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- Os cílios primários regulam os níveis de tiorredoxina redutase 1 e de yH2AX em resposta a níveis elevados de glucosePublication . Marques, Rira; Paiva, Mariana; Ginete, Catarina; Nolasco, Sofia; Marinho, Susana H.; Veiga, Luisa; Brito, Miguel; Soares, Helena; Carmona, BrunoA diabetes caracteriza-se por uma anormal capacidade de controlar o nível de glucose na corrente sanguínea, podendo levar a outras complicações, tais como hipertensão, doenças cardiovasculares, e retinopatia. A desregulação dos níveis de glucose na retina tem demonstrado aumentar os níveis de peróxido de hidrogénio, conduzindo a uma rutura na barreira sanguínea da retina, uma das causas de retinopatia diabética. O cílio primário é um organelo que demonstrou ter um papel no controlo do equilíbrio energético e da homeostase da glucose. Defeitos na estrutura e função dos cílios podem resultar no desenvolvimento de várias doenças, conhecidas como ciliopatias, e que incluem fenótipos como obesidade e diabetes. Neste trabalho pretendemos estudar o papel do aumento dos níveis de glucose na montagem de cílios primários em culturas de células do epitélio pigmentar da retina (RPE-1), bem como o papel dos cílios na resposta celular aos níveis elevados de glucose. Para isso, suplementámos os meios de crescimento das células RPE-1 com diferentes concentrações de glucose (5 mM, 25 mM e 5 mM de glucose + 20 mM manitol). Estas células também foram induzidas a montar cílios antes ou depois da suplementação com a glucose. Neste estudo observámos que a suplementação de glucose não afetou o número de células ciliadas, sendo que o comprimento dos cílios foi menor em células suplementadas com 25 mM de glucose. Também avaliámos os níveis nucleares de tiorredoxina redutase 1 (TXNRD1), uma das principais enzimas intervenientes na resposta ao stress oxidativo desencadeado pela hiperglicemia, e de γH2AX, um marcador celular de quebras no DNA e de senescência celular. Observámos que os níveis nucleares de TXNRD1 e de γH2AX são afetados pela adição de glucose e que a existência de cílios modula a resposta das células em resposta a níveis elevados de glucose. Estes resultados mostram que a presença de cílios primários afeta drasticamente a resposta celular às elevadas concentrações de glucose que provavelmente induzem o stress oxidativo, podendo ter um papel crucial no desenvolvimento de retinopatia diabética.
- Are genetic modifiers the answer to different responses to hydroxyurea treatment? A pharmacogenetic study in sickle cell anemia Angolan childrenPublication . Ginete, Catarina; Delgadinho, Mariana; Santos, Brígida; Pinto, Vera; Silva, Carina; Miranda, Armandina; Brito, MiguelSickle cell anemia (SCA) is an inherited disease affecting the hemoglobin that is particularly common in sub-Saharan Africa. Although monogenic, phenotypes are markedly heterogeneous in terms of severity and life span. Hydroxyurea is still the most common treatment for these patients, and the response to treatment is highly variable and seems to be an inherited trait. Therefore, identifying the variants that might predict hydroxyurea response is important for identifying patients who will have a poorer or non-response to treatment, and the ones that are more prone to suffer from severe side effects. In the present pharmacogenetic study, we analyzed the exons of 77 genes described in the literature as potentially associated with hydroxyurea metabolism in Angolan children treated with hydroxyurea and evaluated the drug response considering fetal hemoglobin levels, other hematological and biochemical parameters, hemolysis, number of vaso-occlusive crises and hospitalizations. Thirty variants were identified in 18 of those genes as possibly associated with drug response, five of them in gene DCHS2. Other polymorphisms in this gene were also associated with hematological, biochemical, and clinical parameters. Further research examining the maximum tolerated dose and fixed-dose with a larger sample size is necessary to corroborate these findings.
