene in the breathing zone of workers exceeded the threshold limit value, and 24 workers exceeded the biological exposure index. A strong and significant correlation was found between styrene environmental concentrations and urinary metabolites. Higher SCE rate (P<0.01) was observed in exposed workers than in controls. Besides, significant correlations were obtained for SCE rate with both environmental and internal exposure parameters (r=0.496, P<0.01 and r=0.511, P<0.01, respectively). Results from MN test and comet assay showed slight and non-significant increases related to the exposure. Our data seem to support previous studies reporting genotoxicity associated with occupational exposure to styrene, excluding the confounding influence of smoking, although caution must be taken in the interpretation of these results since the significance of an increase in SCE rate is still unclear.
- Evidences of large deletions in patients with the Lysosomal Storage Diseases Mucolipidosis type II and III: experimental approaches for picking up both homozygous and heterozygous casesPublication . Coutinho, Maria Francisca; Encarnação, Marisa; Carvalho, Filipa; Lacerda, Lúcia; Willbrand, Flemming; Ribeiro, Helena; Prata, Maria João; Alves, SandraBackgroung/Objectives: Mucolipidosis II and III are rare genetic diseases in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is reduced or absent. GlcNAc-phosphotransferase is a multimeric enzyme encoded by two genes: GNPTAB and GNPTG. Although a wide spectrum of mutations in GNPTAB has recently been reported to cause ML II and III alpha/beta, large deletions have not yet been reported. Furthermore, some previously reported patients present only one heterozygous mutation while the second one is still missing. Here we present evidence that some of these cases may be due to the difficulty to detect large heterozygous deletions through direct sequencing. Methods: We developed a semiquantitative approach by multiplex PCR and capillary electrophoresis, co-amplifying two fragments in each PCR reaction: one corresponding to GNPTAB exons and the other to an internal control fragment. Results: After applying this technique to perform a genomic screen in two Portuguese patients in whom a heterozygous missense was identified while the second mutation was missing, we found that in both patients the last exons (20 and 21) of the GNPTAB gene were missing in one of the alleles. We also found and characterize a gross homozygous deletion involving exon 19 in one Danish patient. Conclusion: Here we present a molecular approach that turned possible to identify for the first time large deletions in the GNPTAB, underlying mucolipidoses in patients who were both homozygous and heterozygous for such alterations. Our semiquantitative PCR based method is a valuable tool that allows the screening of large deletions. Consequently, we propose that cases with only one heterozygous mutation detected through direct sequencing methods, should be further screened for the possible presence of a large heterozygous deletion.
- Genotypes and antimicrobial resistant phenotypes of Neisseria gonorrhoeae in Portugal (2004-2009)Publication . Florindo, Carlos; Pereira, Rui; Boura, Márcia; Nunes, Baltazar; Paulino, Albertina; Gomes, João Paulo; Borrego, Maria J.OBJECTIVES: To determine the antibiotic phenotype and MAST-genotype distribution of Neisseria gonorrhoeae isolates in Portugal between 2004 and 2009, and to evaluate specific associations between MAST-genotypes and sexual orientation, age and antibiotic resistance. METHODS: A total of 236 N gonorrhoeae isolates were typed through N gonorrhoeae multiantigen sequence typing (NG-MAST). The degree of polymorphism and the phylogenetic relatedness among NG-MAST sequence types (STs) were evaluated with MEGA4 software on concatenated sequences of por and tbpb alleles. Etest was used to determine the susceptibility to ceftriaxone, ciprofloxacin, penicillin and spectinomycin. RESULTS: No isolates displayed resistance to spectinomycin and ceftriaxone, whereas 79.1% and 37.4% were resistant to penicillin and ciprofloxacin, respectively. A total of 104 different STs (one per 2.3 isolates) were found; the most common were ST210 (8.1%) and ST225 (7.6%). STs formed two major groups separated by 159.8 (SE 8.9) nucleotide differences, yielding several subgroups, one of them including the worldwide-prevalent ST225. The probability of ciprofloxacin resistance among isolates within this subgroup was 73.5-fold higher than for the remaining isolates. Indeed, for the genetically closest subgroup, which includes the most prevalent ST (ST210), only 8.0% of isolates were resistant to ciprofloxacin. There was a non-homogeneous distribution per year for ST225 (p<0.001), ST210 (p=0.011) and ST2 (p=0.007). CONCLUSIONS: The heterogeneous ST scenario may represent the 'tip of the iceberg', reflecting a high number of undiagnosed and unreported gonorrhoea cases. A laboratory-based national surveillance of N gonorrhoeae infections is necessary to provide a broader spectrum of isolates that will allow the sexual network situation in Portugal to be established.
