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- Normalization strategies for real-time expression data in Chlamydia trachomatisPublication . Borges, V.; Ferreira, R.; Noguerira, P.; Nunes, A.; Borrego, M.J.; Gomes, João PauloSince Chlamydia trachomatis is a genetically non-tractable pathogen, transcriptomics assumes a fundamental role for the better understanding of its biology. However, the suitability of endogenous controls for normalization of transcriptomic data in this bacterium still needs validation. We aimed to assess the stability of 10 genes for their potential use as endogenous controls in qPCR at both normal and stress (antibiotic treatment) growth conditions throughout the developmental cycle of three strains with different cell-appetence. Normalization was performed using the quantified bacterial genomes. We also tested the applicability of two widely used softwares (geNorm and Normfinder) to our data. For all strains, we found that 16SrRNA was the most stably expressed gene throughout the normal developmental cycle, but it was highly unstable under antibiotic exposure, suggesting prudence when using ribosomal genes as endogenous controls in expression experiments involving stress environments. The geNorm and Normfinder algorithms revealed contrasting results and seem inappropriate for the selected pool of genes. Considering the multiplicity of experimental conditions, there should be an in loco validation of endogenous controls, where 16SrRNA appears to be in the front line. Alternatively, normalization of expression data against genomic DNA, which is less influenced by experimental constraints (especially relevant for intracellular organisms) and stress conditions, likely constitutes a good option. The present study constitutes the first evaluation of putative endogenous controls for real-time expression assays in C. trachomatis
- Era uma vez…os super-micróbios voadoresPublication . Cano, M.
- BaSeFood Newsletter, Issue 4Publication . Sanches-Silva, A.; Albuquerque, T.G.; Costa, H.S.
- Molecular characterization of human T cell leukemia virus type 1 subtypes in a group of infected individuals diagnosed in Portugal and SpainPublication . Pádua, E.; Rodés, B.; Pérez-Piñar, T.; Silva, A.F.; Jiménez, V.; Ferreira, F.; Toro, C.Over the past decade, Portugal and Spain received large numbers of immigrants from HTLV-1 endemic areas. Our aim was to investigate the diversity of subtypes circulating in these two countries and the introduction of new variants. We performed a molecular analysis of HTLV-1 strains in patients diagnosed since 1998. LTR and env proviral sequences from 26 individuals were analyzed to generate phylogenetic trees along with reference HTLV-1 subtypes from several geographic origins. Epidemiological and clinical data were recorded. Most subjects were immigrants (57.7%) from South America and Africa. All isolates belonged to the cosmopolitan A subtype. Most carried the transcontinental subgroup A, but five subjects carried subgroup D and one carried subgroup C, previously unreported in Europe. HTLV strains showed separate clusters linked to the patients' geographic origin. Although subjects with HTLV-1 infection tend not to be engaged in high-risk practices, silent dissemination of a broad diversity of HTLV-1 viruses may still occur.
- Supramolecular organizations in the aerobic respiratory chain of Escherichia coliPublication . Sousa, Pedro M.F; Silva, Sara T.N.; Hood, Brian L.; Charro, Nuno; Carita, João N.; Vaz, Fátima; Penque, Deborah; Conrads, Thomas P.; Melo, Ana M.P.The organization of respiratory chain complexes in supercomplexes has been shown in the mitochondria of several eukaryotes and in the cell membranes of some bacteria. These supercomplexes are suggested to be important for oxidative phosphorylation efficiency and to prevent the formation of reactive oxygen species. Here we describe, for the first time, the identification of supramolecular organizations in the aerobic respiratory chain of Escherichia coli, including a trimer of succinate dehydrogenase. Furthermore, two heterooligomerizations have been shown: one resulting from the association of the NADH:quinone oxidoreductases NDH-1 and NDH-2, and another composed by the cytochrome bo3 quinol:oxygen reductase, cytochrome bd quinol:oxygen reductase and formate dehydrogenase (fdo). These results are supported by blue native-electrophoresis, mass spectrometry and kinetic data of wild type and mutant E . coli strains.