- Identification of genetic variants in 65 obesity related genes in a cohort of Portuguese obese individualsPublication . Ginete, Catarina; Serrasqueiro, Bernardo; Silva-Nunes, José; Veiga, Luisa; Brito, MiguelObesity is a major public health problem, which has a strong genetic component that interplays with environmental factors. Several genes are known to be implicated in the regulation of body weight. The identification of alleles that can be associated with obesity is a key element to control this pandemic. On the basis of a Portuguese population, 65 obesity-related genes are sequenced using Next-Generation Sequencing (NGS) in 72 individuals with obesity, in order to identify variants associated with monogenic obesity and potential risk factors. A total of 429 variants are identified, 129 of which had already been associated with the phenotype. Comparing our results with the European and Global frequencies, from the 1000 Genomes project, 23 potential risk variants are identified. Six new variants are discovered in heterozygous carriers: four missense (genes ALMS1-NM_015120.4:c.5552C>T; SORCS1-NM_001013031.2:c.1072A>G and NM_001013031.2: c.2491A>C; TMEM67-NM_153704.5:c.158A>G) and two synonymous (genes BBS1-NM_024649.4:c.1437C>T; TMEM67-NM_153704.5:c.2583T>C). Functional studies should be performed to validate these new findings and evaluate their penetrance and pathogenicity. Regardless of no cases of monogenic obesity being identified, this kind of investigational study is important when we are still trying to understand the etiology and pathophysiology of obesity. This will allow the identification of rare variants associated with obesity and the study of their prevalence in specific populational groups.
- Comparative efficacy and safety of pharmacological interventions for managing sickle cell disease complications in children and adolescents: a systematic review with network meta-analysesPublication . Tonin, Fernanda; Ginete, Catarina; Ferreira, Joao; Delgadinho, Mariana; Fernandez-Llimos, Fernando; Brito, MiguelObjectives: Sickle cell disease (SCD), an inherited hemoglobinopathy that causes anemia, severe pain, and vaso-occlusive crisis (VOC), is currently recognized as a global public health concern, being the leading cause of pediatric stroke. Our aim was to synthesize the evidence on the efficacy and safety of interventions for managing SCD in this population. Methods: A systematic review with searches in PubMed, Scopus, and Web of Science was performed (April-2022). Randomized controlled trials comparing disease-modifying agents in SCD patients under 18 years old were included. For each outcome of interest, data were pooled by means of Bayesian network meta-analyses with the surface under the cumulative ranking curve analyses (SUCRA). Results were reported as odds ratio (OR) with 95% credibility intervals (CrI). Results: Seventeen trials (1982-2022) mostly from African countries (65%) and North America (53%), assessing the effect of different interventions’ regimens (hydroxyurea [n=6 trials], L-arginine [n=3], antiplatelets [n=2], immunotherapy/monoclonal antibodies [n=2], sulfates [n=2], docosahexaenoic acid [n=1], niprisan [n=1]) and placebo were included. No statistical differences among treatments were found for the main outcomes. SUCRA revealed that immunotherapy/monoclonal antibodies and hydroxyurea 20 mg/kg are potentially more effective against acute chest syndrome (83% and 76% probabilities, respectively), VOC (71% and 80%, respectively) and needing of transfusions (72% and 75%, respectively), while L-arginine (100-200 mg/kg) and placebo were more prone to these events. Although therapies were overall considered safe, antiplatelet and sulfates may lead to more discontinuations and severe adverse events (uncertainty evidence). Results were similar between age subgroups (<10 years vs. 10-19 years). Conclusions: The available evidence on the effect of drugs on managing SCD in children and adolescents is insufficient and weak. No clear definition for some outcomes exists. Hydroxyurea may remain the standard of care for this population, however, long-term well-designed, and well-reported trials comparing new immunotherapy/monoclonal antibodies should be performed.
