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- Exposure to mycotoxins in the Portuguese adult populationPublication . Maris, Elias; Namorado, Sónia; Chen, A.; Pero-Gason, Roger; De Boevre, Marthe; De Saeger, Sarah; Silva, Maria João; Alvito, PaulaMycotoxins are toxic fungal metabolites commonly found in food, posing health risks such as immunosuppression, carcinogenicity, and endocrine disruption. Despite regulatory limits, chronic low-level exposure remains a concern. Understanding real-life exposure in populations is essential for effective risk assessment. This study aims to investigate mycotoxin exposure among young adults in Portugal, contributing to evidence-based public health interventions. This study leveraged data and biospecimens from the INSEF-ExpoQuim survey, a cross-sectional study nested within thPortuguese National Health Examination Survey (INSEF). Data was collected via REDCap-assisted telephone interviews, covering sociodemographic and exposure-relevant variables. A subset of 295 first morning urine samples was collected from adults aged 28–39 years between May 2019 and March 2020. Urine samples were analyzed by a newly optimized and validated LC-MS/MS method targeting 40 mycotoxins and/or their corresponding metabolites in urine. Urinary creatinine was measured using a validated colorimetric method to allow adjustment and standardization of mycotoxin concentrations, ensuring accurate exposure assessment and comparability. This methodological approach enabled a robust characterization of mycotoxin exposure in a representative Portuguese population cohort.The study included 58% females and 42% males. Most participants had medium to high education, and urbanization was nearly evenly split between towns/suburbs (36.9%) and rural areas (35.9%), with fewer living in cities (27.1%). The majority were employed, and sampling was primarily conducted in summer and autumn. The number of mycotoxin co-exposures in the Portuguese population ranged from 0 to 5, with two simultaneous exposures being most common (n = 160). Among the 40 mycotoxins analysed, deoxynivalenol and tenuazonic acid were most frequently detected, with frequency of detection of 85% and 96%, respectively. This study offers robust biomonitoring data on mycotoxin exposure in Portuguese young adults using a validated LC-MS/MS method. The high prevalence of deoxynivalenol and tenuazonic acid suggests low level dietary contamination. These findings support the need for continued monitoring and the integration ofhuman biomonitoring into national food safety strategies. Detailed sociodemographic analyses are planned to further clarify exposure patterns and enable targeted public health interventions.
- Is Antisense Oligonucleotide-Mediated Exon Skipping a Potential Therapeutic Approach for Mucolipidosis II?Publication . Gonçalves, Mariana; Moreira, Luciana; Encarnação, Marisa; Duarte, Ana Joana; Gaspar, Paulo; Santos, Juliana Inês; Coutinho Maria Francisca; Prata, Maria João; Omidi, Maryam; Pohl, Sandra; Silva, Frederico; Oliveira, Paula; Matos, Liliana; Alves, SandraIntridution: Mucolipidosis II (ML II) is a Lysosomal Storage Disorder caused by N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT) deficiency, which impairs lysosomal hydrolases trafficking. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs) to promote targeted skipping of GNPTAB exon 19, which harbors c.3503_3504del, the most frequent disease-causing variant. Previously, in ML II patients’ fibroblasts, we tested ASOs to induce exon 19 skipping, successfully generating an in-frame mRNA1. Now, our aim is to determine if this in-frame transcript leads to increased GlcNAc-PT levels. Methodology: First, the GlcNAc-PT activity was measured in fibroblasts, but activity levels were similar in ML II and control fibroblasts (treated/non-treated) showing that the assay is not proper to measure endogenous levels. To overcome this, we designed 3 constructs: a WT (full GNPTAB cDNA), a del_ex19 (without exon 19) and a mutant (with the mutation c.3503_3504del) that were transfected in HEK293T cells. Then GlcNAc-PT expression was analyzed by Western Blot (WB). Also, we measured the activity of several hydrolases and evaluated the expression of α-galactosidase A (α-Gal) by WB after ASO treatment. To further validate this therapy we also generated a novel GlcNAc-PT antibody in rabbits. Results: Our results showed that HEK293T cells were able to express all the constructs. The WB of both WT and del_ex19 constructs showed bands corresponding to the α/β precursor. However, only the WT construct expressed the β-subunit, suggesting that there is no GlcNAc-PT activity in the absence of exon 19. As expected, in the delTC construct WB no α/β precursor band was detected. We also observed a slight increase in the activity of various lysosomal hydrolases in ML II fibroblasts after treatment. However, only the α-Gal values were statistically significant, but the WB analysis for this enzyme did not reveal any band in ASO-treated ML II cells. We also developed a novel antibody for GlcNAc-PT. Preliminary results showed a β-subunit band both in control and patient fibroblasts (unexpected), but in overexpression both WT and del_ex19 constructs presented α/β precursor bands. So, further assays are needed to assess its specificity. Conclusion: Our ASO-based approach effectively promotes exon 19 skipping. However, this strategy, as far as we have been able to prove, is not able to restore any GlcNAc-PT enzymatic activity. Further validation, including co-localization studies are planned to clarify these findings.