- Molecular characterization of five patients with homocystinuria due to severe methylenetetrahydrofolate reductase deficiencyPublication . Urreizti, R.; Moya-García, A. A.; Pino-Ángeles, A.; Cozar, M.; Langkilde, A.; Fanhoe, U.; Esteves, C.; Arribas, J.; Vilaseca, M.A.; Pérez-Dueñas, B.; Pineda, M.; González, V.; Artuch, R.; Baldellou, A.; Vilarinho, L.; Fowler, B.; Ribes, A.; Sánchez-Jiménez, F.; Grinberg, D.; Balcells, S.Methylenetetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism. Disturbed function of the enzyme results in hyperhomocysteinemia and causes severe vascular and neurological disorders and developmental delay. Five patients suspected of having non-classical homocystinuria due to MTHFR deficiency were examined with respect to their symptoms, MTHFR enzyme activity and genotypes of the MTHFR gene. All patients presented symptoms of severe central nervous system disease. Two patients died, at the ages of 15 months and 14 years. One patient is currently 32 years old, and is being treated with betaine and folinic acid. The other two patients, with an early diagnosis and a severe course of the disease, are currently improving under treatment. MTHFR enzyme activity in the fibroblasts of four of the patients was practically undetectable. We found four novel mutations, three of which were missense changes c.664G>T (p.V218L), c.1316T>C (p.F435S) and c.1733T>G (p.V574G), and the fourth was the 1-bp deletion c.1780delC (p.L590CfsX72). We also found the previously reported nonsense mutation c.1420G>T (p.E470X). All the patients were homozygous. Molecular modelling of the double mutant allele (p.V218L; p.A222V) revealed that affinity for FAD was not affected in this mutant. For the p.E470X mutation, the evidence pointed to nonsense-mediated mRNA decay. In general, genotype–phenotype analysis predicts milder outcomes for patients with missense changes than for those in which mutations led to severe alterations of the MTHFR protein.
- Cytotoxic and morphological effects of microcystin-LR in in vitro models - a comparision between HepG2, Vero-E6, MDCK and CaCo-2 cell linesPublication . Menezes, Carina; Alverca, Elsa; Dias, Elsa; Sam-Bento, Filomena; Paulino, Sérgio; Pereira, PauloMicrocystin-LR (MCLR), a potent hepatotoxin, is transported selectively into the cells throught specific membrane polypeptides mostly present in the liver. These transporters are also expressed in the brain, kidneys and intestine, although the toxicity of MCLR in these cell types is still poorly understood. In this study, morphological, ultrastructural and biochemical analyses were performed in hepatic, renal and intestinal cell lines in order to evaluate the toxicity of MCLR obtained from semi-purified cyanobacterial extracts. Our results show that after 24h of exposure, MCLR induces the viability decrease of all cell lines, in a concentration-dependent manner. HepG2 cells are the most susceptible, followed by Vero, MDCK and CaCo-2. Ultrastructural analyses show that subcytotoxic concentrations of MCLR induce the formation of large cytoplasmic vacuoles, particularly in the HepG2 cell line. Immunofluorescence microscopy reveals that these vacuoles are enriched in LC3B protein, suggesting autophagy as an early cellular response of HepG2 and Vero cells to MCLR. At cytotoxic MCLR concentrations, lysossomal dysfunction in both cell lines occurs prior to mitochondrial disruption, as demonstrated by the specific labeling with Acridine Orange and Rhodamine-123. This suggests that besides mitochondria, lysossomes may also be an MCLR-early target. Immunolocalization and western blot analysis of the endoplasmic reticulum anti-apoptotic protein GRP94 show distinct MCLR-induced effects in Vero and HepG2 cells: re-localization of GRP94 within Vero cells and decrease of GRP94 expression in the HepG2 cell line. These results demonstrate that all the studied cell lines are susceptible to MCLR although with cell type specificity and differential organelle targeting.