- Genotypic diversity among Angolan children with sickle cell anemiaPublication . Delgadinho, Mariana; Ginete, Catarina; Santos, Brígida; Miranda, Armandina; Brito, MiguelBackground. Sickle cell anemia (SCA) is an inherited blood disorder that affects over 300,000 newborns worldwide every year, being particularly prevalent in Sub-Saharan Africa. Despite being a monogenic disease, SCA shows a remarkably high clinical heterogeneity. Several studies have already demonstrated the existence of some polymorphisms that can provide major clinical benefits, producing a mild phenotype. Moreover, the existence of distinct haplotypes can also influence the phenotype patterns of certain populations, leading to different clinical manifestations. Our aim was to assess the association between polymorphisms in genes previously related to SCA disease severity in an Angolan pediatric population. Methods. This study analyzed clinical and biological data collected from 192 Angolan children. Using NGS data, we classified the HBB haplotypes based on four previously described SNPs (rs3834466, rs28440105, rs10128556, and rs968857) and the genotype for the SNPs in HBG2 (rs7482144), BCL11A (rs4671393, rs11886868, rs1427407, rs7557939), HBS1L-MYB (rs66650371) and BGLT3 (rs7924684) genes. Results. The CAR haplotype was undoubtedly the most common HBB haplotype in our population. The HbF values and the ratio of gamma chains were statistically significant for almost all of the variants studied. We reported for the first time an association between rs7924684 in the BGLT3 gene and gamma chains ratio. Conclusions. The current findings emphasize the importance personalized medicine would have if applied to SCA patient care since some of the variants studied might predict the phenotype and the overall response to treatment
- The nuclear levels of thioredoxin reductase 1, gamma-H2AX, and yap are modulated by primary cilia in response to high glucose levelsPublication . Marques, Rita; Paiva, Mariana; Ginete, Catarina; Nolasco, Sofia; Marinho, Susana H.; Veiga, Luisa; Brito, Miguel; Soares, Helena; Carmona, BrunoDiabetes is a condition characterized by impaired regulation of blood glucose levels, leading to various complications such as hypertension, cardiovascular disease, and retinopathy. Diabetic retinopathy (DR), caused by a disrupted retinal blood barrier, is associated with oxidative stress resulting from dysregulated glucose levels in the retina. The primary cilium, an organelle involved in energy balance and glucose homeostasis, has been implicated in the development of various diseases known as ciliopathies, which include overlapping phenotypes such as obesity, diabetes, and retinopathy. This study aims to investigate the impact of high glucose levels on primary cilia assembly in retinal pigment epithelium (RPE-1) cell cultures and explore the role of cilia in the cellular response to high glucose levels. RPE-1 cells were grown in media supplemented with different glucose concentrations (5 mM, 25 mM, and 5 mM glucose + 20 mM mannitol), and cilia assembly was induced before or after glucose supplementation. The results revealed that glucose supplementation did not affect the number of ciliated cells, but cells supplemented with 25 mM glucose exhibited shorter cilia. To understand the role of cilia in response to high glucose levels, the nuclear levels of thioredoxin reductase 1 (TRXR1), a key enzyme involved in combating oxidative stress triggered by hyperglycemia, were evaluated. Additionally, γH2AX, a marker of DNA breaks and cellular senescence, and YAP, a Hippo pathway effector, were examined. It was observed that glucose supplementation, particularly at high levels (25 mM), influenced the nuclear levels of TRXR1, γH2AX, and YAP. Notably, the presence of cilia modulated the cellular response to high glucose levels, modulating the levels of these proteins. These preliminary findings indicate that primary cilia significantly influence the cellular response to high glucose concentrations, which are known to induce oxidative stress and potentially contribute to the development of DR.
- Effects of Quercetin in transcriptional and post-transcriptional regulation of fetal hemoglobinPublication . Canteiro, Beatriz; Mendes, Maria; Jacques, Filipa; Delgadinho, Mariana; Oliveira, Ketlyn; Ginete, Catarina; Gomes, Mário; Ribeiro, Edna; Brito, Miguel; Gomes, Anita Q.Hemoglobinopathies are a group of inherited blood disorders that primarily affect red blood cells. The most common type is known as sickle cell anemia (SCA). It is characterized by mutations in the HBB gene, which encodes the β-subunit of human hemoglobin, giving rise to hemoglobin S (HbS). When deoxygenated, HbS polymerizes in the red blood cell, giving it a sickle shape and making it rigid and fragile. Fetal hemoglobin (HbF) is the major genetic modulator of the hematologic and clinical features of sickle cell disease, an effect mediated by its exclusion from the sickle hemoglobin polymer. Fetal hemoglobin genes are genetically regulated, and the level of HbF and its distribution among sickle erythrocytes is highly variable. Currently, therapies that induce HbF are promising, such as hydroxyurea (HU). However, due to high costs for underdeveloped countries and the adverse side effects, it is important to test alternative products and develop new compounds, such as Quercetin, a natural flavonoid present in plants that has antioxidant and anti-inflammatory properties.
