Browsing by Issue Date, starting with "2019-03"
Now showing 1 - 10 of 56
Results Per Page
Sort Options
- Systematic investigation of environmental exposures in young adults with declining kidney function in a population at risk of Mesoamerican Nephropathy (MeN)Publication . Smpokou, E.T.; González-Quiroz, M.; Martins, Carla; Alvito, Paula; Le Blond, J.; Glaser, J.; Aragón, A.; Wesseling, C.; Nitsch, D.; Pearce, N.; Norman, J.; Lindh, C.; Morton, J.; Caplin, B.Objectives: There is an epidemic of Mesoamerican Nephropathy (MeN) in Central America, where sugarcane production is prominent. Numerous causes have been proposed, but to date limited evidence supports any one hypothesis. A nested case-control study using biosamples from a rural, community-based follow-up study of 350 young adults from Northwest Nicaragua at risk of MeN, was conducted with the aim of characterizing the associations between urinary concentrations of metals, pesticides and mycotoxins, and decline in kidney function. Methods: Urine samples collected at baseline (pre-sugarcane harvest) and at the first 6-month follow-up (post-sugarcane harvest) visit were tested. Twelve metals and metalloids (Al, total As, Cd, Co, Cr, Cu, Hg, Mn, Pb, Se, Si and Sr) were analysed by inductively-coupled plasma-mass spectrometry. Twelve pesticides or their metabolites (2,4-D, 3-PBA, 4F3PBA, CFCA, DCCA, ETU, glyphosate, MCPA, OH-PYM, 5-OH-TBZ, TEB-OH and TCP), and two mycotoxins (OTA and CIT), were analysed by liquid chromatography coupled to mass spectrometry. Differences in the creatinine-corrected urinary concentrations of the measured exposures between outcome groups (participants with stable versus declining kidney function over a 2-year follow-up period) were examined. Results: Elevated levels of aluminium and total arsenic as well as metabolites of several pesticides were detected across the population. No differences were identified between the declining and stable groups in the levels of metals or pesticides tested. OTA and CIT were below the limit of detection. Conclusions: These findings provide evidence against the tested metals, metalloids, pesticides and mycotoxins as the primary cause(s) of MeN in Nicaragua.
- Compliance of the PNAEQ Preanalytical scheme with the ISO 15189:2012Publication . Cardoso, Ana; Ventura, Catarina; Viegas, Silvia; Correia, Helena; Faria, Ana PaulaSince 2007 PNAEQ the Portuguese External Quality Assessment Program, has implemented the EQA program to evaluate the preanalytical phase PNAEQ established a consortium with Labquality and ECAT Foundation that allowed the evaluation of more items. Although the accreditation of clinical laboratories by ISO 15189 2012 is not mandatory in Portugal, the implementation of good practices is essential to ensure that the collection, handling and transport of samples ensures correct analytical results Laboratories must have tools to detect, monitor, reduce and eliminate errors in Preanalytical phase. The participation in these schemes provided by PNAEQ allows the evaluation and information of the direct impact on the sample quality, technician safety, analytical results and thereafter on the patient health, providing schemes that allow the compliance with the management and technical requirements of ISO 15189 2012. The aim of this work is to inform clinical laboratories that the participation on a EQA scheme on Preanalytical phase allows the compliance with the normative reference and evidence the good laboratory practices.
- Evaluation of Portuguese Community Health Projects and initiatives within the European and National Healthy Cities NetworkPublication . Rito, Ana; Cardoso, Rafael; Portugal, Inês; Baleia, Joana; Bica, MargaridaIntroduction: Following the World Health Organization European Healthy Cities Network, the Portuguese Healthy Cities Network was formally created by municipalities equally committed to promote equity, health and quality of life through local action. Objectives: To evaluate the health promotion strategies and initiatives implemented at municipality level in the Portuguese Healthy Cities Network and to confirm if these were in line with the requirements of the Health 2020 policy integrated on Phase VI of European Healthy Cities Network (2014-2018). Methodology: An exploratory-descriptive methodological design was used and a semi-structured questionnaire, developed by the World Health Organization Regional Office for Europe, was applied to the 29 municipalities of the Portuguese Healthy Cities Network (2013) invited to participate. 22 (75.8%) healthy cities met the criteria and were included. Results: Programmes on promotion of physical activity were the most frequently implemented across the Portuguese Healthy Cities Network (81.8%). All municipalities (100%) reported that children (>5 years) were the main targeted group of Portuguese Healthy Cities Network initiatives, followed by elderly (95.5%), adolescents (86.4%) and adults (86.4%). Low levels (27% – 32%) of initiatives that engaged other stakeholders, were reported as well as there was lack of research projects related to health matters and established partnerships by the scientific community. Overall, there was a perception of a positive impact of the Portuguese Healthy Cities Network programmes as 50% of the municipalities reported a remarkable improvement in health and quality of life of the population. Conclusions: Although life-course initiatives addressing the major burden of diseases were implemented, a more comprehensive approach is needed to follow Health 2020 principles. Development and reinforcement of the Portuguese Healthy Cities Network programmes is still a challenge. It should cover different population groups in order to tackle social inequalities and it also demands new partnerships, new forms of communication, as well as monitoring and evaluation mechanisms in place.