- Portuguese food composition database quality management systemPublication . Oliveira, L. M.; Castanheira, I. P.; Dantas, M. A.; Porto, A. A.; Calhau, Maria AntóniaBACKGROUND: The harmonisation of food composition databases (FCDB) has been a recognised need among users, producers and stakeholders of food composition data (FCD). To reach harmonisation of FCDBs among the national compiler partners, the European Food Information Resource (EuroFIR) Network of Excellence set up a series of guidelines and quality requirements, together with recommendations to implement quality management systems (QMS) in FCDBs. The Portuguese National Institute of Health (INSA) is the national FCDB compiler in Portugal and is also a EuroFIR partner. INSA's QMS complies with ISO/IEC (International Organization for Standardisation/International Electrotechnical Commission) 17025 requirements. The purpose of this work is to report on the strategy used and progress made for extending INSA's QMS to the Portuguese FCDB in alignment with EuroFIR guidelines. SUBJECTS/METHODS: A stepwise approach was used to extend INSA's QMS to the Portuguese FCDB. The approach included selection of reference standards and guides and the collection of relevant quality documents directly or indirectly related to the compilation process; selection of the adequate quality requirements; assessment of adequacy and level of requirement implementation in the current INSA's QMS; implementation of the selected requirements; and EuroFIR's preassessment 'pilot' auditing. RESULTS: The strategy used to design and implement the extension of INSA's QMS to the Portuguese FCDB is reported in this paper. The QMS elements have been established by consensus. ISO/IEC 17025 management requirements (except 4.5) and 5.2 technical requirements, as well as all EuroFIR requirements (including technical guidelines, FCD compilation flowchart and standard operating procedures), have been selected for implementation. The results indicate that the quality management requirements of ISO/IEC 17025 in place in INSA fit the needs for document control, audits, contract review, non-conformity work and corrective actions, and users' (customers') comments, complaints and satisfaction, with minor adaptation. Implementation of the FCDB QMS proved to be a way of reducing the subjectivity of the compilation process and fully documenting it, and also facilitates training of new compilers. Furthermore, it has strengthened cooperation and trust among FCDB actors, as all of them were called to be involved in the process. CONCLUSIONS: On the basis of our practical results, we can conclude that ISO/IEC 17025 management requirements are an adequate reference for the implementation of INSA's FCDB QMS with the advantages of being well known to all members of staff and also being a common quality language among laboratories producing FCD. Combining quality systems and food composition activities endows the FCDB compilation process with flexibility, consistency and transparency, and facilitates its monitoring and assessment, providing the basis for strengthening confidence among users, data producers and compilers.