- Trihalomethanes in Lisbon Indoor Swimming Pools: Occurrence and Determining FactorsPublication . Silva, Z.; Rebelo, H.; Silva, M.M.; Alves, A.; Cabral, C.; Almeida, A.C.; Aguiar, F.; Oliveira, A.; Nogueira, A.; Pinhal, H.; Matos, A.; Ramos, C.D.; Pacheco, P.; Aguiar, P.; Cardoso, A.S.The presence of water disinfection by-products (DBPs) in swimming pools constitutes today a public health concern, particularly because swimming is an activity used by a high percentage of the population, namely elderly and young children. Moreover, several adverse short-term and long-term health effects have been associated with these compounds (Lakind et al., 2010; Zwiener et al., 2007). Water disinfection methods are used in all swimming pools, namely in public pools, to ensure an adequate and effective protection of users against microbiological pathogens. Chlorination is the most common disinfection method used worldwide, because it is low cost, easy to use, efficient against a broad spectrum of microorganisms, and enables the maintenance of a residual protection. The use of chlorine based treatment techniques has one strong drawback, which is the generation of several DBPs, potentially harmful products, that can be absorbed by ingestion, inhalation and absorption through the skin (Nieuwenhuijsen et al., 2009; Caro and Gallego, 2007). DBPs comprise several compounds that are formed through the reaction of chlorine with organic matter present in water. Amongst DBPs, the most relevant and better characterized are trihalomethanes (THMs): chloroform (CF), bromoform (BF), bromodichloromethane (BDCM), and chlorodibromomethane (DBCM). These compounds have been associated with health effects such as respiratory, ocular and cutaneous symptoms and also with some long-term health effects such as bladder cancer and adverse reproductive outcomes (Lee et al., 2009; Zwiener et al., 2007). Accurate exposure assessments to THMs in indoor pool environment is particularly difficult because their formation depends on many factors such as water and air temperature (Tw and Tair), humidity (Hu), pH, free residual chlorine (FrCl), total organic content and number of pool users (Lee et al., 2009). In Portugal a specific legislation to assess the quality of swimming pool waters is inexistent. The guideline value used in this study for total THMs (TTHMs) was the one established in the Portuguese Law 306/2007, for drinking water quality - 100 μg/L. Some other water parameters determined in swimming pools such as Tw turbidity (Turb), pH, FrCl, total residual chlorine, conductivity (Cond), permanganate index (COD) and isocyanuric acid were assessed by Portuguese Law 5/97, regarding technical and safety conditions of closed environments with water diversions. WHO guidelines for safe recreational water environments (2006) were also used to assess some water and air parameters in pools. Standard 62.1 (2006) from American Society of Heating, Refrigerating, and Air-Conditioning Engineers (ASRHAE) was also used to assess pool air parameters. The main goal of this investigation project was to study the occurrence, distribution and determinants of THMs in indoor swimming pools. This will enable an evaluation of swimming pool users’ exposure to THMs. In order to achieve this goal, the characterization of water quality of 30 Lisbon indoor swimming pools, using chorine based treatment techniques, was made during a six month period. Because of laboratory working restrictions, CF concentration in pool air was studied only in 6 swimming pools, at the same period. Several parameters such as TTHMs concentration, CF, BDCM, DBCM, BF concentration in water, FrCl, combined residual chlorine (CrCl), pH, Tw and Tair, Hu, COD, Cond, Turb and chloride (Cl) were determined in each pool, once a month. THMs water sampling was made in duplicates in two pool water spots, physicochemical parameters were determined in one of the THMs water sampling spots and THMs air sampling was made in two pool spots, considered to be representative of the swimming pool air. All sampling procedures were performed by specialized sampling technicians from Lisbon Health Centres Group (ACES). Statistical analysis was performed using the SPSS 17.0 software. Descriptive analysis was applied to all variables in order to determine mean, median, standard deviation, minimum and maximum. Student’s t-test and Mann-Whitney tests were used to compare numerical variables and Spearman’s rank correlation coefficient were used to examine correlation between all environmental parameters. TTHMs water level ranged from 10 to 160 μg/L, while CF water level typically ranged from 5 to 150 μg/L, with occasional higher levels detected. Regarding other THMs levels in pool water, BDCM ranged from 0.5 to 15 μg/L, DBCM from 0.4 to 10 μg/L and BF was obtained in concentrations less than 2 μg/L. Moreover, in some