National Institute of Health Dr. Ricardo Jorge
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Translational control of the human erythropoietin expression via an upstream open reading frame in cardiac tissue
No full textCellular stress activates an integrated stress response, which includes rapid changes in global and gene-specific translation. Translational regulation of specific transcripts mostly occurs at mRNA translation initiation and is mediated via different cis-acting elements present in the mRNA 5’ untranslated region (5’UTR), such as upstream open reading frames (uORFs). uORFs modulate translation of the main ORF by decreasing the number and/or efficiency of scanning ribosomes to reinitiate at the start codon of the main ORF.
Human erythropoietin (EPO) is a glycoprotein synthesized and released mainly from the kidney, which has a key role in hematopoiesis. However, recent studies have revealed that EPO is a multifunctional molecule produced and utilized by many tissues that rapidly responds to different cell stress stimuli and tissue injuries. The 5’UTR sequence of the human EPO mRNA has one uORF with 14 codons, which is conserved among different species, indicating its potential role in translational regulation. To test whether EPO expression is translationally regulated in response to ischemia in cardiac tissue, reporter constructs containing the normal or mutant EPO 5’UTR fused to the Firefly luciferase cistron were expressed in H9c2 (heart myoblasts) and C2C12 (muscle myoblasts) cell lines. Luminometry assays revealed that the EPO uORF represses translation of the main ORF in both cell lines. Under chemical ischemia, EPO uORF-mediated translation repression is specifically released in muscle cells. In response to chemical hypoxia, translational derepression occurs in both cell lines. We are currently exploring additional mechanisms through which EPO cardioprotection effects are regulated at the translational level.This research was supported by Fundação para a Ciência e a Tecnologia – FCT (PTDC/BIM-MED/0352/2012) and Instituto Nacional de Saúde Doutor Ricardo Jorge.N/
The interplay between nonsense-mediated mRNA decay (NMD) and the unfolded protein response (UPR) in response to myocardial infarction
No full textNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and degrades mRNAs carrying premature translation-termination codons (PTCs), protecting the cell from potentially harmful truncated proteins. Furthermore, recent studies have demonstrated that NMD is also a mechanism of gene expression regulation. This feature is reflected on its ability to regulate the cell response to many stress conditions, such as endoplasmic reticulum (ER) stress, hypoxia, reactive oxygen species, and nutrient deprivation. Stress conditions, specifically ER stress, has been related to myocardial infarction, a pathological state that occurs during ischemia, where nutrient and oxygen deprivation in the heart causes aggregation of proteins in the ER and the activation of the the three arms (ATF6, IRE1α and PERK) of the unfolded protein response (UPR) to mitigate the stress and avoid cell death. Given that NMD was seen to be able to regulate the UPR and to protect cells from death during ER stress, in this work we intend to study the impact of NMD in the PERK-mediated response to ER stress induced by ischemia during myocardial infarction, and its impact to the pathophysiology of this disease. For this purpose, differentiated H9c2 cells will be used as a model of cardiomyocytes, which will help us to dissect the crosstalk between NMD and UPR in myocardial infarction-mimicking conditions. By now, we have already established the differentiation protocol for the H9c2 cell line in order to obtain mature cardiac-like cells, and we are now optimizing and establishing the experimental conditions to further develop this project.This project is partially supported by Fundação para a Ciência e Tecnologia (UID/Multi/04046/2013 to BioISI from FCT/MCTES/PIDDAC).N/
Suppression therapy as novel approach for genetic diseases and cancer
No full textPremature translation-termination codons (PTCs or nonsense codons) can arise from mutations in germ or somatic cells. The introduction of a PTC into an mRNA can trigger nonsense-mediated decay (NMD), an important mRNA surveillance mechanism that typically recognizes and degrades mRNAs containing PTCs to prevent the synthesis of C-terminally truncated proteins potentially toxic for the cell. The physiological importance of NMD is manifested by the fact that about one third of genetic disease-associated mutations generate PTCs, including beta-thalassemia.