- Pandemic Influenza Virus Surveillance in Portugal: The Laboratory Network for the Diagnosis of Influenza A(H1N1)2009 InfectionPublication . Guiomar, Raquel; Pechirra, Pedro; Gonçalves, Paulo; Cordeiro, Rita; Conde, Patrícia; Arraiolos, Ana; Batista, Inês; Paixão, Eleonora; Nunes, Baltazar; Furtado (on behalf of the Laboratory Network for the Diagnosis of Influenza A(H1N1)2009 Infection), CristinaIn April 2009 a new influenza A(H1N1) virus of swine origin disseminated throughout the world, resulting in the first pandemic of the XXI century. To face the increasing number of diagnosis being requested, a National Laboratory Network for Influenza Surveillance of the new influenza A(H1N1)pandemic virus was activated in Portugal. This is a descriptive study of the Influenza-like Ilness (ILI) cases reported by this network. Association between the variables was evaluated by chi-squared test. Over 62089 ILI cases were notified, 25594 (41.2%) cases were laboratory confirmed A(H1N1)pdm virus, from week 17/2009 to week 15/2010. In the week 33 (summer) were detected 1039 (4.1%) positive cases for A(H1N1)pdm virus although the winter peak occurred in week 46 with 3131 (12.5%) A(H1N1)pdm positive cases. In the age group of 5-14 years old were detected the majority of positive cases 9983 / 15785 (63.0%) opposite in the elderly group (>65 years old) was detected the lower number of A(H1N1)pdm positive cases, 280/2361 (11.0%). The distribution by gender accounts 40.4% of female and 42.3% of male positive cases. The signs and/or symptoms present were analysed revealing that headache (49.7%), cough and myalgies (46.5%) and odinophagia (46.1%) were statistically associated with A(H1N1)pdm positive cases. The chronic pulmonary disease seemed to be more associated with laboratory confirmed A(H1N1)pdm cases. Ninety five strains were isolated and antigenically characterised, 45 were taken for genetic analysis (HA and NA gene). All the strains were antigenically and genetically like the pandemic vaccine strain. It was detected only one strain with the mutation H275Y in the neuraminidase, resistant to oseltamivir. This Laboratory network was an important tool to monitories and control the evolution of the pandemic.
- Development of NIPBL locus-specific database using LOVD: from novel mutations to further genotype-phenotype correlations in Cornelia de Lange SyndromePublication . Oliveira, Jorge; Dias, Cristina; Redeker, Egbert; Costa, Eurico; Silva, João; Lima, Margarida Reis; Den Dunnen, Johan T.; Santos, RosárioThe establishment of Locus Specific Databases (LSDB) is a crucial aspect for the Human Genetics field and one of the aims of the Human Variation Project. We report the development of a publicly accessible LSDB for the NIPBL gene (http://www.lovd.nl/NIPBL) implicated in Cornelia de Lange Syndrome (CdLS). This rare disorder is characterized by developmental and growth retardation, typical facial features, limb anomalies, and multiple organ involvement. Mutations in the NIPBL gene, the product of which is involved in control of the cohesion complex, account for over half of the patients currently characterized. The NIPBL LSDB adopted the Leiden Open Variation database (LOVD) software platform, which enables the comprehensive Web-based listing and curation of sequence variations and associated phenotypical information. The NIPBL-LOVD database contains 199 unique mutations reported in 246 patients (last accessed April 2010). Information on phenotypic characteristics included in the database enabled further genotype–phenotype correlations, the most evident being the severe form of CdLS associated with premature termination codons in the NIPBL gene. In addition to the NIPBL LSDB, 50 novel mutations are described in detail, resulting from a collaborative multicenter study. Hum Mutat 31:1216–1222, 2010. © 2010 Wiley-Liss, Inc.