- Paediatric cerebral vasculopathy in sickle cell anaemia: contribution of genetic modifiersPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rita; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle cell anaemia (SCA) arises from homozygosity for the mutation c.20A>T in the HBB gene. However, it shows a multifactorial-like behaviour with high heterogeneity of clinical features. Cerebral vasculopathy (CVA), namely paediatric ischemic stroke, is one of its most devastating consequences. The risk of CVA development, specifically stroke or silent cerebral infarction, may be modulated by underlying genetic modifiers, for example those affecting vascular homeostasis. In this study, we aimed to investigate the impact of variants in genes related with endothelial adhesion (VCAM1 and ITGA4) and nitric oxide metabolism (NOS3) on CVA in a group of 70 SCA children well characterized according to their CVA degree. In addition, the effect of the same genetic variants on biochemical/haematological biomarkers of chronic haemolysis was also analysed. Moreover, we also evaluated the putative additional modulating role of variants previously identified as stroke risk factors by genome-wide associated studies: GOLGB1 Y1212C, ENPP1 K173Q and PON1 Q192R. Molecular analysis was performed using PCR, PCR-RFLP, next-generation sequencing and Sanger sequencing. SPSS software was used for statistical analyses and association studies. One of the seven VCAM1 promoter haplotypes found and the VCAM1 promoter rs1409419_T showed association with moderate to high time-averaged mean of maximum velocity in the middle cerebral artery. The same association was observed for the variant ENPP1 K173Q. On the other hand, we observed that ITGA4 variants rs113276800_A and rs3770138_T were associated with stroke events. As for NOS3, one of the six haplotypes and the intron 4 VNTR_4b allele (5 repeats) were associated with lower risk of silent cerebral infarction. Chronic haemolysis biomarker levels also seemed to be influenced by genetic variants. LDH levels was higher in the presence of VCAM1 promoter rs1409419_T and one VCAM1 haplotype but lower in patients with one of the two ITGA4 haplotypes found. Genetic modulation also occurred in total bilirubin levels, which were higher in association with VCAM1 rs3783613_C allele. Our results, namely the association between specific variants with certain cerebral vasculopathy predictors further enhances their putative modulating effect on SCA paediatric stroke risk, severity and prognosis. These findings provide additional clues on the SCA pathophysiology and uncover features in these genes that may prove to be crucial as potential therapeutic targets.
- The use of a modified U1 snRNA as a therapeutic strategy to correct a 5’ splice-site mutation in Mucopolysaccharidosis IIIC: in vitro steps towards an in vivo approachPublication . Santos, J.I.; Matos, L.; Rocha, M.; Coutinho, M.F.; Prata, M.J.; Alves, S.Genetic therapy directed towards the correction of RNA missplicing is being investigated not only at basic research level but even in late-stage clinical trials. Many mutations that change the normal splicing pattern and lead to aberrant mRNA production have been identified in Lysosomal Storage Disorders (LSDs). The Mucopolysaccharidosis IIIC (MPS IIIC) is a LSD caused by mutations in the HGSNAT gene, encoding an enzyme involved in heparan sulphate degradation. Splicing mutations represent one of the most frequent (~20%) genetic defects in MPS IIIC. Approximately 55% corresponds to 5' splice-site mutations which thus constitute a good target for mutation specific therapeutic approaches. Recently, we demonstrated in fibroblast cells that a modified U1snRNA vector designed to improve the definition of exon 2 5’ss of the HGSNAT can restore splicing impaired by the mutation c.234+1G>A.(Matos et al., 2014). Presently our goal is to evaluate in vivo the therapeutic potential of the modified U1snRNA by testing it in mice expressing the human splicing defect. For this, in a first step we tried to generate full-length splicing competent constructs of wild-type (wt) and c.234+1G>A HGSNAT by cloning the wt or the mutated HGSNAT splicing-competent cassettes into the pcDNA 3.1 backbone. According to the protocol reported by other researchers (Pinotti et al., 2009), plasmid vectors will be used to promote transient expression of the human HGSNAT wt or mutant alleles in mice. Here, we describe the cloning process followed to obtain the aforementioned splicing constructs. During the cloning steps different difficulties were found as, for example, in fragments amplification, ligation, and obtainment of bacterial transformants. Even so, positive bacterial colonies were obtained, selected, and amplified by colony PCR. However, DNA sequencing data showed the presence of different nucleotide point alterations in the obtained clones, invalidating its use for further steps. Therefore, plasmid constructs were ordered commercially. Now we are performing its transfection in Hep3B/COS-7 cells to confirm that they recapitulate the splicing process observed in wt and patient cDNA being thus ready to be expressed in mice to test the therapeutic effect of the modified U1snRNA. This work shows the different steps and difficulties of the cloning process to obtain HGSNAT expression constructs towards testing of an in vivo U1snRNA therapeutic approach.