- The role of UPF1 cap-independent translation in colorectal cancerPublication . Lacerda, Rafaela; Menezes,Juliane; Elias, Adriana; Sousa, Sofia de; Romão, LuísaColorectal cancer (CRC) is one of the deadliest diseases worldwide with projections pointing towards an increase for the next two decades. Translation dysregulation of many genes contributes to CRC development, and here we are studying the role of translation dysregulation of up-frameshift 1 (UPF1) in CRC. This protein is involved in many cellular mechanisms such as nonsense-mediated mRNA decay, cell cycle progression, or telomere maintenance and homeostasis. It also works as a tumour suppressor protein in most cancers but not in CRC, in which UPF1 plays an oncogenic role. We used the Xena platform to perform in silico analyses that revealed UPF1 protein overexpressed in CRC, contrary to several other analysed cancers. Besides, UPF1 protein levels are increased in CRC compared to the counterpart normal tissues. Experimentally, we confirmed that UPF1 protein expression is maintained in different CRC cell lines under normal conditions or endoplasmic reticulum (ER) stress. To understand the mechanism underlying such maintenance, we used a bicistronic reporter construct to test whether UPF1 5’ untranslated region (UTR) can mediate alternative translation initiation and we concluded that such sequence drives cap-independent translation initiation, in both normal and stress conditions. Deletional and mutational analyses of UPF1 5’UTR showed that nucleotides 1–100 [stem-loop (SL) I] and 151–275 (SL III) — out of 275 nucleotides — are the minimal required sequences for the cap-independent translation initiation mechanism to work properly. Using RNA antisense oligonucleotides (ASOs) targeting UPF1 SL I and III, we observed a reduced UPF1 expression in HCT116 (CRC) cells, supporting the functional role of SL I and SL III in mediating cap-independent translation. Altogether, these results highlight the importance of cap-independent translation initiation in UPF1 expression regulation, in conditions that mimic the tumour microenvironment, and this might be used as a therapeutic target.
- Variants of the PKLR gene are associated with rates of hospitalization in Angolan children with sickle cell anemiaPublication . Manco, Licínio; Santos, Brígida; Faustino, Paula; Ginete, Catarina; Brito, Miguel; Arez, Ana PaulaBackground: Sickle cell anemia (SCA), caused by the c.20 A>T (p.Glu6Val) mutation in the HBB gene, is one of the most prevalent hereditary diseases in Sub-Saharan Africa. Patients with the HbSS genotype exhibit variable phenotypic expressions and disease severity, often reflected in hospitalization rates. Reduced activity of pyruvate kinase (PKR, gene: PKLR), a key enzyme in glycolysis, impacts SCA pathophysiology through 2,3-DPG accumulation and ATP deficit. This study analyzed the association of three PKLR variants with hospitalization and clinical parameters in Angolan children with SCA. Material and Methods: Sixty-three SCA children (3–12 years) were monitored in a prospective study (2019–2022). Polymorphisms rs8177970 and rs3020781 (intron 3) and rs1052177 (3' UTR) in the PKLR gene, were analyzed via PCR-RFLP. Allele frequencies and Hardy-Weinberg equilibrium (HWE) were assessed using Arlequin 3.5, and SPSS 27 was used for statistical analyses. Association studies were compared using the Mann-Whitney U test, combining heterozygous and homozygous individuals for the minor allele into a single group. Results: Minor allele frequencies for rs8177970, rs3020781, and rs1052177 were 0.214, 0.221, and 0.377, respectively, with all genotype distributions consistent with the HWE. Polymorphism rs1052177 was significantly associated with hospitalization rates (p=0.025), while rs8177970 showed a near-significant association (p=0.068). No associations were found with hematological parameters. Conclusion: This study conducted in SCA children from Angola found potential links between two PKLR SNPs and hospitalization rates. The PKLR gene may act as a genetic modifier of the clinical progression of SCA, as these SNPs can affect gene expression levels.