Repositório Científico do Instituto Nacional de Saúde
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Unravelling the genetic basis of complex clinical cases of hemoglobinopathies
Publication . Silva, Eduarda; Pena, Rita; Miranda, Armandina; Costa, Alcina; Gaspar, Gisela; Lopes, Pedro; Faustino, Paula
Hemoglobinopathies encompass all genetic diseases of hemoglobin (Hb), the iron-containing oxygen-transport protein present in red blood cells. They occur due to mutations in globin genes or in their regulatory regions, and are classified as Hb variants and thalassemias. The aim of this work was to identify the molecular lesions in the origin of complex cases of hemoglobinopathies and understand the underlying pathophysiological mechanisms.
We investigated 15 clinical cases suspected of having one or more hemoglobinopathy, presenting with atypical hematological phenotypes. The study included the search for alterations in beta- and alpha-globin gene clusters by PCR, Gap-PCR, Sanger sequencing, and Multiplex Ligation-dependent Probe Amplification. In silico analyses were performed using Polyphen-2, SIFT, and varSeak.
Two beta-thalassemia carriers with abnormally low HbA2 level were found to have double heterozygosity for a mutation in HBB gene (c.92+1G>A, c.92+6T>C) and a delta-chain Hb variant (Hb A2-Yialousa). Another case was justified by a novel large deletion, which removes the entire beta-globin gene cluster as well as the olfactory receptor genes, OR52A1 and OR51V1. Changes in HbA2 values were also justified by a deletion that eliminates the HBD (Corfu deletion) or by the presence of the HbA2´variant. Atypically high levels of fetal Hb were explained by alterations in promoters of HBG genes (HBG1:c.-248C>G, HBG1:c.-228T>C, HBG2:c.-211C>T) or by deletions that remove both HBD and HBB (HPFH-1, HPFH-2). An even more complex case was originated by triple heterozygosity involving the Southeast Asian alpha-thalassemia deletion, the alpha-chain variant Hb Westmead, and the beta-chain variant HbE. As far as we know, this is the first case in which the three alterations were found in the same individual.
Individuals presenting abnormal phenotypes due to more than one hemoglobinopathy may be misdiagnosed if not correctly studied. Unravelling the genetic basis of complex clinical cases allows a better referral to genetic counselling, improves the understanding of the pathophysiology of the disease and its modifying factors, and may reveal new therapeutic targets.
Systematic review on toxicological effects of platinum nanoparticles: towards their use as safe biomedical tools
Publication . Touzani, Assia; Ramos-Pan, Lucía; Fraga, Sónia; Fernández-Bertólez, Natalia; Laffon, Blanca; Valdiglesias, Vanessa
Platinum nanoparticles (PtNP) have received considerable attention in the nanomedicine field due to their magnetic, catalytic, and optical properties. However, the potential toxicity of PtNP has not been properly evaluated yet, and current information on the possible risks related to their use is still limited. On this basis, the main objective of this systematic review was to gather available data on PtNP biological behaviour and potential harmful effects, as well as to highlight the gaps of knowledge that need to be filled in to progress in their use in clinical practice. A total of 441 studies were obtained and reviewed from the initial search; 108 fulfilled the selection criteria and were included in the revision. Mainly in vitro but also in vivo studies were reported using a variety of biological systems and animal models, with no data from human epidemiological studies published so far. All these studies were extensively evaluated to provide useful information on the PtNP biocompatibility and their potential to be employed for medical purposes. In particular, information on the physicochemical features of the PtNP influencing their biological behaviour, methods employed for toxicity evaluation, biological systems used, and outcomes addressed were analysed and discussed. In general, the results obtained showed a good biocompatibility of these NP, although some of them detected significant toxicity highly dependent of size, concentration/dose, coating, or exposed biological system. Furthermore, anticancer or protective effects were also described for PtNP in several revised studies. These findings encourage to continue exploring the benefits of PtNP for clinical practice.