- Avaliação do Impacte da Poluição Atmosférica na Saúde: Uma aplicação aos concelhos de Matosinhos, Maia, Valongo e LisboaPublication . Nicolau, Rita; Machado, AusendaEste estudo visou a estimação de efeitos na mortalidade e no internamento hospitalar diário ocasionados pela exposição de curto prazo a diversos poluentes atmosféricos, nomeadamente, partículas em suspensão na atmosfera com dimensão inferior a 10 μm (PM10), dióxido de azoto (NO2), dióxido de enxofre (SO2), ozono (O3) e monóxido de carbono (CO). O impacte de cada poluente sobre a mortalidade e sobre os internamentos foi avaliado em três concelhos da Área Metropolitana do Porto (Maia, Valongo e Matosinhos), e no concelho de Lisboa com base em dados relativos ao período 2000-2004 (mortalidade) e 2000-2007 (internamentos hospitalares). Metodologia: Os modelos identificados para cada concelho, procuraram investigar a possível associação existente entre a mortalidade diária, ou os internamentos hospitalares diários, e a concentração média diária de cada poluente, com controlo de aspectos temporais (tais como, o dia, ano, mês, semestre, feriados e fins de semana) e de efeitos ocasionados por variáveis de confundimento e/ou modificadoras (tais como, a temperatura atmosférica e períodos de actividade gripal sazonal). Para modelar esta associação utilizaram-se regressões de Poisson desenvolvidas a partir de Modelos Aditivos Generalizados (GAM). As contagens diárias de óbitos e de internamentos hospitalares (decorrentes de admissões às urgências) foram inicialmente agregadas por concelhos de residência dos indivíduos. A análise destes eventos foi estratificada pelos seguintes grupos de doença: i. todas as causas excepto causas externas, ii. doenças do aparelho circulatório, iii. doenças do aparelho respiratório. As estimativas do efeito de cada poluente foram expressas em termos do risco relativo (de morte ou de internamento hospitalar) atribuível a um incremento de 10 µg/m3 na concentração diária do poluente. Resultados: O presente estudo comprovou que os poluentes atmosféricos analisados (PM10, NO2, SO2, O3 e CO) tiveram efeitos estatisticamente significativos na mortalidade e no internamento hospitalar dos residentes nos concelhos estudados (Matosinhos, Maia, Valongo e Lisboa). Os efeitos identificados correspondem a acréscimos do número médio diário de óbitos ou de internamentos. Observou-se que os efeitos na saúde foram substancialmente maiores nas doenças dos aparelhos respiratório e circulatório do que no total de doenças. Em cada grupo de doença, verificou-se a existência de maiores riscos de internamento na população jovem (≤14 anos), riscos intermédios para os idosos (≥65 anos) e menores riscos para a totalidade da população. Demonstrou-se que os efeitos na mortalidade na população com 65 e mais anos foram superiores aos estimados para a população geral. A investigação não permitiu contudo comprovar a existência de efeitos de maior magnitude na mortalidade de jovens, devido ao reduzido número de óbitos neste grupo etário.
- New nutritional data on traditional foods for European food composition databasesPublication . Costa, H.S.; Vasilopoulou, E.; Trichopoulou, A.; Finglas, P.; Participants of EuroFIR Traditional Foods Work PackageBackground/Objectives: There are many different cultures within Europe, each with its own distinct dietary habits. Traditional foods are the key elements that differentiate the dietary patterns of each country. Unfortunately, in most countries, there is little information on the nutritional composition of such foods. Therefore, there is a need to study traditional foods to preserve these elements of European culture and, if possible, enrich and improve dietary habits across the continent. The Traditional Foods work package within the European Food Information Resource (EuroFIR) project aimed to provide new nutritional data on traditional foods for use in national food composition tables. Subjects/Methods: A EuroFIR consensus-based method with standardised procedures was applied for the systematic study of traditional foods and recipes in selected European countries. Traditional foods were selected on the basis of the EuroFIR definition of the term ‘traditional food’ and prioritised according to specific criteria. From the prioritised list, the five traditional foods per country to be investigated were selected to represent a full course meal. Protocols with guidelines for the recording of traditional recipes, the collection, preparation and distribution of laboratory samples, as well as quality requirements for laboratory selection, were developed to establish a common approach for use by all countries for the acquisition of reliable data. Results: The traditional character of the selected foods has been documented and traditional recipes have been recorded. Chemical analyses to determine the nutritional composition of 55 traditional foods were performed and the data were evaluated and fully documented according to EuroFIR standards. Information on food description, the recipe, component identification, sampling plan, sample handling, analytical method and performance was collected for each of the 55 investigated traditional foods. Conclusions: This common methodology for the systematic study of traditional foods will enable countries to further investigate their traditional foods and to continue to update their national food composition databases and EuroFIR’s food databank system.