- Follow-up of fatty acid β-oxidation disorders in expanded newborn screening eraPublication . Janeiro, Patrícia; Jotta, Rita; Ramos, Ruben; Florindo, Cristina; Ventura, Fátima V.; Vilarinho, Laura; Tavares de Almeida, Isabel; Gaspar, AnaFatty acid β-oxidation (FAO) disorders have a wide variety of symptoms, not usually evident between episodes of acute decompensations. Cardiac involvement is frequent, and severe ventricular arrhythmias are suspected of causing sudden death. Expanded newborn screening (ENS) for these disorders, hopefully, contribute to prevent potentially acute life-threatening events. In order to characterize acute decompensations observed in FAO-deficient cases identified by ENS, a retrospective analysis was performed, covering a period of 9 years. Demographic data, number/type of acute decompensations, treatment, and follow-up were considered. Eighty-three clinical charts, including 66 medium-chain acyl-CoA dehydrogenase deficiency (MCADD), 5 carnitine-uptake deficiency (CUD), 3 carnitine palmitoyltransferase I and II (CPT I/II) deficiency, 5 very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), and 4 multiple acyl-CoA dehydrogenase deficiency (MADD) cases were reviewed. Nineteen patients had acute decompensations (1 CPT I, 1 CPT II, 3 MADD, 14 MCADD). Six patients developed symptoms previously to ENS diagnosis. Severe clinical manifestations included multiple organ failure, liver failure, heart failure, and sudden death. Long-chain FAO disorders had the highest number of decompensations per patient. Conclusion: Despite earlier diagnosis by ENS, sudden deaths were not avoided and acute decompensations with severe clinical manifestations still occur as well.
- Evaluation of a gene-by-gene approach for prospective whole-genome sequencing-based surveillance of multidrug resistant Mycobacterium tuberculosisPublication . Macedo, Rita; Pinto, Miguel; Borges, Vítor; Nunes, Alexandra; Oliveira, Olena; Portugal, Isabel; Duarte, Raquel; Gomes, João PauloWhole-genome sequencing (WGS) offers unprecedented resolution for tracking Mycobacterium tuberculosis transmission and antibiotic-resistance spread. Still, the establishment of standardized WGS-based pipelines and the definition of epidemiological clusters based on genetic relatedness are under discussion. We aimed to implement a dynamic gene-by-gene approach, fully relying on freely available software, for prospective WGS-based tuberculosis surveillance, demonstrating its application for detecting transmission chains by retrospectively analysing all M/XDR strains isolated in 2013-2017 in Portugal. We observed a good correlation between genetic relatedness and epidemiological links, with strongly epilinked clusters displaying mean pairwise allele differences (AD) always below 0.3% (ratio of mean AD over the total number of shared loci between same-cluster strains). This data parallels the genetic distances acquired by the core-SNV analysis, while providing higher resolution and epidemiological concordance than MIRU-VNTR genotyping. The dynamic analysis of strain sub-sets (i.e., increasing the number of shared loci within each sub-set) also strengthens the confidence in detecting epilinked clusters. This gene-by-gene strategy also offers several practical benefits (e.g., reliance on freely-available software, scalability and low computational requirements) that further consolidated its suitability for a timely and robust prospective WGS-based laboratory surveillance of M/XDR-TB cases.