Application of a real-time reverse transcription polymerase chain reaction for rapid detection of Escherichia coli in drinking water: an EU representative study
Publication . Gómez, Livia; Brandão, João; Navarro, Anna; Martines, Antonino; Sanseverino, Isabella; Porcel-Rodríguez, Elena; Marinov, Dimitar; Ableitner, Oksana; Allerberger, Franz; Atsma, Adrie; Briancesco, Rossella; Cenov, Arijana; Cíchová, Marianna; Coccia, Anna Maria; Libera, Simonetta Della; De Maeyer, Katrien; De Vries, Hendrik Jan; Elsinga, Goffe; Heijnen, Leo; Hokajärvi, Anna-Maria; Janská, Veronika; Hrabar, Jerko; Huse-Kutowska, Monika; Jozic, Slaven; Klatovsky, Stefanie; Kornschober, Christian; Kohls, Katharina; Krom, Adrie; Luden, Katrin; Mikula-Pratschke, Claudia; Pitkänen, Tarja; Poje, Mateja; Prokšová, Miloslava; Retelj, Matjaz; Rozwadowska, Beata; Rytkönen, Annastiina; Sarekoski, Anniina; Schlager, Sabine; Springer, Burkhard; Stojan, Iva; Stroobach, Eline; Šućić, Neven; Valério, Elisabete; Van Pelt, Gabi; Vranken, Jerom; Vrdoljak Tomaš, Ana; Vukić Lušić, Darija; Włos, Ewa; Wubbels, Gerhard; Ziombska, Maria; Zelenik, Katja; Ziuziakowski, Marcin; Lettieri, Teresa
The microbiological quality of water intended for human consumption in the EU is regulated by the recast Drinking Water Directive 2020/2184 (DWD), which sets strict parametric values for intestinal enterococci and Escherichia coli (E. coli), allowing no more than zero colony-forming units per 100 mL. Detection and enumeration of E. coli typically rely on culture-based reference methods or the most probable number approach, which require 1–2 days to produce results—potentially delaying timely action during contamination events. In contrast, molecular techniques can deliver results within hours. The DWD permits the use of alternative methods if they are as reliable as the reference method and developing and validating such methods requires multiple laboratory trials in line with ISO standard 16140-2. Following this, we conducted a representative EU study to validate a molecular method based on real-time reverse transcription polymerase chain reaction for rapid E. coli detection in drinking water. In a concerted action, the first of its kind for drinking water, nineteen laboratories across ten Member States participated. To ensure consistency, drinking water was artificially contaminated with E. coli. The alternative method showed slightly lower sensitivity than the reference method (91.1 % vs. 97.2 %) but delivered much faster results, making it a valuable screening tool. It can support rapid decision-making during contamination events, reducing the risk of waterborne outbreaks and protecting public health. For reliable routine performance, appropriate training in this alternative method is strongly recommended.
Anti-corrosion and cytotoxicity properties of inorganic surface treatments on Mg1Ca biodegradable alloy
Publication . Neves, C.S.; Sousa, I.; Freitas, M.A.; Moreira, L.; Costa, C.; Teixeira, J.P.; Fraga, S.; Silva, R.M.; Silva, R.F.; Starykevich, M.; Scharnagl, N.; Zheludkevich, M.L.; Ferreira, M.G.S.; Tedim, J.
In this work biodegradable Mg1Ca alloy underwent surface modification using hydroxyapatite (HAp), aluminium oxide (Al2O3), and treatments with phosphoric (H3PO4), hydrofluoric (HF), and acetic (CH3COOH) acids. The resulting surface-treated Mg substrates were assessed in terms of phase content and chemical composition through X-ray diffraction (XRD) and glow discharge optical emission spectrometry (GDOES). Additionally, atomic force microscopy (AFM) and scanning electron microscopy (SEM) were employed to examine the surface's topography and structure, while the corrosion behavior and cytotoxicity were surveyed using electrochemical impedance spectroscopy (EIS), alongside WST-1 reduction and lactate dehydrogenase (LDH) release assays on L929 mouse fibroblasts. The findings indicated that the surfaces of all samples were uniformly structured, while chemical analysis of the treated surfaces suggested the presence of mostly thin films. Furthermore, EIS results highlighted that the HAp-treated Mg1Ca alloy exhibited superior corrosion resistance, and the cytotoxicity assessment of Mg1Ca-HAp and Mg1Ca-H3PO4 alloys showed minimal cytotoxic effects on mouse fibroblasts, compared to other treated surfaces, suggesting enhanced biocompatibility of those two surface treatments. Overall, this constitutes the first comparative study of different surface treatments developed on biodegradable Mg1Ca alloy, aiming to identify optimal modification strategies for biomedical applications.
Incidental nanoparticle characterisation in industrial settings to support risk assessment modelling
Publication . Moreno-Martín, Veronica; López, Maria; Bou, David; Fraga, Sónia; Teixeira, João Paulo; López-Lilao, Ana; Sanfélix, Vicenta; Monfort, Eliseo; Viana, Mar
Research on nanoparticle (NP) release and potential exposure can be assessed through experimental field campaigns, laboratory simulations, and prediction models. However, risk assessment models are typically designed for manufactured NP (MNP) and have not been adapted for incidental NP (INP) properties. A notable research gap is identifying NP sources and their chemical, physical, and toxicological properties, especially in real-world settings. This work aims to provide insights into the release and physico-chemical properties of INP while contributing to improving models for INP release. INP release was evaluated through a case study in a ceramic tile firing facility, where aerosol (10 nm - 10 μm) properties were determined. The Control Banding (CB) Nanotool model was applied to test outputs based on provided input parameters. Results: demonstrate the constant generation and release of INP during tile firing, with NP concentrations up to 68711/cm³ and mean diameters of 37 nm, with 95% smaller than 100 nm. Particle morphology was mostly spherical, suggesting nucleation from precursor gases as the main formation mechanism. INP chemical composition was driven by primary ceramic components, while trace elements like Ni and Ti exhibited sizedependent patterns. In vitro cell viability tests indicated low to medium cytotoxicity of PM2 aerosols, decreasing human alveolar epithelial cell viability in a concentration-dependent manner. Applying the risk model with varying input parameters revealed that the risk level (RL) based on severity scores decreased when aerosol size distribution data were used, illustrating the model’s sensitivity to input variables. We conclude on the need for comprehensive experimental datasets to support risk assessment models and achieve effective risk management strategies in real-world scenarios.