- Effects of Carica papaya leaf extracts in transcriptional regulation of fetal hemoglobinPublication . Mendes, M.; Canteiro, Beatriz; Delgadinho, Mariana; Oliveira, Ketlyn; Ginete, Catarina; Gomes, Mário; Ribeiro, Edna; Brito, Miguel; Gomes, Anita Q.Purpose: Sickle cell disease (SCD) is one of the most common human genetic disorders, which is caused by a single point mutation (Glu6Val) on the HBB gene. Currently, one of the treatments for this global health problem involves the induction of fetal hemoglobin (HbF). There are some drugs on the market that pharmacologically induce HbF, namely Hydroxyurea (HU), however, their safety concerns and the expensive cost in low- and middle-income countries limit their use. In this context, it is essential to study novel fetal hemoglobin-inducing compounds that have fewer adverse effects and are widely available, such as natural compounds. Therefore, the main aim of this work was to evaluate the effects of Carica Papaya methanolic leaf extracts (CPMLE) in HbF reactivation.
- How hydroxyurea alters the gut microbiome: a longitudinal study involving Angolan children with sickle cell anemiaPublication . Delgadinho, Mariana; Ginete, Catarina; Santos, Brígida; Fernandes, Carolina; Silva, Carina; Miranda, Armandina; Vasconcelos, Jocelyne Neto de; Brito, MiguelSickle cell anemia (SCA) is an inherited hematological disorder and a serious global health problem, especially in Sub-Saharan Africa. Although hydroxyurea (HU) is the leading treatment for patients with SCA, its effects on the gut microbiome have not yet been explored. In this context, the aim of this study was to investigate this association by characterizing the gut microbiome of an Angolan SCA pediatric population before and after 6 months of HU treatment. A total of 66 stool samples were obtained and sequenced for the 16S rRNA gene (V3-V4 regions). Significant associations were observed in alpha and beta-diversity, with higher values of species richness for the children naïve for HU. We also noticed that children after HU had higher proportions of several beneficial bacteria, mostly short-chain fatty acids (SCFAs) producing species, such as Blautia luti, Roseburia inulinivorans, Eubacterium halli, Faecalibacterium, Ruminococcus, Lactobacillus rogosae, among others. In addition, before HU there was a higher abundance of Clostridium_g24, which includes C. bolteae and C. clostridioforme, both considered pathogenic. This study provides the first evidence of the HU effect on the gut microbiome and unravels several microorganisms that could be considered candidate biomarkers for disease severity and HU efficacy.
- Effects of quercetin in transcriptional regulation of fetal hemoglobinPublication . Canteiro, B.; Mendes, M.; Delgadinho, Mariana; Oliveira, K.; Ginete, Catarina; Gomes, M.; Brito, Miguel; Gomes, Anita Q.; Ribeiro, EdnaPurpose: Sickle cell disease (SCD) is a genetic blood disorder that affects the shape and transport of red blood cells (RBCs) in blood vessels, leading to various clinical complications. The pharmacological reactivation of Fetal Hemoglobin (HbF) is considered to be a viable therapeutic method in SCD. In this regard, hydroxyurea (HU), a powerful ribonucleotide reductase inhibitor, is being employed as a HbF-inducing pharmaceutical. However, its cytotoxicity, carcinogenic potential, and variable effects limit its use. Thus, a major challenge today is to identify new agents, with high HbF-inducing activity, low cytotoxicity, and available in low- and middle-income countries, such as natural compounds. Quercetin, a natural flavonoid, has been identified as a potential HbF inducer. The main aim of this work was to evaluate Quercetin's role in the reactivation of fetal hemoglobin (HbF) by analyzing the expression of globin and HbF regulatory/silencing genes.