In recent years, a novel therapeutic approach entitled suppression therapy has been developed based on low molecular weight compounds to induce the translation machinery to recode a PTC into a sense codon, the so called “readthrough”. Here, by using a model of constructs containing the firefly luciferase gene as a reporter gene for beta-globin transcripts that result from PTCs, we intend to prove the principle that the suppression therapy can restore enough beta-globin protein to outweight the disease manifestations of beta-thalassemia. Our preliminary results show that both the aminoglycoside G418 and non-aminoglycoside PTC124 do not seem to be able to suppress the nonsense mutation at codon 26 or 39 of the human beta-globin mRNA in cultured HeLa cells, as reflected on the firefly luciferase activity and protein levels assessed by bioluminescence assays and Western blot, respectively. Regarding future directions, a deeper study on the use of G418 and PTC124 as efficient suppression agents for the treatment of PTC-associated diseases will be performed as it offers a major potential to treat a wide range of inherited pathologies.This work is partially supported by Fundação para a Ciência e aTecnologia (PTDC/BIM-MEC/3749/2014).N/
An endoribonuclease of the YicC-like family delays sporulation via sRNA degradation in Clostridioides difficile
No full textClostridioides difficile CD25890 is a YicC-like endoribonuclease involved in regulating sporulation initiation, a process critical for the host-host transmission of this anaerobic pathogen. Using comparative transcriptomics we identified a small RNA, SQ528, that accumulates at higher levels in a CD25890 deletion mutant and we show that purified CD25890 cleaves SQ528 in a metal-dependent manner. Moreover, the overexpression of SQ528 increases sporulation under certain nutritional conditions phenocopying a CD25890 deletion mutant. CD25890 is an hexamer in solution and in vivo. An N-terminal domain, which self-interacts as assessed by size exclusion chromatography and a two hybrid assay, is essential for oligomerization of CD25890. A C-terminal domain harbours residues H230, E254, and E258, conserved among orthologues, important for catalysis. AlphaFold2 modelling and cryo-EM suggest an elongated barrel-like structure with an internal cavity lined with basic residues that may aid in RNA binding. We show that CD25890 forms a complex with polynucleotide phosphorylase which combines the endoribonuclease activity of the first with the exonucleolytic activity of the latter and leads to the complete degradation of SQ528. This study identifies a native substrate for the YicC-family of ribonucleases and advances our understanding of the role of CD25890 in sporulation initiation in C. difficile.This project was supported by award PTDC/BIA-MIC/29293/2017 to M.S. and has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 857203 to C.V.R. This study was financially supported by the Portuguese Fundação para a Ciência e Tecnologia (FCT), Projects MOSTMICRO-ITQB with references UIDB/04612/2020, and LS4FUTURE Associated Laboratory (LA/P/0087/2020). D.M. is the recipient of a PhD fellowship (PD/BD/143148/2019) within the scope of the PhD program INTERFACE funded by FCT. B.A.S. is the recipient of the FCT PhD4COVID grant SFRH/BD/08066/2020. This work benefited from access to the Instruct Image Processing Centre, an Instruct-ERIC centre. Financial support was provided by “Instruct-ERIC (PID 19564 and 20782)”. The microscopy data were acquired at BIC, ITQB-NOVA, Oeiras, Portugal with equipment funded by FCT, project PPBI-POCI-01-0145-FEDER-022122. Mass spectrometry data were generated by the Mass Spectrometry Unit (UniMS), ITQB/iBET, Oeiras, Portugal
Update of the safety assessment of N,N‐bis(2‐hydroxyethyl)alkyl(C8‐C18)amines (FCM No 19) and N,N‐bis(2‐hydroxyethyl)alkyl(C8‐C18)amine hydrochlorides (FCM No 20) for their use in plastic materials and articles intended to come into contact with food