pools, DBCM and BF were not detected. From these results, it can be concluded that there was a large variation in TTHMs and CF water levels between the pools and that CF was the THM obtained in higher concentrations. These results are in agreement with studies presented by other authors (Lee et al., 2009; Chu and Nieuwenhuijsen, 2002). In air, CF level ranged from 40 to 200 μg/m3, with occasional higher levels detected. Tw presented less variation (27 to 31 ºC) than Tair (21 to 33 ºC), although sporadic higher values were found. FrCl and Cond presented higher values than the guidelines from DR 5/97 only in a small number of pools. Turb and COD values were within the guidelines from DR 5/97. pH varied from 7 to 8.5, with only a small number of pools having values higher than 7.8. As referred in WHO guidelines, pH should be maintained between 7.2 and 7.8 for chlorine disinfectants to ensure efficient disinfection. Using the statistical tests mentioned before, it was possible to obtain some interesting results. There was a clear positive linear correlation between CF water concentration (CFW) and TTHMs water concentration (R>0.98, p<0.01), good correlation other THMs, there was a clear positive linear correlation between BDCM and DBCM water concentration (R>0.78, p<0.01) and good correlation between TTHMs water concentration and Tw (R>0.45, p<0.05). These results are in agreement with Lee et al. (2009) which obtained positive linear correlations between CFW and COD and with Chu and Nieuwenhuijsen (2002) which also obtained correlations between TTHMs water concentration and Tw. In conclusion, there appears to be good water and air quality in the studied Lisbon swimming pools. Regarding THMs levels in water, some pools presented high TTHMs and CF concentrations. Moreover, CF air concentrations were also high in some swimming pools. These results clearly demonstrate that THMs monitoring is particularly important and that, in a near future, this should be extended to other DBPs. Furthermore, for a correct and conscious use of swimming pools, appropriate and targeted information about safe practices should be provided to pool users.
- Molecular Assessment of X-Linked Intellectual Disability: A Gene Cluster Based ApproachPublication . Oliveira, Bárbara; Jorge, Paula; Criado, Maria BegoñaIntellectual disability (ID) is a common form of cognitive impairment, responsible by a significant number of limitations in intellectual function and adaptive behavior. X-linked intellectual disability represents a common cause of monogenic mental retardation affecting mostly males. Among the genetic causes involved, mutations in FMR1, AFF2 and ARX genes emerge as the major causes. FMR1 and AFF2 genes contain polymorphic repetitive regions susceptible to suffer dynamic mutations, a process that may give rise to pathogenic expansions. In ARX gene, the second exon represents a mutational hot spot as holds repetitive regions that codify to alanines. Among the mutations described, variations in polyalanine tracts, namely the c.429_452dup24, are among the most frequent. Aiming the characterization of a population at risk for ID, a multiplex molecular screening was performed regarding mutational hotspots in FMR1, AFF2 and ARX genes in intellectually-disabled individuals and in a control population. A Triplet-primed PCR technique was optimized to evaluate [AGG] interspersion pattern in FMR1 repetitive regions to assess allele stability. Furthermore, ARX exon 2 size variations were characterized by sequencing analysis. Allelic frequencies of FMR1 and AFF2 genes in the Portuguese population are similar to other Caucasian populations. The present work shows that the increase of triplet content in FMR1 gene is independent of variations in AFF2 triplet content (Pearson’s R: ρ >0,05), although, the occurrence of 30 repeat-sized alleles (for FMR1) and 14 repeat-sized alleles (for AFF2) is the most frequent combination (13,4%) (X2: ρ<0,05). FMR1 alleles with the first interruption after nine or ten [CGG] triplets demonstrated to have different origins (X2: ρ <0,05; Pearson’s R: ρ <0,05). A strong correlation among the 29 repeat-sized alleles and the CTC SNP haplotype and between the 30 repeat-sized alleles and the TTT lineage (X2: ρ <0,05) was observed. In ARX analysis the c.429_452dup24 mutation was identified in three individuals from two different families and the polymorphic variant c.441_464del24 in other three patients. Small insertions codifying for alanines were also detected (c.304GCG[11] and c.304GCG[13]) with a pathogenic effect yet to be determined. The importance of studying FMR1 alleles in terms of [AGG] interruption and genetic background is evident for an accurate clinical diagnosis. ARX mutations of known pathogenicity, although few frequent, are a strong indication that this genes should be included in routine screening of ID.