- Transcriptomics profiling of Niemann-Pick type C patients: activation of the unfold protein response in a specific casePublication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo-Min; Cardoso, Maria Teresa; Chaves, Paulo; Gaspar, Paulo; Santos, Juliana Inês; Ribeiro, Isaura; Quelhas, Dulce; Lacerda, Lúcia; Leão-Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, SandraBackground: Niemann-Pick type C (NP-C) is a neurodegenerative Lysosomal Storage Disease (LSD) with a heterogeneous clinical presentation secondary to abnormal intracellular accumulation of cholesterol. We have studied a patient with clinical diagnosis of NP-C but presenting inconclusive results regarding biomarkers testing and molecular analysis. To better characterize this patient, we have performed NGS-based technologies (targeted DNA sequencing and single cell-RNA sequencing). Methods: For the molecular diagnosis we used a NGS gene panel followed by the analysis of cDNA (in the patient and both parents). Latter, we have used massively parallel single cell RNA-seq (MARS-Seq) to address gene profiling changes and better characterize the pathomechanisms related to specific disease-causing mutations in this patient as well as in two NPC patients. The most prominent hits from this transcriptomics analysis were validated by qRT-PCR. Results and Discussion: Using our targeted NGS panel we identified two novel mutations in NPC1 gene (p.V505G; p.V562V). Next, through cDNA analysis of one of the patient parents we were able to understand the impact of the V562V silent mutation located in the middle of the exon 11. This mutation leads to exon 11 skipping giving origin to an out-of-frame transcript and eliciting the nonsense-mediated decay pathway. This mechanism contributed to the almost absence of the mutant transcript in the patient. Thus, we were not able to easily detect it in the sequencing electropherogram of the patient which turned the molecular diagnosis more challenging. By its turns, apparently the presence of the other mutation (the missense V505G) impairs the proper NPC protein folding leading to its ER retention. In fact, the MARS-Seq analysis of this patient showed that a number of genes were upregulated and a significant number of the highly enriched genes are related to the unfold protein response (UPR).
- Molecular identification of clinical and environmental avian Aspergillus isolatesPublication . Sabino, Raquel; Burco, Julia; Valente, Joana; Veríssimo, Cristina; Clemons, Karl V.; Stevens, David A.; Tell, Lisa A.Aspergillosis causes high morbidity and mortality in avian species. The main goal of this study was to use molecular techniques to identify Aspergillus species collected from different avian species with aspergillosis. A subsample of those isolates was also screened for resistance to itraconazole. Over a 2-year period, clinical samples were recovered from 44 birds with clinical signs of the disease, clinical pathology results suspicious of aspergillosis, or from birds that died from Aspergillus spp. infection. Environmental sampling was also performed in seabird rehabilitation centers and natural seabird environments. Seventy-seven isolates (43 clinical and 34 environmental) were identified as Aspergillus fumigatus sensu stricto. No cryptic species from the Fumigati section were detected. Two environmental isolates were identified as Aspergillus nidulans var. dentatus and Aspergillus spinulosporus. None of the Aspergillus isolates tested were resistant to itraconazole. Our study emphasizes the dominant association of Aspergillus fumigatus sensu stricto in avian mycoses and shows the lack of itraconazole resistance in the studied isolates.
- Impact of cooking methods and malting on amino acids content in amaranth, buckwheat and quinoaPublication . Motta, Carla; Castanheira, Isabel; Bryan Gonzalesb, Gerard; Delgado, Inês; Torres, Duarte; Santos, Mariana; Matos, Ana SofiaThis study reports the effect of boiling, steaming and malting on the amino acid composition of the pseudocereals amaranth, buckwheat and quinoa. For all pseudocereals the foremost amino acid was glutamic acid, presenting 13.2% in both raw and malted (2.2 g/100 g), and 15.6% in steamed (2.5 g/100 g) amaranth; 10.8% in steamed (2.0 g/100 g) and 17.6% in boiled (2.1 mg/100 g) quinoa; 15.4% in malted (2.3 g/100 g) and 21.2% in raw (2.8 g/100 g) buckwheat. Almost all amino acids present in the three pseudocereals evinced a significant increase of the retention values in malted samples, except in amaranth and quinoa for cysteine and glutamic acid, respectively. Histidine and aromatic amino acids presented the highest values of amino acid scores. Cluster analysis allowed to identify the pseudocereals with the highest nutritional protein quality, were boiled and malted quinoa and raw and malted buckwheat were included. Malting process revealed to be the method that produce more effect on the amino acid content for all pseudocereals.