No full textThe European Commission asked EFSA to review whether the authorisation of N,N‐bis(2‐hydroxyethyl)alkyl(C8‐C18)amine (FCM No 19) and N,N‐bis(2‐hydroxyethyl)alkyl(C8‐C18)amine hydrochlorides (FCM No 20) is still in accordance with Regulation (EC) No 1935/2004, as provided for in Article 12(3). The FCM Panel concluded that some uses of the substance N,N‐bis(2‐hydroxyethyl)alkyl(C8‐C18)amine (FCM No 19) are not in accordance with this Regulation, since the migration is likely to exceed the current SML(T) of 1.2 mg/kg food under certain conditions of use. Based on the provided data, the FCM Panel concluded that the FCM substance No 19, N,N‐bis(2‐hydroxyethyl)alkyl(C8‐C18)amine, is not of safety concern for the consumer if (i) the substance is used at up to 0.1% w/w as polymer production aid and as processing aid to manufacture polyolefin materials and articles of thickness up to 1 mm that are intended for contact with all types of food except infant foods. This exception for infant foods and the restriction for maximum thickness do not apply to caps of bottles; (ii) the migration does not exceed 5 mg/kg food; (iii) the source of the alkyl group is either from hydrogenated vegetable oil or synthetic from ethylene oligomers with a high degree of linear structure and (iv) the impurities do not exceed 5% w/w. As they bear unsaturation, PFAEO‐coco, PFAEO‐oleyl, PFAEO‐HT, PFAEO‐T and PFAO‐C18 do not fall within the scope of the FCM substance No 19. The information related to these substances was only considered supportive for FCM substance No 19. If they were intended to be used to manufacture FCMs, a proper application following the EFSA Guidance documents should be submitted. No uses of the FCM substance No 20, N,N‐bis(2‐hydroxyethyl)alkyl(C8‐C18)amine hydrochlorides, were claimed and no information was provided to support that the current authorisation is in accordance with the Regulation (EC) No 1935/2004
Plano de Atividades 2025
No full textConselho Diretivo do INSA: Fernando de Almeida (Presidente), Cristina Abreu Santos (Vogal).
Plano de atividades de 2025 homologado.Plano de Atividades do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA) para o ano de 2025. O INSA é um organismo público integrado na administração indireta do Estado, sob a tutela do Ministério da Saúde. Enquanto Instituto Público de interesse estratégico nacional, tem por missão contribuir para ganhos em saúde pública, enquanto Laboratório do Estado e Laboratório Nacional de Referência. Tendo por contexto o Plano Estratégico 2024-2026 e os desafios estratégicos identificados, o Plano de Atividades para 2025, reflete as prioridades institucionais e respetivos indicadores, enquanto instrumento orientador para as atividades a desenvolver no corrente ano
Rapid antigen testing and COVID-19 vaccine effectiveness in primary health care in Portugal: exploring the potential for bias
No full textA disponibilização dos testes rápidos de antigénio (TRAg) permitiu que o diagnóstico de infeção por SARS-CoV-2 possa ser feito antes ou durante uma consulta médica. Este conhecimento pode condicionar a procura de cuidados e por outro lado, o Médico de Família (MF) pode ter informação sobre o estado de infeção antes do recrutamento do doente para os estudos de efetividade vacinal. Estudos com desenho de caso-controlo teste-negativo (TND), tradicionalmente usados para estimar a efetividade da vacina contra a COVID-19, podem assim incorrer em viés se a realização dos TRAg e a consulta com o MGF variarem consoante o estado vacinal e o resultado do autoteste. Este estudo descreve os padrões de autoteste em cuidados de saúde primários e as diferenças entre indivíduos que realizaram autoteste e não, nos cuidados de saúde primários em Portugal, avaliando o potencial de viés nos estudos de efetividade vacinal contra a COVID-19. Foram incluídos indivíduos com 60 ou mais anos com Infeção Respiratória Aguda (IRA) que consultaram um MGF entre setembro de 2022 e maio de 2023. Foram recolhidos dados demográficos, de vacinação, testagem, sinais e sintomas e comorbilidades. Dos 166 indivíduos incluídos (21 casos, 145 controlos), 67 (40%) realizaram TRAg. Estes foram mais frequentemente mulheres (72% vs. 67%) e mais jovens (94% entre 60-79 anos vs. 87%). Os indivíduos que realizaram autotestes tiveram uma taxa de positividade para SARS-CoV-2 de apenas 10%, comparado com 14% entre aqueles que não realizaram autoteste. Além disso, apresentavam menor prevalência de doenças crónicas (64% vs. 79%) e foram menos vacinados na campanha vacinal sazonal (39% vs. 53%). Os resultados sugerem uma possível associação negativa entre vacinação e TRAg. A elevada proporção de indivíduos que realizaram TRAg (40%) pode gerar viés no estudo de EV, reforçando a necessidade de estudos com maior dimensão e estimativas de efetividade vacinal estratificadas de acordo com esta variável.Rapid antigen self-tests allow SARS-CoV-2 diagnosis without consulting a General Practitioner (GP). A test-negative design (TND) study estimating COVID-19 vaccine effectiveness may be biased if self-testing and GP consultation differ by vaccination status and self-test results. This study describes self-testing patterns and differences between self-testers and non-self-testers in primary care in Portugal to assess potential bias. Using TND data (September 2022–May 2023), we included patients aged 60+ with Acute Respiratory Infection (ARI) consulting a GP. GPs collected data on demographics, vaccination, self-test use, and clinical status. Cases were RT-PCR positive for SARS-CoV-2; controls were negative. Of the 166 patients included (21 cases, 145 controls), 67 (40%) performed a self-test. These individuals were more likely to be female (72% vs. 67%) and younger (94% aged 60–79 vs. 87%). Those who self-tested had a lower SARS-CoV-2 positivity rate (10% vs. 14%), a lower prevalence of chronic conditions (64% vs. 79%), and were less frequently vaccinated in the seasonal vaccination campaign (39% vs. 53%). Results suggest a potential negative association between vaccination and self-testing. Given the high self-testing rate (40%), bias may be significant. The small sample size limited further analysis, highlighting the need for larger studies with vaccine effectiveness estimates stratified by self-testing
Impact of secondhand smoke on the human proteome: searching for early health-risk biomarkers
No full textOs não-fumadores expostos ao fumo do cigarro passivo ou, simplesmente fumo passivo (FP), apresentam um risco acrescido de desenvolver diversas doenças graves. No entanto, os mecanismos moleculares que explicam estes efeitos continuam pouco esclarecidos, o que reforça a necessidade de identificar biomarcadores capazes de avaliar o risco associado a esta exposição.
Neste estudo, analisámos o proteoma do epitélio nasal e do plasma de indivíduos não-fumadores saudáveis expostos ao FP no local de trabalho, num contexto ainda enquadrado pela Lei n.º 37/2007, utilizando uma abordagem proteómica ‘shotgun’ por espectrometria de massa.
No epitélio nasal, observámos um aumento de proteínas envolvidas em vias centrais do metabolismo energético, como a Gliceraldeído-3-fosfato desidrogenase (GAPDH) e a Triosefosfato isomerase (TPI1), sugerindo uma possível reprogramação metabólica induzida pela exposição. Identificámos também uma diminuição da tubulina beta-4B (TUBB4B), relacionada com a organização do citoesqueleto, e um aumento da proteína anti-apoptótica SERPINB3, apontando para alterações em processos de morte e sobrevivência celular.
No plasma, destacaram-se o aumento da Butirilcolinesterase (BChE) e a diminuição da Proteína de ligação à vitamina D (GC), ambas associadas à resposta a xenobióticos e a processos de lesão tecidular. Foram ainda detetadas alterações em proteínas reguladoras da inflamação sistémica, como C1R, C1QC, HRG e PROS1. A expressão diferencial de APOA4 e SERPINF2 sugere, adicionalmente, a ativação de mecanismos relacionados com risco aterotrombótico.
Em conjunto, estes resultados contribuem para aprofundar a compreensão das vias biológicas que ligam a exposição ao fumo passivo ao risco acrescido de cancro e de doenças cardiovasculares, e apresentam um conjunto promissor de potenciais biomarcadores para avaliação do risco associado à exposição ao FP.Non-smokers exposed to secondhand smoke (SHS) have an increased risk of developing several serious diseases. However, the molecular mechanisms underlying these effects remain poorly understood, underscoring the need to identify biomarkers capable of assessing the risk associated wi th this exposure.
In this study, we analysed the nasal epi thel ial and plasma proteomes of healthy non-smokers exposed to SHS in the workplace, in a context still regulated by Law No. 37/2007, using a shotgun proteomics approach by mass spectrometry.