- Transcriptomic analysis of plasmid and plasmid-related chromosomal ORFs in C. trachomatis strains with different cell-appetencePublication . Ferreira, R.; Borges, V.; Nunes, A.; Borrego, M.J.; Gomes, João PauloDespite the undergoing chromosomal size-reduction of Chlamydia trachomatis, almost all strains maintain the conserved 7,5kb plasmid. It has been recently considered a virulence factor, as plasmid-bearing strains evidenced a higher ability to successfully colonize epithelial cells and sustain infection than plasmidless strains. However, the biological role of the eight plasmid ORFs remains poorly characterized. Thus, we aim to correlate the relative expression of each plasmid ORFs with the number of plasmids per genome as well as with the relative expression of chromosomal genes that seem to be regulated by the plasmid, during the whole Chlamydia developmental cycle. Both the transcriptomic analysis of all the selected ORFs and the determination of the plasmid copy number were performed by qPCR in prototype and currently circulating strains with distinct cell-appetence and ecological success, throughout Chlamydia developmental cycle. So far, our results indicate: 1) huge differences (up to 100-fold) in the expression levels between plasmid ORFs; 2) for the same ORF, different expression levels and profiles among strains; 3) variation in the number of plasmids per genome during the infectious cycle; 4) the higher expression level of the plasmid ORFs do not seem to correlate with a higher number of plasmids per genome. These results, together with our ongoing transcriptomic survey of the plasmid-related chromosomal genes, will contribute to shed some light on the molecular function of the plasmid ORFs in chlamydial biology.
- Molecular features underlying the higher ecological success of C. trachomatis E and F genotypesPublication . Nunes, A.; Ferreira, R.; Borges, V.; Borrego, M.J.; Gomes, João PauloIn the light of the >98% genomic similarity among Chlamydia trachomatis serovars, the higher worldwide ecological success of E and F is enigmatic. We intend to provide a quick overview of the molecular data that distinguish these from the remaining strains. Examples are: - E and F possess a similar chromosomal genetic make-up distinct from the remaining genotypes. Some loci linked to this independent co-segregation comprehend membrane proteins, hypothetical virulence factors, and regulatory regions (published data). - Some loci reveal nonrandom mutational patterns, where mutations exclusive of E and F are clustered in specific protein domains, likely promoting strains functional and/or structural attributes (published data). - Based on data from a worldwide survey, MOMP of E and F exhibit the lowest mutation rate (22.3-fold lower), implying more fitted antigenic profiles to deal with host immunity (published data). - The likelihood of E and F strains to undergo genetic recombination is about 12-fold lower than that of the other genotypes (P<10-2), suggesting a putative clonal evolution, where superimposed favorable clones may be strongly maintained in vivo (preliminary data from our lab). - Strains E and F do not seem to originate higher infectious load in vivo, when compared with other genital genotypes (published data). Full-genomic data from multiple and diverse clinical isolates will be essential to decipher the secret behind the higher ecological success of E and F strains.
- Meat handlers training in Portugal: A survey on knowledge and practicePublication . Gomes-Neves, Eduarda; Cardoso, Carla Sofia; Araújo, Ana Cristina; Costa, José Manuel Correia daProfessional training for meat handlers is an European Community food law requirement in order to apply HACCP principles and achieve food safety goals. A self-administered questionnaire designed to assess “Knowledge” and “Practice” of public hygiene measures was completed by meat handlers (MH) (n = 159) in slaughterhouses in Portugal. A significant proportion of the group (72.7%) has had professional training in two different areas: Good Practice in Food Industry (12.03%) and Work Safety and Hygiene (22.8%); 37.9% of the respondents have had training in both areas. However 24.5% of the subjects have never had training. Meat handlers with professional training in Good Practice in Food Industry (GPFI) and in both areas (BT) have had the highest proportions of correct answers in Knowledge (66.92 ± 16.36 and 67.26 ± 21.05, respectively) and Practice questions (70.53 ± 17.47 and 68.67 ± 22.58, respectively). The results of this study point to the need to improve training, particularly in Good Practice in Food Industry, thus enabling meat handlers to achieve more correct answers in Knowledge and Practice. The development of evaluation criteria for the effectiveness of professional training is crucial to protect Public Health.
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