In the nasal epi thel ium, we obser ved an increase in proteins involved in key energy metabol ism pathways, such as Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and Triosephosphate Isomerase (TPI1), suggesting a possible metabolic reprogramming induced by exposure.
We also identified a decrease in beta-tubul in 4B (TUBB4B), related to cytoskeletal organisation, and an increase in the anti-apoptotic protein SERPINB3, indicating alterations in cell death and survival pathways.
In plasma, we found an elevation of Butyrylcholinesterase (BChE) and a reduction of Vitamin D-binding Protein (GC), both linked to xenobiotic response and tissue injury processes. We also detected changes in regulators of systemic inflammation, such as C1R, C1QC, HRG, and PROS1. Additionally, the differential expression of APOA4 and SERPINF2 suggests activation of mechanisms related to atherothrombotic risk.
Together, these findings deepen our understanding of the biological pathways linking secondhand smoke exposure to the increased risk of cancer and cardiovascular disease, and highlight a promising set of potential biomarkers for assessing SHS- related health risk
Trichinellosis: study of a human population potentially exposed to infection in Portugal, 2023-2024
No full textA triquinelíase é uma zoonose parasitária de origem alimentar causada por nemátodas do género Trichinella, frequentemente transmitida através do consumo de carne curada, mal cozinhada ou inadequadamente congelada contendo larvas infetantes. Esta doença tem um impacto significativo na saúde pública e constitui um desafio global no âmbito da segurança alimentar, tendo sido detetada em animais silvestres de 66 países. Em Portugal, a triquinelíase é uma doença de declaração obrigatória, não havendo registo de qualquer caso humano desde 1987. Esta ausência contrasta com a situação observada em países vizinhos, como Espanha, que partilha práticas alimentares e perfis epidemiológicos semelhantes ao nosso país.
Este estudo tem como objetivo avaliar a presença de anticorpos do tipo imunoglobulina G (IgG) anti-Trichinella spp. em 200 indivíduos potencialmente expostos ao consumo de carne mal cozinhada ou não inspecionada. A deteção foi realizada através do ensaio de imunoabsorção enzimática (ELISA), sendo os resultados positivos ou equívocos posteriormente confirmados por imunoblot.
Dos 200 indivíduos em estudo foi confirmado um caso positivo, resultando numa seroprevalência de 0,5%. Além disso, o caso confirmado apresentou reatividade específica para os antigénios de Toxocara sp., sugerindo uma possível reatividade cruzada ou co- infeção.
A baixa seroprevalência de anticorpos contra Trichinella spiralis observada na população estudada, sugere que a triquinelíase humana é rara em Portugal. No entanto, existe a possibilidade de um subdiagnóstico e consequente subnotificação o que, por si só, reforça a importância de vigilância ativa e continuada das zoonoses parasitárias.Trichinellosis is a foodborne parasitic zoonosis caused by nematodes of the genus Trichinella, of ten transmitted through the consumption of
cured, undercooked or inadequately frozen meat containing infective larvae. This disease has a significant impact on public health and poses a global challenge to food safety, it has been detected in wild animals in 66 countries. In Portugal, trichinellosis is a notifiable disease and no human cases have been reported since 1987. This contrasts wi th the situation in neighbouring countries such as Spain, which has similar food practices and epidemiological profiles to our country.
The aim of this study is to assess the presence of immunoglobul in G (IgG) antibodies against Trichinella spp. in 200 individuals potentially exposed to the consumption of undercooked or uninspected meat. Detection was performed using an enzyme- linked immunosorbent assay (ELISA), with positive or equivocal results subsequently confirmed by immunoblot.
Of the 200 individuals in the study, one positive case was confirmed, resulting in a seroprevalence of 0.5%. Additionally, the confirmed case exhibited specific reactivity to Toxocara sp. antigens, indicating potential cross- reactivity or a co infection.
The low seroprevalence of antibodies against Trichinella spiralis in the studied population suggests that human trichinellosis is rare in Portugal.
However, underdiagnosis and underreporting are possible, which highlights the importance of actively and continuously surveilling parasitic zoonose